Cance-r··-:·.
`Research
`
`"<> '
`
`•
`
`November 15, 2010 • Volume 70 • Number 22 -
`
`CLINICAL STUDIES
`
`8994
`
`The Ability of Bio markers to Predict
`Systemic Progression in Men with
`High-Risk Prostate Cancer
`Treated Surgically Is Dependent on
`ERG Status
`R. Jeffrey Karnes, John C. Cheville,
`Cristiane M. Ida, Thomas J. Sebo, Asha A. Nair,
`· Hui Tang.Jan-Marie Munz, Farhad ~osari,
`and George Vasmatiys
`·
`
`Precis: The study presents TOP2A ps a
`superior prognostic marlrer for prostate
`cancer and shows that its predictive ability is
`dependent on cancer subtype.
`
`Repeat Dose Study of the Cancer
`Chemopreventive Agent Resveratrol
`in Healthy Volunteers: Safety,
`Pharmacokinetics, and Effect on the
`Insulin-like Growth Factor Axis
`Victoria A. Brown, Ketan R. Patel,
`Maria Viskaduraki, James A. Crowell,
`Marjorie Perloff, Tristan D. Booth,
`Grygoriy Vasilinin, Ananda Sen,
`Anna Maria Schinas, Gianfranca Piccirilli,
`Karen Brown, William P. Steward,
`Andreas J. Gescher, and Dean E. Brenner
`
`Precis: This study di!firi1 suiJ.able doses,
`accompanying blood levels, and potential
`efficacy biomarkers which might help to
`optimize future canw· chemoprevention
`intervention studies a/the red wine .
`constiJ.uent resveratrol
`
`BREAKING ADVANCES
`8971 I Highlights from Recent
`
`Cancer Literature
`
`REVIEWS
`
`8973
`
`Breast Cancer Stem Cells:
`Something Out of Notching?
`Hannah Harrison, Gillian Farnie,
`Keith R. Brennan, and Robert B. Clarke
`
`8977
`
`Turning on a Fuel Switch of Cancer:
`hnRNP Proteins Regulate Alternative
`Splicing of Pyruvate Kinase mRNA
`Mo Chen,Jian Zhang, andJames L Manley
`
`9003
`
`PRIORITY REPORTS
`
`8981
`
`8988
`
`Inhibition of Glutaminase
`Preferentially Slows Growth of
`Glioma Cells with Mutant IDBI
`Meghan J. Seltzer, Bryson D. Bennett,
`Avadhut D. Joshi, Ping Gao, Ajit G. Thomas,
`Dana V. Ferraris, Takashi Tsukamoto,
`Camilo J. Rojas, Barbara S. Slusher,
`Joshua D. Rabinowitz, Chi V. Dang,
`and Gregory J. Riggins
`
`Precis: IDHJ mutations create a higher
`dependence of glioma cells on o,.-kctoglutaratc,
`relative to normal cells, that could be
`therapeutically exp/oiled by targeting
`glutamine metabolism
`
`Heat Shock Protein 60 Regulation of
`the Mitochondrial Permeability
`Transition Pore in Tumor Cells
`Jagadish C. Ghosh, Markus D. Siegelin,
`Takchiko Dohi, and Dario C. Altieri
`
`l'recis: Findings implicate a chaperone network
`in controlling miluchondrial permeability
`transition and apaptusis iJ, cancer cells.
`
`A Journal of the American Association for Cancer Research
`
`V
`
`Miltenyi Ex. 1043 Page 1
`
`

`

`L}fftl
`
`Multiple Injections of Electroporated Autolog·ous T Cells
`Expressing a Chimeric Antigen Receptor Mediate
`Regression of Human Disseminated Tumor
`Yangbing Zhao 1. Edmund Moon2
`, Cannine Carpenito 1, Chrystal M. Paulos 1, ~i~ojun· Liu\":.:
`Andrea L. Brennan 1, Anne Chew 1, Richard G. Carroll 1. John Scholler 1,
`·
`·.
