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427. Therapeutic Vaccination With Irradiated GM-CSF Gene-Transduced Cancer Side Population Cells Effectively Suppress Tumor Growth and Lung Metastasis
CANCER-IMMUNOTHERAPY II 427. Therapeutic Vaccination With Irradiated GM-CSF Gene-Transduced Cancer Side Population Cells Effectively Suppress Tumor Growth and Lung Metastasis
Chika Sakamoto, Hiroyuki Inoue, Megumi Narusawa, Yumiko Matsumura,1 Shohei Miyamoto,1 Makoto Inoue,2 Koichi Takayama,3 Mamoru Hasegawa,2 Yoichi Nakanishi,3 Kenzaburo Tani.1 1 Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2DNAVEC Corporation, Tsukuba, Japan; 3Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu Unversity, Fukuoka, Japan. 1
3
1
It has been shown that autologous GM-CSF gene-transduced tumor vaccine therapy (GVAX) is safe, and has substantial antitumor effects against various cancers. However, its efficacy is limited, presumably because cancer stem cells (CSCs) can contrive to escape from host’s antitumor immunity. To what extent and how CSCs in tumors evade antitumor responses driven by GM-CSF-based antitumor immunity remains elusive. Therefore, it is important to explore whether CSCs are susceptible to antitumor immunity and to develop CSCs-based GVAX therapy towards more efficacious combinatorial immunotherapeutic modalities. Here we hypothesized whether enriched CSCs subpopulation had the susceptibility to GMCSF-induced antitumor immunity. We used the Hoechst 33342 dye-based side population (SP) to isolate CSCs-enriched population from a 4T1 murine breast cancer cell line. We confirmed that 4T1-SP cells exhibited several stem cell properties such as higher colony formation ability in vitro and tumorigenicity in vivo. Additionally, the expression levels of signal transducer and activator of transcription-3, vascular endothelial growth factor and PD-L1/B7-H1 were higher in 4T1-SP cells than 4T1-non-SP cells, illustrating that 4T1-SP cells had resistance to chemotherapy and immunosuppressive phenotype. We transduced mouse GM-CSF gene into 4T1-SP cells using non-transmissible Sendai virus vector of SeV/dF/GM. Despite these progressive malignant characteristics of 4T1-SP cells, GM-CSF gene-transduced 4T1-SP (4T1-SP/GM) cells remarkably impaired their tumorigenicity compared with control cells. In vivo depletion assay showed that the impaired tumorigenicity was partially dependent on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Notably, therapeutic vaccinations using irradiated 4T1-SP/GM (ir.4T1-SP/GM) cells markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with that of the controls including irradiated 4T1-non-SP/GM cells, with tolerability. Furthermore, GMCSF gene-transduced cell vaccinations comprising primary 4T1-SP cells isolated from 4T1-SP cell-derived subcutaneous tumors also elicited substantial antitumor effects. Moreover, vaccinations with ir.4T1-SP/GM cells decreased the lung metastases compared to those with control cells. Tumor suppression during therapeutic vaccination was accompanied by robust accumulation of mature dendritic cells at vaccination sites and systemic Th1-based cellular immunity evidenced by IFN-γ ELISPOT assay. Collectively, we demonstrated that therapeutic vaccinations with ir.4T1-SP/GM cells induced effective antitumor immunity in syngeneic mouse models, providing a promising prospect to improve the efficacy of GVAX therapy.
