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396 BARF1 Specific T Cells for the Adoptive Immunotherapy of EBV Positive Nasopharyngeal Carcinoma Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell T[.]
CANCER - IMMUNOTHERAPY II (59%±6%) than control T cells (11%±8%) even at the 5:1 effectorto-target ratio in 51Cr release assays Furthermore, in long-term coculture assays, CAR.MCSP+ T cells efficiently and consistently eliminated several MCSP+ targets including melanoma (SEMNA and CLB, residual tumors: 0.1%±0.06% and 0.1%±0.1, respectively), mesothelioma (PH1 and MILL: 3.8%±3.1%; 3.2%±5%), head and neck carcinoma (PCI-30, 0.5%±0.5%), and basal breast carcinoma (UACC-812 and MDA-MB-231: 5.7% ±6.1%; 3.1% ±2.5%, respectively) while having no effect on a MCSP– targets (38% ±10%) As expected all tumor cells expanded in coculture with control T cells The antitumor activity of CAR.MCSP+ T cells was paralleled by release of Th1 cytokines, such as IL2 (from 6±10 pg/μL to 190±98 pg/μL) and IFNg (from 105±48 pg/μL to 3710±975 pg/μL) upon coculture with different MCSP+ tumors Both CAR.MCSP transgenic CD4+ and CD8+ cells proliferated in response to SEMNA tumor cells as compared to control T cells, as assessed by CFSE dilution assays We also generated a third generation CAR encoding CD28 and 4-1BB endodomains However, since this construct did not show superior function in vitro as compared to the CD28 endodomain we selected the latter for the following in vivo experiments Using NSG mice (n=10/group) either melanoma (SEMNA) or head and neck carcinoma (PCI-30) or basal breast carcinoma (UACC-812) cells were engrafted s.c Across all tumor models, mice treated with CAR.MCSP+ T lymphocytes consistently showed tumor control (753mm3±350mm3; 18.5mm3±10mm3; 28mm3±13mm3) as compared to mice receiving control T lymphocytes (7126mm3±2500mm3; 190mm3±75mm3; 166 mm3±64mm3) by days 40-50 post tumor engraftment In summary, CAR.MCSP-redirected T cells can be used for the treatment of a variety of solid tumors 395 Tumor Recurrences Share Immunogenic Antigens across Both Tumor Types and Primary Treatments Which Can Be Therapeutically Targeted with VSV-cDNA Libraries Nicolas Boisgerault,1 Jose Pulido,1 Timothy Kottke,1 Oliver Donnelly,2 Esteban Celis,3 Jill Thompson,1 Rosa Diaz,1 Kevin Harrington,4 Hardev Pandha,5 Peter Selby,2 Alan Melcher,2 Richard Vile.1 Mayo Clinic, Rochester; 2University of Leeds, Leeds, United Kingdom; 3H Lee Moffitt Cancer Center, Tampa; 4The Institute of Cancer Research, London, United Kingdom; 5University of Surrey, Guildford, United Kingdom cDNA libraries expressed from the highly immunogenic Vesicular Stomatitis Virus (VSV) platform can treat established tumors in both prostate and melanoma models Viral stocks can be delivered systemically, not have to target tumor and generate potent T cell responses against a variety of different tumor associated antigens (TAA) which, cumulatively, lead to rejection of well established subcutaneous tumors Following different suboptimal primary treatments of tumor bearing mice in which initial tumor regression is followed by aggressive recurrence, VSV-cDNA libraries constructed from these recurrent tumors can, if administered early enough, prevent tumor recurrence However, the VSV-cDNA libraries which are active against primary tumors are ineffective against recurrences, showing that the antigenic profile of recurrent tumors is substantially different from that of the primary tumors Using an in vitro splenocyte-based assay, we cloned TAA from VSV-cDNA libraries of recurrent melanoma and prostate tumors, generated by different primary treatments, which fall into three clearly defined classes In the first, (treatment-specific TAA) proteins are lost/ gained in recurrences, relative to primary tumors, which are highly specific to the nature of the primary treatment (such as loss of the HSVtk protein from recurrences derived from initial HSVtk/ Ganciclovir treatment) The second class (tumor type-specific TAA) contains proteins which are lost/gained in recurrences only of certain S152 histological types (such as a variant of CD44 which is acquired by relapsed TC2 prostate tumors but not B16 melanoma recurrences) Finally, the VSV-cDNA technology has identified a class of proteins (recurrence-specific TAA) which are present in recurrences of different tumor types and independently of the nature of the primary treatment These recurrence-specific TAA are associated with control of DNA replication and progression through the cell cycle, such as Topoisomerase IIa, cdc7 kinase and YB-1 Significantly, we have shown that these proteins can serve as genuine recurrent-specific TAA, in that mice cured by recurrent-specific VSV-cDNA libraries contain T cells which recognize epitopes from these proteins Finally, we have used these data to show that it is possible to treat recurrences both of different tumor types, and generated from different primary treatments, with a cocktail of recurrence-specific TAA expressed from VSV These data suggest that 1) early recurrences across tumor and treatment types share common molecular pathways by which they evolve in vivo and 2) that by identifying these common, recurrencespecific antigens it may be possible to devise both immunotherapeutic, and chemotherapeutic, strategies to treat recurrent tumors across a variety of histological types and primary treatments 396 BARF1-Specific T Cells for the Adoptive Immunotherapy of EBV-Positive Nasopharyngeal Carcinoma Mamta Kalra,1 Minhtran C Ngo,1 Ulrike Gerdemann,1 Ann Leen,1 Chrystal Louis,1 Cliona M Rooney,1 Stephen Gottschalk.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Background: The majority of nasopharyngeal carcinomas (NPC) are positive for Epstein-Barr virus (EBV), and the outcome for patients with recurrent/refractory disease remains poor Treatment of NPC patients with EBV-specific cytotoxic T cells (EBV-CTLs) has been promising, resulting in clinical responses However, the antitumor activity of EBV-CTLs in patients with bulky disease was limited This lack of efficacy may simply be quantitative in that current methods of CTL generation induce limited T-cell responses to the EBV antigens selectively expressed in NPC (EBNA1, LMP1, LMP2, BARF1) While T-cell responses to EBNA1, LMP1, and LMP2 have been studied in detail and clinical studies with EBNA1-, LMP1-, and LMP2-specific T cells are in progress, little is known about BARF1specific T-cell responses The goal of the present study was to (i) characterize BARF1-specific T-cell responses in EBV-positive healthy donors and NPC patients, (ii) map immunogenic T-cell epitopes, and (iii) expand BARF1-specific T cells for the adoptive immunotherapy of EBV-positive NPC Methods: Peripheral blood mononuclear cells (PBMCs) from EBV-positive healthy donors (n=16) and NPC patients (n=7) were stimulated with a BARF1 overlapping peptide library consisting of 53 15-mer peptides After 8-10 days of culture, T-cell responses were determined by IFN- Elispot assay using either the entire library or BARF1 peptide mini-pools, each containing 10 to 13 peptides Subsequently, in responding donors, BARF1-specific T-cell lines were generated by consecutive rounds of stimulation with BARF1-loaded autologous dendritic cells (DCs) for epitope mapping and functional analysis Results: Twelve (68%) of 16 healthy donors and (71%) of NPC patients showed T-cell reactivity towards BARF1 Of these BARF1-positive subjects, 9/12 healthy donors and 4/5 patients recognized distinct peptide pools The majority of CD8-restricted epitopes were mapped to the N- (pool 1) and C-termini (pool 4), while CD4-restricted epitopes were located in the middle of BARF1 (pools and 3) Detailed screening of reactive pools in four healthy donors resulted in the identification of 17 immunodominant peptides For one of the CD8-restricted epitopes we have so far mapped the minimal peptide length and determined that it is restricted through HLA*B1562 Conclusion: T-cells specific for CD4- and CD8-restricted BARF1 epitopes are present in PBMCs of 70% of Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy CANCER - IMMUNOTHERAPY II EBV-seropositive healthy donors and EBV-positive NPC patients, and can be expanded ex vivo Targeting BARF1 in addition to the other EBV antigens expressed in NPC may improve the efficacy of adoptive immunotherapy approaches 397 IL-12 Increases Immune Responses Induced by a Novel pDNA Prostate Cancer Immunotherapy Approach in Non-Human Primates Bernadette Ferraro,1 Amritha Balakrishnan,1 Jewell N Walters,1 Devin J Myles,1 Jian Yan,2 Amir S Khan,2 Niranjan Y Sardesai,2 David B Weiner.1 University of Pennsylvania School of Medicine, Philadelphia, PA; Inovio Pharmaceuticals, Inc., Blue Bell, PA Prostate cancer (PCa) remains a significant public health problem Current treatment modalities for PCa can be useful, but may be accompanied by deleterious side effects and often not confer long-term control Accordingly, additional modalities, such as immunotherapy, may represent an important approach for PCa treatment Delivery of DNA vaccines with electroporation (EP) has shown promising results for prophylactic and therapeutic targets in a variety of species, and recently in humans (Bagarazzi et al., Sci Transl Med 2012 Oct 10;4(155)) Application of this technology for PCa immunotherapy strategies has been limited to single antigen and epitope targets We sought to test the hypothesis that a broader collection of antigens would improve the breadth and effectiveness of a PCa immune therapy approach We concurrently explored if the molecular adjuvant IL-12 would increase the magnitude of these responses To this end, we developed highly optimized DNA vectors encoding prostate-specific antigen (PSA), and prostate-specific membrane antigen (PSMA), and six-transmembrane epithelial antigen of the prostate (STEAP) as a three-pronged approach to immune therapy of PCa Vaccine immunogenicity was evaluated in rhesus macaques (NHPs) NHPs (n=4/group) received immunizations of DNA alone (DNA) or DNA with IL-12 (DNA+12) delivered with EP at weeks 0, 6, 12, and 24 Immune responses were evaluated at weeks 0, 14, 20 and 26 DNA alone elicited modest IFN production by ELISpot at weeks 14 (153 SFU), 20 (55 SFU) and 26 (79 SFU) IL-12 significantly increased IFN production at weeks 14 (612 SFU), 20 (293 SFU) and 26 (394 SFU) Further characterization of immune responses by flow cytometry demonstrated that at week 26 DNA+12 increased the level antigen-specific CD8+ T cells (0.43%) compared to DNA (0.19%) Importantly, DNA+12 promoted CD8+ T cells with the potential for cytotoxic and effector function, as evidenced by antigen-specific CD107a (0.34%) and T-bet (0.28%), respectively There was also a strong humoral response as determined by PSA- and PSMA-specific seroconversion These data support further study of these immunogens as part of a novel approach to immune therapy of PCa adoptively-transferred T cells can be protected from the inhibitory effects of TGF through the transgenic expression of a truncated, dominant-negative receptor (DNRII), which blocks transmission of TGF signal We have now extended this strategy by converting the inhibitory signal into an activation stimulus for T cells We generated a chimeric cytokine receptor expressing the exodomain of TGFRII linked to the endodomain of toll-like receptor (TLR) and GFP (RIID4) We transduced primary T cells with RIID4 and obtained 69.3±6.0% transduction which was stable for >60 days of culture To address whether transgenic expression of RIID4 protected against TGF, we modified 2G.CAR-PSCA T cells to co-express either the dominant negative DNRII or RIID4 receptors These T cells were then stimulated weekly with PSCA+ tumor cells (K562-PSCA) with or without exogenous TGF1 (5ng/mL) In the absence of TGF1, 2G.CAR-PSCA, 2G.CAR-PSCA(DNRII) or 2G.CAR-PSCA(RIID4) T cells proliferated at similar levels for 30 days (6.4x102, 2.5x103, 5.9x103 fold, respectively) But in the presence of TGF1, 2G.CARPSCA T cells did not expand, and cultures failed within weeks In contrast, transgenic expression of DNRII or RIID4 protected the cells from the inhibitory