`;_
`Bruce L. Levine1, Steven M. Albelda2
`, and Carl H. June1
`
`·. •.,
`
`Abstract
`
`Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive
`T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production
`have lifl)ited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be
`gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe
`platform for adoptive immunotherapy, w.e first optimized the vector backbone for RNA in vitro transcription
`to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could
`be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells m1:(cid:173)
`diated regression of large vascularized flank mesothelioma tumors in NOD/scid/'yc(-/-) mic~. Dramatic tu(cid:173)
`mor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been
`growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated
`with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes
`showing that autologous T cells from cancer patients can be engineered to provide an effective therapy
`for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are
`a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that
`may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the ther(cid:173)
`apeutic index of T cells engineered to express powerful activation domains without the associated safety
`concerns of integrating viral vectors. Cancer Res; 70{22); 9053-61. ©2010 AACR.
`
`1.
`
`Introduction
`
`Adoptive transfer of CTLs has shown great promise in
`both viral infections and cancers. After many years of disap(cid:173)
`pointing results with chimeric antigen receptor (CAR) T-cell
`therapy, improved culture systems and cell engineering tech(cid:173)
`nologies are leading to CAR T cells with more potent anti(cid:173)
`tumor effects (1). Results from recent clinical trials indicate
`improved clinical results with CARs introduced with retrovi(cid:173)
`ral vectors (2, 3). Perhaps not surprisingly, these CAR T cells
`
`Authors' Affiliations: 1Abramson Family Cancer Research Institute and
`Department of Pathology and Laboratory Medicine and 2Thoracic
`Oncology Research Laboratory and Division of Pulmonary, Allergy, and
`Critical Care Medicine, University of Pennsylvania School of Medicine,
`Philadelphia, Pennsylvania
`Note: Supplementary data for this article are available at Cancer
`Research Online (http://cancerres.aac~ournals.org/).
`Y. Zhao and E. Moon contributed equally to this work
`Corresponding Authors: Carl H. June or Yangbing Zhao, University of
`Pennsylvania, 551 BAB 2/3, 421 Curie Boulevard, Philadelphia, PA
`19104-6160. Phone: 215-746-5133; Fax: 610-646-8455; E-mail: cjune@
`exchange.upenn.edu or yangbing@exchange.upenn.edu.
`doi: 10.1158/0008-5472.CAN-10-2880
`©2010 American Association for Cancer Research.
`
`also exhibit enhanced toxicity (4, 5). Recent editorials have
`discussed the need for safer CARs ( 6, 7).
`The receptor transfer strategies described above used ret(cid:173)
`roviral vector transduction that results in stable genomic in(cid:173)
`tegration of the transgene. This allows for constitutiv~
`expression of the transgenic receptors. f-1'owever, the integra(cid:173)
`tion of the provirus into the genome bears the risk of inser(cid:173)
`tional mutagenesis and, at least theoretically, malignant
`transformation of the transduced cells. In addition, stable ex(cid:173)
`pression of the transgene may be a disadvantage when unin(cid:173)
`tended cross-reactivity of the transgenic immunoreceptor
`results in severe adverse ~ffects as reported recently (5, 8).
`Here, we report that by combining a robust T-cell culture
`system (9) with the optimized mRNA CAR electroporation
`protocol described herein, we have developed a platform
`that has the potential to increase the therapeutic window
`with CARs that contain increasingly potent signaling do(cid:173)
`mains.• Using good manufacturing practice (GMP)-grade
`RNA encoding a CAR against m esothelin, a glycosyl(cid:173)
`pbosphatidylinositol-linked molecule that is overexpressed
`on ovarian and pancreatic cancer and mesothelioma (10),
`we show robust a ntitumor effects in preclinical models.
`Most notably, significantly prolonged survival and reduced
`tumor burden was observed in treated mice compared with
`
`www.aacrjournals.org
`
`Al({ AmeiicanA.ssocialio11 for Cmu:er Research
`
`9053
`
`Miltenyi Ex. 1043 Page 2
`
`

`

`control groups, even when using autologous T cells from a
`patient with advanced metastatic cancer. Electroporation of
`T cells with optimized RNA CARs provides a novel and
`cost-efficient platform for the treatment of cancer without
`the associated safety concerns of integrating gene vectors.
`
`Materials and Methods
`
`Construction of in vitro transcription mRNA
`vectors for CARs
`Mesothelin (ssl} and CD19-specific CARs (11, 12) were op(cid:173)
`timized as described in detail in Supplementary Materials
`and Methods.
`
`RNA in vitro transcription
`Three RNA in vitro transcription (IV'!') systems were used
`to optimize RNA expression in T cells as described in detail
`in Supplementary Materials and Methods.