S164
428. Design of Novel Multifunctional Chimeric Antigen Receptors (Tag/CARs) for Cancer Immunotherapy
Lingfeng(Steven) Liu,1 Daniel Sommermeyer,1 Paula Kosasih,1 Stanley R. Riddell.1,2 1 Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of Washington, Seattle, WA. An advance in cancer immunotherapy is the use of autologous T cells engineered to express a synthetic chimeric antigen receptor (CAR) that is specific for a tumor cell surface molecule, with the most impressive clinical results observed in targeting CD19 on B cell leukemias and lymphoma. The structure of the CAR typically includes an scFv, a spacer domain, and intracellular signaling modules to trigger T cell effector function, but lacks sequences that allow rapid selection, in vivo tracking and regulation of CAR-T cells. We designed a novel multiple-functional CAR that incorporates one or more 8 amino acid StrepTag II (ST II) sequences in the extracellular portion of the CAR (Tag/CAR), and demonstrated that these sequences enable in vitro selection, activation/expansion, and in vivo tracking of CAR+ T cells using StrepTactin or anti-STII mAb labeled fluorophores or beads. This novel design is effective for scFvs specific for multiple tumor targets including CD19 and ROR1, and T cells engineered with such STII CARs exhibit comparable in vivo antitumor activity as T cells engineered with CARs that lack STII. The STII and linker sequences were designed to minimize immunogenicity as predicted by HLA peptide binding motifs. This innovation in synthetic CAR structure illustrates the potential to incorporate design features that provide control over the manufacturing, in vivo tracking and potentially the in vivo regulation of CAR T cells.
429. A Human HER2-Specific TCR Confers Potent Anti-Tumor Effector Functions in Genetically Engineered Primary Cytotoxic Lymphocytes
Jenessa B. Smith,1 Evripidis Lanitis,1 Denarda Dangaj,1 Seleeke Flingai,1 Mathilde Poussin,1 Shuwen Xu,3 Brian J. Czerniecki,3 Yong F. Li,4 Paul F. Robbins,4 Daniel J. Powell.1,2 1 Ovarian Cancer Research Center, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA; 2Abramson Cancer Center, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 3Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA; 4Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD. ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T-lymphocytes (CTLs) specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8+ T-cells with specificity for the ErbB2369-377 peptide, we performed one round of in vitro peptide stimulation of CD8+ T-cells isolated from an HLA-A2+ patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2reactive T-cells with strong ErbB2-specific, anti-tumor effector functions. We then stimulated these ErbB2-reactive T-cells with ErbB2+ HLA-A2+ tumor cells in vitro and sorted tumor-activated ErbB2369-377 peptide T-cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2369-377 peptide specificity. Primary human CD8 + T-cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2369-377 peptide Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Chika Sakamoto, Hiroyuki Inoue, Megumi Narusawa, Yumiko Matsumura,1 Shohei Miyamoto,1 Makoto Inoue,2 Koichi Takayama,3 Mamoru Hasegawa,2 Yoichi Nakanishi,3 Kenzaburo Tani.1 1 Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2DNAVEC Corporation, Tsukuba, Japan; 3Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu Unversity, Fukuoka, Japan. 1
3
1
It has been shown that autologous GM-CSF gene-transduced tumor vaccine therapy (GVAX) is safe, and has substantial antitumor effects against various cancers. However, its efficacy is limited, presumably because cancer stem cells (CSCs) can contrive to escape from host’s antitumor immunity. To what extent and how CSCs in tumors evade antitumor responses driven by GM-CSF-based antitumor immunity remains elusive. Therefore, it is important to explore whether CSCs are susceptible to antitumor immunity and to develop CSCs-based GVAX therapy towards more efficacious combinatorial immunotherapeutic modalities. Here we hypothesized whether enriched CSCs subpopulation had the susceptibility to GMCSF-induced antitumor immunity. We used the Hoechst 33342 dye-based side population (SP) to isolate CSCs-enriched population from a 4T1 murine breast cancer cell line. We confirmed that 4T1-SP cells exhibited several stem cell properties such as higher colony formation ability in vitro and tumorigenicity in vivo. Additionally, the expression levels of signal transducer and activator of transcription-3, vascular endothelial growth factor and PD-L1/B7-H1 were higher in 