impact of this cytokine (7.4 and 21 fold at weeks of culture, respectively) To determine whether there were long term differences between DNRII- and RIID4-modified cells we monitored cell expansion and found that only RIID4-modified T cells were able to expand for >60 days in the presence of TGF1 (3.0x105 fold) while DNRII cells began to contract after 30 days in culture (0.72 fold) Administration of TGF1 also selected 2G.CAR-PSCA(RIID4) T cells, leading to an enrichment in this transgenic cell population over time (from 63.6% to 93.3%) This modification appears to be safe since the administration of TGF1 alone was insufficient to drive transgenic T cell proliferation (0.04 fold) and the withdrawal of antigenic stimulation resulted in T cell contraction (0.02 fold) Finally, to address whether this modification could improve the anti-tumor activity of CAR-T cells, we co-cultured 1x106 fireflyluciferase-Du145 cells, which express PSCA and produce TGF1, with 1x105 2G.CAR-PSCA, 2G.CAR-PSCA(DNRII) or 2G.CARPSCA(RIID4) T cells After days we observed superior control of tumor growth by RIID4-expressing T cells compared with DNRII or CAR alone conditions (Total Flux; 9±0.1x109, 10±1x109, 20±1x109 p/s, respectively) Thus, suggest that RIID4 not only protects cells from the inhibitory effects TGF but converts this cytokine signal into one which is stimulant 399 Hypomethylating Agent 5-aza Cytidine Exposure To Improve AP1903 Treatment for Apoptosis Induction of iCasp9/∆CD19 Gene Modified T Cells 398 Transgenic Expression of a Novel Immunosuppressive Signal Converter on T Cells Elodie Bole-Richard,1 Jean-Marie Certoux,1 Idir Idirene,1 Laurent Jossot,1 Eric Deconinck,1,2 Fabrice Larosa,2 Etienne Daguindau,2 Christophe Borg,1,2 Pierre Tiberghien,1 Christophe Ferrand,1 Marina Deschamps.1 UMR1098-EFSBFC-SFR FED 4234, Besancon, France; Hematology, CHU Jean Minjoz, Besancon, France Chimeric antigen receptor (CAR)-transduced T cells are promising tools for the treatment of cancers To extend this therapeutic modality to prostate cancer we have constructed a 2nd generation CAR targeting the tumor antigen PSCA (2G.CAR-PSCA), which provides cells with the ability to kill PSCA+ prostate tumor cells (51.7±1.2% specific lysis of Du145 cells at 20:1 E:T) However, many tumors including prostate cancer secreting TGF, which inhibits in vivo T cell proliferation, activation and function Our group has previously demonstrated that Haematopoietic transplantation may result in serious complications, notably graft versus host disease (GvHD) T-lymphocyte depletion of the bone marrow graft is able to prevent GvHD, while increasing the risk of rejection and reducing the anti-leukemic effect An alternative issue is to use a suicide gene system that allows in-vivo conditional T cell depletion Based on our experience reporting drawbacks of the suicide gene HSV-tk expressing donor T cells, we investigate the use of human derived inducible caspase (iCasp9/ iC9) and truncated CD19 (CD19) expressing T-cell This allows rapid apoptosis after exposure to a Chemical Inducer of Dimerization (CID; AP1903, Bellicum Pharmaceuticals) As reported in-vivo by others, we found a drug responsiveness of some iC9/CD19+ gene modified cells (GMC) after CID in-vitro exposure Such findings Norihiro Watanabe,1 Usanarat Anurathapan,1 Malcolm K Brenner,1 Helen E Heslop,1 Ann M Leen,1 Cliona M Rooney,1 Juan F Vera.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S153 ... Application of this technology for PCa immunotherapy strategies has been limited to single antigen and epitope targets We sought to test the hypothesis that a broader collection of antigens would... antigen receptor (CAR)-transduced T cells are promising tools for the treatment of cancers To extend this therapeutic modality to prostate cancer we have constructed a 2nd generation CAR targeting... part of a novel approach to immune therapy of PCa adoptively-transferred T cells can be protected from the inhibitory effects of TGF through the transgenic expression of a truncated, dominant-negative