`
`T-cell culture
`Anonymous healthy donors donated lymphocytes at the
`University of Pennsylvania Apheresis Unit after informed
`consent under an Institutional Review Board- approved pro(cid:173)
`tocol, and T cells were purified by elutriation. In some experi(cid:173)
`ments, we used cryopreserved T cells and tumor cells from
`the same patient. "Patient 108" had malignant mesothelioma.
`As part of an earlier clinical trial, this patient underwent leu(cid:173)
`kapheresis and had tumor cells generated from his malignant
`pleural effusion. T cells were activated by addition of CD3/
`CD28 beads (Invitrogen) and a single cycle of stimulation
`as described (9). For the experiment shown in Fig. 5, patient
`108 T cells were stimulated with irradiated artificial antigen(cid:173)
`presenting cells expressing 4-lBBL and loaded with anti-CD3
`monoclonal antibody (mAb} OKT3 and CD28 mAb 9.3 as
`described (13). T cells were maintained at a density of
`0.8 x 106 to 1 x 106 cells/mL in RPMI 1640 with 10% FCS
`and 1% penicillin-streptomycin (RIO) after bead stimulation.
`
`RNA electroporation of T cells
`Activated T cells were electroporated on day 10 of culture
`as described in Supplementary Materials and Methods.
`
`Flow CTL
`A slightly modified version of a flow cytometry cytotoxicity
`assay was used (14).
`
`Mouse xenograft studies
`Studies were performed as previously described with cer(cid:173)
`tain modifications (15, 16) as described in Supplementary
`Materials and Methods.
`
`Statistical considerations
`Analysis was performed with STATA version 10 (Stata(cid:173)
`Corp) or Prism 4 (GraphPad Software}. In vitro data represent
`means of duplicates, and comparisons of means were made
`via Mann-Whitney test. For comparison among multiple
`groups, Kruskal-Wallis analysis was performed with Dunn
`
`.r·: .
`. .
`multiple comparison tests to compare individual groups. Sur-
`vival curves were compared using the, log-rank test with a
`Bonferroni correction for comparing multiple curves.
`
`Results
`
`Electroporation of ~A C~s mediates variable
`expression in stinutlated T cells
`We have previously reported that anti~m~sothelin ssl scFv
`CARs with c,ombinations of CD3~, CD28, and 4-lBB activa(cid:173)
`tion domains are highly and stably expressed in T cells when
`introduced using lentiviral vector technology (11). Human
`T cells were activated for 10 days as previously described (9),
`and as the cells returned to a near resting state, they were
`electroporated with RNA encoding the ssl scFv with the pre(cid:173)
`viously described combinations of signaling moieties. We
`found that the level of transgene expression was not uniform
`(Supplementary Fig. SI}, as T cells electroporated with CAR
`bearing CD3~ alone (ssl-z) showed the highest transgene
`expression, followed by nearly e~uivalent levefs of ssl-28z
`(CD28 + CD3~} and ssl-bbz (4-lBB + CD3~} expression.
`Because "second-generation" CARs containing costimulation
`domains seem superior in several preclinical and early-stage
`clinical trials when expressed with viral vector systems (11,
`12, 17, 18), we decided not to optimize expression of the
`"first-generation" ssl-z CAR Rather, the second-generation
`ssl-bbz and CD19-bbz CARs were chosen for further optimi(cid:173)
`zation using RNA electroporation because they are being
`tested in a clinical trial using lentiviral vector technology
`( Clinicaltrials.gov NCT00891215).
`
`Optimization of Ri~A constructs improves transgene
`expression in stimulated T cells
`Structural modification of noncoding regions by incorpo(cid:173)
`ration of two repeats of 3' untranslated regions (UTR} from
`[3-globulin and longer poly(A) sequences has been shown to
`enhance RNA stability, translational efficiency, and the
`function of RNA-transfected dendritf cells (19). However,
`these strategies have not been systematically evaluated in
`RNA-electroporated T cells. To test if this approach applies
`to human T lymphocytes, we modified our IVT vector
`(pGEM-sslbbz.64A) by adding 5'UTR (SP163) or 3'UTR
`[two ·repeats of 3'UTR derived from human [3-globin
`(2bgUTR} or a prolonged poly(A) (150A} sequence as shown
`in Fig. lA]. The SP163 translational enhancer is derived
`from the 5'UTR of the vascular endothelial growth factor
`gene and is reported to increase expression levels 2- to
`5-fold compared with promoter alone (20). RNA made from
`these constructs was electroporated into stimulated T cells.
`As shown in Fig. lB, compared with our b asic fVT con(cid:173)
`struct containing a 64-poly(A) tract, addition of 31UTR from
`[3-globulin (2bgUTR) and longer poly(A) (150A} tailing en(cid:173)
`hanced Lhe t.ransgene expression, especially when combined
`(2bgUTR.150A). In contr ast, incorporation of the SPJ63 se(cid:173)
`quence at the 5' encl of ssl-bbz repressed transgene expres(cid:173)
`sion, which might be clue to reduced capping efficiency
`when the SPJ 63 sequence was added.
`
`9054 Cancer Res; 70(22) November 15, 201 O
`
`Cancer Research
`
`Miltenyi Ex. 1043 Page 3
`
`

`

`A
`pGEM-ss1 bbz.2bgUfR.64A
`pGEM-SP163.sslbbz64A
`
`B
`
`• 2bgUTR.64A
`SP163.64A
`2bgUTR150A
`150A
`64A
`
`800
`
`700
`
`600
`
`500
`
`U::: 400
`~
`
`300
`
`200
`
`100
`
`0
`
`-+- 2bgUTR64A
`-o- SP163. 64A
`-+- 2bgUTR. 150A
`....,_ 150A
`-lir- 64A
`-4-NoEP
`
`3.5 h
`
`Day 1
`
`Day 2
`Time after EP
`Figure 1. Optimization of mRNA by modttication of the UTRs confers high-level expression of CARs in electroporated T cells. A, a schematic representation
`of ss1 -bbz construct with different modifications of S'UTR or 3'UTR. pGEM-based IVT vector containing ss1-bbz (pGEM-ss1 bbz.64A) was modified
`as described in Materials and Methods to add a 3'UTR {2bgUTR.64A), a 5'UTR (SP163.64A), a longer poly{A) tail (150A), or both 3'UTR and longer
`poly(A) (2bgUTR.150A). B, RNA made from the modified constructs was electroporated into T cells and the transgene expression was followed by flow
`cytometry. Left, histograms of the transgene expression at day 1 after electroporation; right, mean fluorescence intensity (MFQ of the CAR for 4 d after
`electroporation. Experiments are representative of at least two independent experiments.
`
`Day 3
`
`Day 4
`
`Optimization of the 5' cap structure enhances
`the expression and function of CARs in
`electroporated T cells
`The 5' cap located at the end of mRNA molecule consists
`of a guanine nucleotide connected to the mRNA via a 5' to 5'
`triphosphate linkage. Several cap structures have been de(cid:173)
`scribed, including caps O and 1 (21). Several methods have
`been used to incorporate the 5' cap structure onto the trans(cid:173)
`gene and poly(A) tail construct. Commercially available sys(cid:173)
`tems incorporate cap O or 1 using cotranscriptional or
`enzymatic approaches to produce capped mRNA. This pro(cid:173)
`cess is important to optimize to enhance translational effi(cid:173)
`ciency and because of the considerable expense of the
`various capping systems (see Supplementary Materials and
`Methods). RNA made using the different capping systems
`was electroporated into stimulated T cells, and the transgene
`expression was monitored by flow cytometry (Fig. 2A and B).
`The results showed that the transgene expression of T cells
`electroporated with RNA capped by anti-reverse cap ana(cid:173)
`logue (ARCA) was 3-fold higher than regular cap (RC) ana(cid:173)
`logue capped fu'lfA at 4 hours. The transgene persistence of
`ARCA capped RNA was also improved, as at day 5 after elec(cid:173)
`troporation >50% of the T cells still expressed the CAR as
`shown in Fig. 2B.
`
`www.aacrjournals.org
`
`We next compared enzymatic addition of caps O and I to
`nonenzymatic addition of the ARCA The potential advantage
`of using the capping enzyme (CE) system is that this ap(cid:173)
`proach includes CE and mScript 2' -0-methyltransferase that
`work together to produce the capl structure, which is very
`similar to ARCA and provides superior translation efficiency
`in many in vivo systems. To evaluate the efficiency of cap O or
`1 RNA encoding ssl-bbz, human T cells were electroporated
`with RNA made by ARCA, CE, capl CEs, or CEs plus addi(cid:173)
`tional poly(A). As shown in Fig. 2C, the CAR expression using
`capl RNA electroporation was equivalent to ARCA IVT
`mRNA The transgene expression was further enhanced by
`incorporation of the longer poly(A) tail, consistent with the
`results in Fig. 1.
`One potential functional advantage of optimize~ IVT
`RNA is that CAR expression could be sustained, as transla(cid:173)
`tion of additional CAR could lead to more persistent ex(cid:173)
`pression and overcome downregulation induced by target
`recognition or homeostatic e).-pansion. Activated T cells
`were electroporated with various RNA preparations encod(cid:173)
`ing ssl-bbz, and then cocultured with K562-rneso or control
`K562-CDl9 target cells for 2 days (Supplementary Fig. S2).
`T cells electroporated with ARCA and CEI or CEl+A capped
`ssl-bbz RNA could still maintain their transgene expression
`
`Cancer Res; 70(22) November 15, 2010
`
`9055
`
`1,
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`Miltenyi Ex. 1043 Page 4
`
`

`

`after being stimulated with the K562-meso cell line com(cid:173)
`pared with the same T cells cocultured with control target
`cells. In contrast, T cells electroporated with ssl-bbz RNA
`capped by the RC analogue did not have detectable CAR
`on the surface after cocultured with antigen-bearing target.
`Based on the above results and other data (data not
`shown), we concluded that RNA capped with ARCA or with
`capl and a long poly(A) tail is the best RNA production sys(cid:173)
`tem among the RNAs tested. For large-scale GMP production
`of IVT RNA, when the production cost is also considered,
`cap l is preferred.
`
`In vitro function of optimized IVT RNA CARs
`RNAs prepared from both plasmids bearing parental or
`internal ORF-free CAR sequences were electroporated into
`T cells, and it ':Vas found that the transgene expression
`from the RNAs with internal ORF-free electropQrated T cells
`was equivalent to the T cells electroporated with RNAs y.rith
`parental sequences (Supplementary Fig. S3) at · 20 hours
`after electroporation. However, substantial prolongation ·of
`CAR expression was observed in activated T cells electro(cid:173)
`porated with clinical-grade RNA generated from internal
`ORF:free pD-A.ssl.OF or pD-A.19.OF RNAs using the CE
`system that incorporated both capl and prolonged poly(A)
`into the IVT RNAs (Fig. 3). Transgene expression of the
`optimized IVT RNA could be detected as long as 7 days
`after RNA electroporation for both meso and CD19 RNA
`CARs as shown in Fig. 3C.
`Previous studies have shown that 4-lBB is upregulated on
`cos• T cells after T-cell receptor stimulation (22). We incu(cid:173)
`bated bulk T cells electroporated with ssl-bbz or CD19-bbz
`RNA with target cells expressing either mesothelin or CD19,
`and found robust upregulation of 4-lBB, particularly on cos•
`T cells, which was target specific (Fig. 3A). The T cells expressing
`
`RNA CARs aiso secreted s~bsfanti;,j iiino'~ts of interleukin-2
`(IL-2) and t~anslocated CD107a on target-specific recogni(cid:173)
`tion (Fig. 3B and D). Finally, in a flow-based lytic assay, we
`found that both CD19 (19.OF) and ssl (ssl.OF) CAR RNA(cid:173)
`electroporate'd T cel)s· could specifically lyse target cells
`efficiently (Supi,letn_eritary-fi~. S4).
`. . ·.--·
`RNA-electroporate4 T cells·med.iate regression of
`human d.isseminated:mesothelioma xenografts
`A pilot experiment wa~ _first conducted· to evaluate the
`therapeutic potential of T cells expressing optimized RNA
`CARs in mice bearing large pre-established tumors. Mesothe(cid:173)
`lin-positive tumors were established in NSG mice as previ(cid:173)
`ously reported (11). Sixty-six days after tumor inoculation,
`10 x 106 to 15 x 106 ssl-bbz RNA CAR-electroporated T cells
`from a healthy donor were injected intratumorally, twice
`weekly for 2 weeks. The biweekly administration schedule
`was based on the in vitro expression data shown in Fig. 3.
`As seen in Fig. 4A, the tumors regressed in the mice treated
`with ssl RNA- electroporated T cells, whereas progressive tu(cid:173)
`mor growth was · observed in the control group of _mice. At
`the time the mice were sacrificed> on day 98, tumor size
`was substantially smaller in all of the mice treated with elec(cid:173)
`troporated T cells than that of the mice treated with saline
`(Supplementary Fig. S5). These results indicate therapeutic
`potential of multiple injections of RNA CAR T cells; however,
`they are not as potent in the same tumor model using lenti(cid:173)
`viral transduced T cells, where two intratumoral injections of
`T cells were able to cure most mice (11).
`·
`We developed the Ml08-Luc model to test if RNA CAR(cid:173)
`electroporated T cells are capable of treating mice bearing
`large disseminated tumors. Ml08 parental cells were stably
`transduced with firefly luciferase to allow for biolumines(cid:173)
`cence imaging (BLI), and in preliminary experiments, we
`
`A
`350
`
`300
`
`250
`
`- 200
`u..
`:E 150
`
`100
`
`50
`
`0
`
`4h D1 D2 D3 D4 D5 D6
`Time after EP
`
`B
`C ·* 100
`CII a so
`
`1h
`
`)(
`Cl)
`a, 60
`C
`CII
`~ 40
`C
`E
`I- 20
`';f!.
`
`0
`
`C soo
`
`500
`
`400
`
`;;: 300
`:ii:
`
`200
`
`100
`
`0
`
`,._CE
`...,...No RNA
`
`4h D1 D2 D3
`D4 D5 D6
`Time after EP
`
`.
`
`I .... ARCA
`-B-CE
`..... CE+A
`-e-CE1
`...... CE1+A
`-N-NoRNA
`
`4h
`
`Day 1
`Day 2
`Time after EP
`
`Day 3
`
`Figure 2. Optimization of RNA capping enhances and sustains CAR expression on electroporated T cells. A, T cells were electroporated with IVT RNA
`capped by the indicated capping method, including using RC analogue, ARCA, or CE at a fixed RNA dose of 2.5 µg/100 µLT cells. Transgene expression
`was monitored by measuring MFI using flow cytometry at the indicated times after electroporation (EP). B, T cells from the above experiment were
`monitored by fiow cytometry to determine the fraction of cells expressing the transgene. C, T cells electroporated with IVT RNA encoding ss1-bbz capped
`by different capping methods, including ARCA, CE, CE with addition poly(A) (CE+A), CE system-generated cap1 RNA (CE1), or CE system-generated
`cap1 RNA plus enzymatic poly(A) (CE1+A) at an RNA dose of 10 µg RNA/100 µLT cells. Transgene expression was monitored by flow cytometry (MFI)
`for 3 d after electroporation. Experiments are representative of two independent experiments.
`
`9056 Cancer Res; 70(22) November 15, 2010
`
`Cancer Research
`
`Miltenyi Ex. 1043 Page 5
`
`

`

`ss1.OF
`
`pDrive.ss1
`
`19.OF
`
`pDrive.19
`0.6
`0.1
`
`No EP
`0.2
`
`K562-meso
`
`· ......
`
`. .
`
`C
`100
`80
`to
`~
`20
`
`A
`
`ID
`ID
`.....
`I
`'<t
`
`B 1sooo
`
`12000
`
`::::i'
`E
`O>
`.e, 8000
`N
`I
`~
`
`4000
`
`CDB
`
`■ K562-meso
`ISi K562-CD19
`
`0
`
`~ct, ,~ .,?
`
`"'
`
`i;t ~4
`
`0.1
`
`0.3
`
`. 10'
`
`- 101
`
`11•
`
`11'
`
`K562-CD19
`
`100
`80
`60
`40
`20
`
`10°
`
`101
`
`102
`
`10•
`
`10'
`
`6h
`Day 1
`
`Day2
`
`Day3
`
`Day4
`
`Day5
`
`Day6
`
`Day7
`
`D
`
`• ...
`0 c
`u
`
`CD8
`
`K562-CD19
`
`K562-meso
`
`~It.I
`
`Figure 3. Sustained RNA CAR expression and function using RNA generated from regulatory-compliant vector constructs. Four hours after electroporatlon,
`the T cells electroporated with the indicated RNA were cocultured with K562-meso or K562-CD19 for 16 h. A, antigen-specific T-cell activation was
`detected by the induction of 4-1 BB expression. B, IL-2 production was measured by ELISA. C, stimulated T cells were electroporated with clinical-grade
`RNA (10 µg RNA/100 µLT cells) generated from pD--A.ssl.OF (top) or pD-A.19.0F (bottom) and the transgene expression was monitored at the time
`as indicated. D, 1 d after electroporation, RNA CAR T-cell function was tested by measuring CD107a surface translocatlon after T cells expressing the
`indicated RNA CAR were cocultured for 4 h with K562-CD19 or K562-meso targets. The effector cells were gated on CD3. Experiments are representative
`of at least two independent experiments.
`
`found that NSG mice develop widely disseminated disease
`with progressive ascites and that all mice die or become
`moribund by day 100 (data not shown). NSG mice (n = 18)
`were injected with Ml OS-Luc, and they were randomized into
`three i.p. treatment groups. On day 58 day after tumor injec(cid:173)
`tion, when all mice had large vascularized tumors with
`ascites and metastatic nodules lining the peritoneal cavity,
`ssl-bbz RNA CAR- electroporated T cells from a healthy
`donor were injected (i.p.) into the mice, twice weekly, for 2
`weeks. As a control for CAR specificity, a group of mice was
`injected with CD19-bbz RNA CART cells, and another group
`was treated with saline. Tumor burden in the ssl -bbz RNA
`CAR group progressively decreased from baseline on day
`53. Furthermore, on day 78 after tumor inoculation, the tu(cid:173)
`mor growth in the ssl-bbz RNA CART-cell-treated group
`was significantly repressed (P < 0.01) compared with both
`saline or CD19-bbz RNA T-cell-treated groups (Fig. 4B). In
`a side by side experiment, a mouse treated with ssl -bbz
`CAR T cells expressed using a lentiviral vector exhibited a
`more robust treatment effect (Fig. 4C), similar to our previ-
`
`ously published data (11). However, the ssl-bbz RNA CAR
`T-cell-treated group had a survival ,ivantage and a sigrufi(cid:173)
`cant slo~ng of tumor growth between days 72 and 92, at
`which point all of the control mice died from tumor progres(cid:173)
`sion (Fig. 4C).
`
`RNA CAR-electroporated autologous T cells mediate
`regression of disseminated m'esothelioma
`The above studies indicate that biweekly injections of RNA
`CAR T cells can control advanced flank and i.p. tumors, and
`that the inhibition is dependent on the CAR specificity, as
`T cells expressing the CD19 RNA CAR were not effective.
`However, the T cells in those experiments were obtained
`from healthy donors and were allogeneic to t he tumor.
`Because allogeneic antitumor effects were observed with
`repeated long-term administration of RNA CART cells (data
`not shown), autologous peripheral blood mononuclear cells
`froin the patient from whom the M108 tumor was derived
`were used. T cells were stimulated and electroporated using
`GMP grade RNA. Thirty NSG mice were randomized into
`
`www.aacrjournals.org
`
`Cancer Res; 70(22) November 15, 2010
`
`9057
`
`Miltenyi Ex. 1043 Page 6
`
`

`

`-<>-- Salina
`..... ss1-bbz
`
`400
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`300
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`
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`
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`
`Day 53
`
`Day 78
`
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`
`' 2
`106
`7
`4
`
`2
`105
`7
`4
`
`2
`p/s/cm2/sr
`
`Figure 4. Regression of advanced vascularized
`tumors in mice treated with RNA CAR T cells.
`A, flank tumors were established by M108
`Injection (s.c.) in NOD/scid/yc(-/-) (NSG)
`mice (n = 6). Sixty-six days after tumor
`inoculation, 'rice were randomized to equalize
`tumor burden and treated with ss1 -bbz
`RNA-electroporated T cells. The T cells (1 O x 106
`to 15 x 106) were injected intratumorally every
`4 d for a total of four injections using the same
`healthy donor; mice treated with saline served
`as controls (n = 3). Tumor size was measured
`weekly. B, disseminated i.p. tumors were
`established in NSG mice (n = 6 per group) by
`l.p. injection with 8 x 106 M108-Luc cells.
`Beginning on day 58, RNA CAR-electroporated
`T cells (1 x 107
`) expressing ss1 -bbz were injected
`twice weekly for 2 wk. RNA CAR T cells
`expressing CD19-bbz RNA CAR or saline were
`Injected as controls. On day 78, the luminescence
`signal was significantly decreased in the
`ss1-bbz mice compared with the CD19-bbz
`mice (P < 0.01). C, BU from a single mouse
`treated with a single Injection on day 58 of T cells
`(1 x 107
`) expressing the ss1 -bbz CAR using
`a lentiviral vector. BU data for the experiment
`described in Bare plotted. Bars, SE.*, P < 0.05;
`- , P < 0.01. The ~LI signal in the saline group
`is truncated at the high end due to saturation
`of the imaging system.