4T1-SP cells than 4T1-non-SP cells, illustrating that 4T1-SP cells had resistance to chemotherapy and immunosuppressive phenotype. We transduced mouse GM-CSF gene into 4T1-SP cells using non-transmissible Sendai virus vector of SeV/dF/GM. Despite these progressive malignant characteristics of 4T1-SP cells, GM-CSF gene-transduced 4T1-SP (4T1-SP/GM) cells remarkably impaired their tumorigenicity compared with control cells. In vivo depletion assay showed that the impaired tumorigenicity was partially dependent on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Notably, therapeutic vaccinations using irradiated 4T1-SP/GM (ir.4T1-SP/GM) cells markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with that of the controls including irradiated 4T1-non-SP/GM cells, with tolerability. Furthermore, GMCSF gene-transduced cell vaccinations comprising primary 4T1-SP cells isolated from 4T1-SP cell-derived subcutaneous tumors also elicited substantial antitumor effects. Moreover, vaccinations with ir.4T1-SP/GM cells decreased the lung metastases compared to those with control cells. Tumor suppression during therapeutic vaccination was accompanied by robust accumulation of mature dendritic cells at vaccination sites and systemic Th1-based cellular immunity evidenced by IFN-γ ELISPOT assay. Collectively, we demonstrated that therapeutic vaccinations with ir.4T1-SP/GM cells induced effective antitumor immunity in syngeneic mouse models, providing a promising prospect to improve the efficacy of GVAX therapy.
S164
428. Design of Novel Multifunctional Chimeric Antigen Receptors (Tag/CARs) for Cancer Immunotherapy
Lingfeng(Steven) Liu,1 Daniel Sommermeyer,1 Paula Kosasih,1 Stanley R. Riddell.1,2 1 Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of Washington, Seattle, WA. An advance in cancer immunotherapy is the use of autologous T cells engineered to express a synthetic chimeric antigen receptor (CAR) that is specific for a tumor cell surface molecule, with the most impressive clinical results observed in targeting CD19 on B cell leukemias and lymphoma. The structure of the CAR typically includes an scFv, a spacer domain, and intracellular signaling modules to trigger T cell effector function, but lacks sequences that allow rapid selection, in vivo tracking and regulation of CAR-T cells. We designed a novel multiple-functional CAR that incorporates one or more 8 amino acid StrepTag II (ST II) sequences in the extracellular portion of the CAR (Tag/CAR), and demonstrated that these sequences enable in vitro selection, activation/expansion, and in vivo tracking of CAR+ T cells using StrepTactin or anti-STII mAb labeled fluorophores or beads. This novel design is effective for scFvs specific for multiple tumor targets including CD19 and ROR1, and T cells engineered with such STII CARs exhibit comparable in vivo antitumor activity as T cells engineered with CARs that lack STII. The STII and linker sequences were designed to minimize immunogenicity as predicted by HLA peptide binding motifs. This innovation in synthetic CAR structure illustrates the potential to incorporate design features that provide control over the manufacturing, in vivo tracking and potentially the in vivo regulation of CAR T cells.
429. A Human HER2-Specific TCR Confers Potent Anti-Tumor Effector Functions in Genetically Engineered Primary Cytotoxic Lymphocytes
Jenessa B. Smith,1 Evripidis Lanitis,1 Denarda Dangaj,1 Seleeke Flingai,1 Mathilde Poussin,1 Shuwen Xu,3 Brian J. Czerniecki,3 Yong F. Li,4 Paul F. Robbins,4 Daniel J. Powell.1,2 1 Ovarian Cancer Research Center, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA; 2Abramson Cancer Center, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 3Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA; 4Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD. ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T-lymphocytes (CTLs) specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8+ T-cells with specificity for the ErbB2369-377 peptide, we performed one round of in vitro peptide stimulation of CD8+ T-cells isolated from an HLA-A2+ patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2reactive T-cells with strong ErbB2-specific, anti-tumor effector functions. We then stimulated these ErbB2-reactive T-cells with ErbB2+ HLA-A2+ tumor cells in vitro and sorted tumor-activated ErbB2369-377 peptide T-cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2369-377 peptide specificity. Primary human CD8 + T-cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2369-377 peptide Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy