653 Organ Specific Oncolytic Virus Incorporated Antigen Libraries (OSOVIAL) for the Immunotherapy of Cancer Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene[.]
CANCER - IMMUNOTHERAPY II We also observed a signicant increase in the number of HIV-Gag specic T-cells that secrete IFNγ and IL-2 after ex vivo stimulation with HIV/Gag derived peptides (1600 vs 800 for IFNγ, and 300 vs 120 for IL-2 SFC/106 splenocytes) In addition, an in vivo CTL assay conrmed that vaccination with the EAT-2 overexpressing Ad resulted in a signicantly improved in vivo cytolytic activity of HIV/ Gag specic CD8+ T cells generated after Ad-EAT2 and Ad-HIV/ Gag co-injection (68% vs 39% specic killing) Since both mice and humans express highly homologous EAT2 adaptors, our results suggest that human vaccination strategies that specically facilitate SLAM receptor signaling may provide a more effective vaccine against HIV specically, as well as numerous other vaccine targets in general Cancer – Immunotherapy II 652 A Powerful Retroviral Vector for TCR Gene Therapy: Silencing of Endogenous TCR Improved Expression and Anti-Tumor Reactivity of Transduced Tumor-Specic TCRs in Human Lymphocytes 653 Organ Specic Oncolytic Virus Incorporated Antigen Libraries (OSOVIAL) for the Immunotherapy of Cancer Sachiko Okamoto,1 Hiroaki Ikeda,2 Hiroshi Fujiwara,3 Masaki Yasukawa,3 Hiroshi Shiku,2 Junichi Mineno.1 Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Shiga, Japan; 2Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Mie, Japan; 3Dept of Bioregulatory Medicine, Ehime University Graduate School of Medicine, Toon, Ehime, Japan Jose Pulido,1,2 Timothy Kottke,2 Jill Thompson,2 Peter Selby,3 Alan Melcher,3 Richard Vile.2,3 Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, MN; 2Molecular Medicine, Mayo Clinic, Rochester, MN; 3Cancer Research UK Clinical Centre, St James’ University Hospital, Leeds, United Kingdom Adoptive T cell therapy with genetically engineered lymphocytes to express tumor antigen specic T-cell receptor (TCR) has been shown as an attractive strategy to treat cancer patients However, the limited efcacy of TCR gene therapy has been reported to be associated with the inefcient surface expression of transduced TCRs in T lymphocytes Endogenous TCR competes with introduced TCR for CD3 molecules and reduce the cell surface expression of the introduced TCR In addition, the introduced TCR α and β chains have been reported to mispair with endogenous TCR subunits, resulting in insufcient formation of heterodimers of therapeutic TCR Moreover, misparing of endogenous and transduced TCR subunits may cause the generation of T cells with unexpected specicities, including a risk of autoreactivity In this study, we developed novel “siTCR” retroviral vectors encoding both siRNA constructs that specifically knockdown endogenous TCRs and codon-optimized, siRNA-resistant TCR αβ chains specic for human tumor antigens Human lymphocytes transduced with these vectors exhibited high expression of the introduced tumor-specic TCR on the cell surface accompanied with reduced endogenous TCR at low copy numbers of the integrated vector, resulting in enhanced cytotoxic activity against antigenexpressing tumor cells As the target of this novel strategy is endogenous TCR, siTCR vectors may be a powerful tool for any TCR gene therapy without any dependency of TCR variation To apply this novel siTCR vector strategy to any TCR variations, we also developed universal siTCR retroviral vector cassette, which made possible to construct any siTCR retroviral vector with suitable TCRs This new approach satisfies the following requirements: enhancement of the ectopic TCR heterodimer expression on the cell surface and enhanced biological function at low proviral copy number (which may reduce the risk of mutagenesis); reduction of the endogenous TCR expression, which may effectively reduce TCR mispairing and decrease the risk of inducing self-reactive TCR αβ heterodimers; a universal method that does not depend on TCR variation Therefore, our novel TCR gene therapy may open the new gate for effective immunotherapy in cancer patients AM and RV are joint senior authors We have previously developed an immunotherapy for cancer in which normal cells are intentionally killed in the presence of the potent adjuvant hsp70 to induce autoimmune T cell reactivity In turn, this autoimmune reactivity is effective against tumors expressing normal tissue specic antigens of the same histological type (Cancer Res 2009 69:7767) Using viral vectors to deliver cytotoxic/hsp70 genes to the appropriate normal tissues, we have shown that this approach can effectively treat established melanoma, prostate cancer and pancreatic cancers in murine models However, there are concerns about inducing