`
`* ** ~ 109
`
`A
`
`B
`
`Saline
`
`19-bbz
`
`ss1 -bbz
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`C
`
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`1010
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`108
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`•0• ss1-bbz LV
`
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`
`0
`
`20
`
`80
`60
`40
`Time (d)
`
`100 120
`
`9058
`
`Cancer Res; 70(22) November 15, 2010
`
`Cancer Research
`
`Miltenyi Ex. 1043 Page 7
`
`

`

`three i.p. treatment groups, as depicted in the diagram in
`Supplementary Fig. S6. Mice were inoculated with M108-
`Luc (i.p.) on day 0 and treated with ssl-bbz or CD19-bbz
`RNA CAR T cells or with saline control, and tumor burden
`was monitored by serial BLI and body weight as indicated.
`Therapy was started on day 56 when the tumor was ad(cid:173)
`vanced based on the finding of ascites on physical examina(cid:173)
`tion and high BLI signals. Tumor burden was dramatically
`reduced in the group treated with T cells electroporated with
`ssl-bbz RNA CART cells, whereas the tumor continued to
`grow in the control mice treated with either CD19-bbz
`RNA CAR T cells or saline (Fig. SA and C). Even in this set(cid:173)
`ting, where the T cells are autologous to the tumor, there was
`still a modest CD19 CAR treatment effect, which may be due
`to the RNA backbone, as this is unlikely to be related to the
`CD19 scFv CAR given that there were no B cells in these
`mice. However, after the first six doses of T cells, imaging
`revealed a lower mean change tumor bioluminescence in
`the ssl CAR mice (39%) compared with both the CD19 CAR
`(244%) and the saline mice (237%; P < 0.001). The 50% median
`survival after T-cell injection was significantly (P < 0.05)
`
`greater in the ssl CAR mlc~.(73:'d·a'.ysJ cO~pared with the
`CD19 CAR (62.days) and saline mice (36 days; Fig. SB). After
`the first six doses were given, the mean change in total body
`weight was lower in the ssl RNA CAR mice (1.62 g) compared
`with both the CDI9·CM, (~.21 g) and the saline mice (11.4 g;
`P < 0.00 l; Fig. sC): lµtho.ugh we observed disease stability and
`even "cures~ by imaging in _so~e of the ssl CAR mice, twnor
`eventually recurred. Despite giving an additional eight doses of
`treatment, tumor progression was observed in the ssl CAR
`mice. Thus, repeated injecti(?nS of ssl-bbz RNA CART cells
`can provide a ·survival benefit for advanced diss~minated
`tumors. The mechanism for tumor recurrence in spite of
`continued therapy is under investigation.
`·
`
`Discussion
`
`The goal of these experiments was to determine the ther(cid:173)
`apeutic potential of activated T cells expressing electropo(cid:173)
`rated RNA CARs. The main point of our article is that
`mRNA CARs provide a platform that is expected to be safer
`and more economical than retroviral or lentiviral vectors for
`~
`
`A
`
`Saline
`
`CD19-bbz
`
`ss1-bbz
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`
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`...,._ Saline
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`
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`0 8 25 27 66 74 81 85 91 '8 105 119 126 133
`Study day
`
`Figure 5. Multiple injections of autologous RNA CAR T cells control the growth of advanced disseminated cancer in a xenogeneic mouse model.
`A, NOD/scld/yc(-/ -) mice {n = 30) were injected with 8 x 106 M108-Luc tumor cells (i.p.) and the mice were randomized into three groups before beginning
`therapy with RNA-electroporated autologous T cells (1 07 per injection) expressing ss1-bbz CAR, control CD19-bbz CAR, or saline on day 56 after
`tumor inoculation. Autologous T cells were injected i.p. and images were performed on surviving animals as indicated. Imaging commenced 5 d before the
`start of T-cell treatment. Tumor BU significantly decreased in the ss1 CAR mice (38.6%). compared with both the CD19 CAR {243.6%) and the saline
`mice {237.1%) after the first six doses (P < 0.001). B, Kaplan-Meier analysis. Median survival was significantly greater in the ss1 CAR mice compared with
`the CD19 CAR