intentional destruction of normal tissues to treat malignancy in cases where such damage could have pathological consequences (such as with the pancreas) We have also shown that oncolytic viruses, such as VSV, are powerful immunoadjuvants for induction of T cell responses against tumor associated antigens (TAA), if a) the virus leads to release of TAA in the lymph node (Nat Med 2008 14:37; Clin Cancer Res 2009 15:4374) and b) when the virus itself encodes the TAA (Cancer Res 2007 15:2840) Therefore, we hypothesized that it would be possible to combine induction of autoimmune reactivity against normal tissue specic antigens with highly immunogenic, viral adjuvant-induced antigen presentation, for cancer immunotherapy To achieve this, we cloned cDNA libraries from a variety of sources, including normal tissues, into oncolytic viruses such as VSV We reasoned that the viral-expressed cDNA library would encompass epitopes from most normal antigens which might serve as immunogens for cancer rejection, thereby dispensing with the need for direct killing of the normal tissue In addition, the immunogenicity of the VSV would serve the adjuvant properties of hsp70 We have now shown that intravenous injection of VSV encoding a cDNA library from normal prostate leads to regression of murine prostate tumors in immune competent mice Regressions were immune mediated, as opposed to induced by viral oncolysis Transient inammation was observed in the normal prostate but no evidence of long term autoimmunity was apparent Mice in which tumors were rejected developed potent Th17 responses against prostate tumor cells, as well as normal prostate tissues Moreover, tumors which initially S254 Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy CANCER - IMMUNOTHERAPY II regressed but were not cured, recurred aggressively, with a markedly different histological appearance, and antigenic profile, to the initially implanted tumor cells, suggesting that OSOVIAL-mediated vaccination imposes a stringent immune selection upon tumors This approach dispenses with the need to induce direct damage to normal tissues in situ, allows for wide-ranging in vivo immune selection for antigens which are likely to be effective targets for both autoimmune and anti tumor responses and uses oncolytic viruses as novel immune adjuvants for the immunotherapy of cancer 654 Editing Human Lymphocyte Specicity for Safe and Effective Adoptive Immunotherapy of Leukemia Elena Provasi,1 Pietro Genovese,2 Zulma Magnani,1 Angelo Lombardo,2 Andreas Reik,3 Liu Pei-Qi,3 Oscar Muniz Pello,2 Jurgen Kuball,4 Attilio Bondanza,1 Giulia Casorati,1 Philip D Gregory,3 Claudio Bordignon,1 Michael C Holmes,3 Philip D Greenberg,4 Luigi Naldini,2 Chiara Bonini.1 San Raffaele Scientic Institute, Milan, Italy; 2HSR-TIGET, Milan, Italy; 3Sangamo BioSciences, Richmond, CA; 4Fred Hutchinson Cancer Research Center, Seattle T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors However, the full potential of this approach is limited by a number of technical hurdles including inefcient gene transfer, unstable transgene expression, exhaustion of gene-modied cells and, most importantly, the co-expression in the same cell of the endogenous and tumor-specic TCR The latter results not only in reduction of expression of the introduced tumor-specic TCR, but also can result in acquisition of autoreactive specicities due to mispairing between endogenous and exogenous TCR chains To address these limitations, we designed a novel strategy based on zinc nger nucleases (ZFNs) and lentiviral vectors that allows for the rst time editing of T cell specicity at the DNA level, by combining the disruption of the endogenous TCR β chain gene with the transfer of a tumor-specic TCR We rst stimulated PBL with anti-CD3 and anti-CD28 antibody-conjugated beads, and cultured the cells with low doses of IL-7/IL-15, to target and preserve early differentiated T cells To eliminate expression of the endogenous TCR, activated cells were treated with integrase defective lentiviral vectors (IDLV) carrying a set of ZFNs specic for the constant regions of the TCR β chain Abrogation of surface expression of the TCR/CD3 complex was observed in up to 7% of the treated cells The majority (>80%) of CD3neg lymphocytes expressed CD62L, CD127, CD27 and CD28, a phenotype most consistent with central memory T cells, and could be expanded in culture with IL7 and IL15 as unmodied T cells To re-direct tumor specic T cell activity, we selected a codon-optimized, cysteine-modied TCR specic for the Wilm’s Tumor Antigen (WT1), which is involved in oncogenic transformation in several tumors To promote balanced expression of the introduced α and β TCR chains, we generated lentiviral vectors (LV) encoding both chains under a single PGK bidirectional promoter (PGK-WT1 LV) Sorted CD3neg cells were efciently transduced (>45%) with PGK-WT1 LV, and expressed stable and high levels of tumor-specic TCR, indicating that CD3neg cells are permissive to further genetic manipulation TCR-edited cells were enriched to 90% purity by polyclonal stimulation, indicating that surface expression of the transferred TCR/CD3 complex was functional and sufcient to promote cell expansion TCR-edited cells killed targets pulsed with lower peptide concentrations than unedited T cells, modied by conventional TCR gene transfer, and lysed fresh WT1+/HLA-A2+ primary acute myeloid leukemia blasts These data demonstrate that genetic re-programming of T cell specicity in early differentiated lymphocytes is feasible and functional and represents an attractive approach to improve the therapeutic outcome of cancer immunotherapy *equal contribution Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 655 Constitutive 4-1BBL Expression Dramatically Increases Proliferation, Survival and In Vivo Persistence of CD19-Targeted T Cells, Promoting Complete Tumor Elimination Maud Condomines, Jason Plotkin, John C Markley, Gertrude Gunset, Michel Sadelain Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York, NY Human T cells engineered to express a chimeric antigen receptor (CAR) composed of an antibody-derived scFv fragment linked to the CD3 zeta chain, can recognize and kill tumor cells expressing the targeted membrane antigen However, T cell activating signals have to be enhanced by a second stimulatory signal in order for T cells to avoid anergy, to be fully activated, proliferate and secrete cytokines CD80 and CD86 are the prototypical costimulatory molecules, which bind the CD28 receptor constitutively expressed at the T cell plasma membrane Activation of 4-1BB, a member of the TNFR family expressed by activated T cells, has been shown to efciently enhance CD8 T-cell proliferation and mediate increased tumor rejection in animal models We previously showed that the retroviral transfer of an anti-PSMA CAR (Pz1) along with CD80 and 4-1BB ligand (4-1BBL) into human T cells induced auto-costimulation enabling high in vivo T cell expansion and tumor rejection in a prostate tumor model In this study, we investigated this concept in T cells harboring either a rst generation anti-CD19 CAR (19z1) or a second generation CAR (1928z), which additionally comprises the CD28 cytoplasmic domain We compared the proliferation and anti-tumor abilities of 19z1+, 19z1-4-1BBL+, 19z1-CD80+4-1BBL+, 1928z+, 1928z-41BBL+ transduced T cells in an aggressive pro-B cell leukemia model (NALM-6) After weekly in vitro stimulations on mouse fibroblasts expressing human CD19, 19z1+, 19z1-4-BBL+, 19z1-CD80+41BBL+, 1928z+ and 1928z-4-1BBL+ T cells exerted a 2-fold, 5-fold, 21-fold, 17-fold and 200-fold expansion rate, respectively All T cell groups killed four CD19 expressing cell lines, including NALM-6, with similar efciencies The proportions of central memory CD8 T cells (CD45RA- and CCR7+) observed after one stimulation in the groups expressing 4-1BBL were 40-60% whereas those evaluated in the 19z1 and 1928z groups were only 20-25% One week after IV injection of 1e6 CAR+ T cells into 4-day NALM-6 bearing NOD/ SCID/IL2Rγ null mice, the proportion of human CD8 and CD4 T cells which had accumulated in the bone marrow measured in mice infused with 1928z-4-1BBL+ T cells was 100-fold and 10fold higher than in mice infused with 19z1+ and 1928z+ T cells, respectively The proportions of CD8 and CD4 T cells in the spleen followed those observed in the bone marrow, including a substantial proportion of central memory T cells One month after T cell injection, 1928z-4-1BBL+ CD8 T cells expanded at least 300 times In vivo bioluminescence imaging showed complete tumor regression in the 1928z-4-1BBL+ T cell-treated group within 15 days of T cell infusion, while a partial regression was observed in the other groups Long-term survival studies are on-going Thus, constitutive 4-1BBL expression drives high proliferation of anti-tumor CD8 T cells, which accumulate in the spleen and bone marrow of immuno-compromised, tumor-bearing mice, promoting central memory T cell persistence and tumor eradication S255 ... responses and uses oncolytic viruses as novel immune adjuvants for the immunotherapy of cancer 654 Editing Human Lymphocyte Specicity for Safe and Effective Adoptive Immunotherapy of Leukemia Elena... set of ZFNs specic for the constant regions of the TCR β chain Abrogation of surface expression of the TCR/CD3 complex was observed in up to 7% of the treated cells The majority (>80%) of CD3neg... 4Fred Hutchinson Cancer Research Center, Seattle T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors However, the full potential of this approach