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235. In Vivo Delivery of Interferon-alpha Gene Enhances Tumor Immunity and Suppresses Immunotolerance after Autologous Hematopoietic Stem Cell Transplantation
CANCER - IMMUNOTHERAPY I GM cells. The results showed that mice inoculated with LLC/SeV/ GM cells combined with Imiquimod, an agonist of TLR7, elicited more rapid elimination of tumor challenge than them inoculated with LLC/SeV/GM cells alone. Taken together, we demonstrated that TLR7 agonist enhance GM-CSF-induced antitumor immunity in mouse lung cancer model, suggesting that pDC generating type I IFNs might be a pivotal subpopulation of APCs which mainly contributes to GM-CSF-induced antitumor effects.
234. T Cells Genetically Modified To Express Chimeric Antigen Receptor Specific for EGFR for Glioma
Hillary G. Caruso,1 Sonny Ang,1 Singh S. Harjeet,1 Simon Olivares,1 Laurence J. N. Cooper.1 1 Pediatrics, M.D. Anderson Cancer Center, Houston, TX.
Diffuse Intrinsic Pontine Glioma (DIPG) is an inoperable and nearly uniformly fatal tumor that afflicts children. These tumors overexpress wild type epidermal growth factor receptor (EGFR), and a recent clinical trial had shown that Nimotuzumab, an antiEGFR monoclonal antibody (mAb), has transient and surprisingly moderate clinical benefit for these patients. We seek to extend this antibody therapy to T-cell therapy by introducing a chimeric antigen receptor (CAR) to redirect the specificity of autologous T cells to EGFR. A second-generation EGFR-specific CAR was generated by fusing the specific region (scFv) of Nimotuzumab to T cell activation domains CD28 and CD3ζ and expressed as Sleeping Beauty (SB) transposon. The CAR was electroporated into primary PBMC with a SB transposase enzyme to cut CAR out of the vector and insert it into the genome. T cells expressing EGFR-specific CAR were propagated on irradiated artificial antigen presenting (aAPC) cells modified to express EGFR. The culture system was adapted to provide robust and selective outgrowth of CAR+ T cells by every-seven-day addition of γ-irradiated aAPC in the presence of IL-2 and IL-21. It is this platform that we are using to generate clinical-grade CD19-specific CAR+ T cells. T cells expressing EGFR-specific CAR were detected 24 hours post-electroporation in 30-50% of the PBMC by flow cytometry using Fc-specific Ab which recognizes the IgG4 region of the CAR. EGFRspecific T cells were numerically expanded by 5 logs over 28 days to yield a population of primarily CD8+ T cells with effector-memory phenotype (CCR7neg CD45RO+). We were able to detect re-directed target specificity of the EGFR-specific CAR+ T cells by measuring intracellular IFN-γ production in response to EGFR+ targets.
S92
In aggregate, these data establish that T cells can be re-directed for EGFR based on Nimotuzumab. Given our ability to manufacture clinical-grade CAR+ T cells, we are proceeding with studies to enable EGFR to be targeted in children with DIPG.
235. In Vivo Delivery of Interferon-alpha Gene Enhances Tumor Immunity and Suppresses Immunotolerance after Autologous Hematopoietic Stem Cell Transplantation
Kenta Narumi,1 Takeshi Udagawa,1 Yoshinori Ikarashi,1 Takahiro Ochiya,2 Teruhiko Yoshida,3 Kazunori Aoki.1 1 Division of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan; 2Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; 3Division of Genetics, National Cancer Center Research Institute, Tokyo, Japan. It is known that lymphopenia is followed by spontaneous expansion of the remaining T cells in the periphery to restore the original T-cell pool size and maintain homeostasis. Lymphopenia-induced homeostatic proliferation (HP) of T cells following autologous hematopoietic stem cell transplantation (HSCT) is driven by the recognition of self antigens, and there is an opportunity to skew the T-cell repertoire during the T-cell recovery by engaging tumorassociated antigens (TAAs), leading to an induction of antitumor immunity. However, integration of other immunotherapeutic strategies is necessary to successfully suppress pre-existing malignant tumors, because HP-driven antitumor responses decay gradually since they are vulnerable to a development of tolerance. IFNs have important roles in regulating the innate and adaptive arms of the immune system: up-regulation of major histocompatibility complex class I gene, promotion of the priming and survival of T cells, increase of the cytotoxic activity of natural killer cells and CD8+ T cells, and activation of dendritic cells (DCs). Therefore, in this study, we examined whether HP-induced antitumor activity can be enhanced by IFN-α gene transfer during a physiologic immune reconstitution. From the viewpoint of IFN-α immune therapy also, an autologous HSCT following a preconditioning is expected to introduce a fresh immune system, in which tolerance to tumor cells is not yet induced, and may present a unique opportunity for IFN-α to augment efficacy of the immune therapy. Growth of subcutaneous tumors was significantly suppressed in syngeneic hematopoietic stem cell transplantation (synHSCT) recipient mice, and an IFN-α gene transfer into the tumors resulted in synergistic tumor suppression when administered in the early period of synHSCT. The antitumor effect of the combination therapy was evident even in distant tumors such as liver metastases that were not transduced with the IFN-α vector. There was no significant toxicity in the treated animals. To analyze antitumor immune mechanisms induced by the combination therapy, we isolated CD11c+ cells from the IFN plasmid-injected tumors in synHSCT recipients. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c+ cells and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c+ cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including IL-6. The CD11c+ cells rescued effector T-cell proliferation from regulatory T cell-mediated suppression, and IL-6 may play a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
234. T Cells Genetically Modified To Express Chimeric Antigen Receptor Specific for EGFR for Glioma
Hillary G. Caruso,1 Sonny Ang,1 Singh S. Harjeet,1 Simon Olivares,1 Laurence J. N. Cooper.1 1 Pediatrics, M.D. Anderson Cancer Center, Houston, TX.
Diffuse Intrinsic Pontine Glioma (DIPG) is an inoperable and nearly uniformly fatal tumor that afflicts children. These tumors overexpress wild type epidermal growth factor receptor (EGFR), and a recent clinical trial had shown that Nimotuzumab, an antiEGFR monoclonal antibody (mAb), has transient and surprisingly moderate clinical benefit for these patients. We seek to extend this antibody therapy to T-cell therapy by introducing a chimeric antigen receptor (CAR) to redirect the specificity of autologous T cells to EGFR. A second-generation EGFR-specific CAR was generated by fusing the specific region (scFv) of Nimotuzumab to T cell activation domains CD28 and CD3ζ and expressed as Sleeping Beauty (SB) transposon. The CAR was electroporated into primary PBMC with a SB transposase enzyme to cut CAR out of the vector and insert it into the genome. T cells expressing EGFR-specific CAR were propagated on irradiated artificial antigen presenting (aAPC) cells modified to express EGFR. The culture system was adapted to provide robust and selective outgrowth of CAR+ T cells by every-seven-day addition of γ-irradiated aAPC in the presence of IL-2 and IL-21. It is this platform that we are using to generate clinical-grade CD19-specific CAR+ T cells. T cells expressing EGFR-specific CAR were detected 24 hours post-electroporation in 30-50% of the PBMC by flow cytometry using Fc-specific Ab which recognizes the IgG4 region of the CAR. EGFRspecific T cells were numerically expanded by 5 logs over 28 days to yield a population of primarily CD8+ T cells with effector-memory phenotype (CCR7neg CD45RO+). We were able to detect re-directed target specificity of the EGFR-specific CAR+ T cells by measuring intracellular IFN-γ production in response to EGFR+ targets.
S92
In aggregate, these data establish that T cells can be re-directed for EGFR based on Nimotuzumab. Given our ability to manufacture clinical-grade CAR+ T cells, we are proceeding with studies to enable EGFR to be targeted in children with DIPG.
235. In Vivo Delivery of Interferon-alpha Gene Enhances Tumor Immunity and Suppresses Immunotolerance after Autologous Hematopoietic Stem Cell Transplantation
Kenta Narumi,1 Takeshi Udagawa,1 Yoshinori Ikarashi,1 Takahiro Ochiya,2 Teruhiko Yoshida,3 Kazunori Aoki.1 1 Division of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan; 2Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; 3Division of Genetics, National Cancer Center Research Institute, Tokyo, Japan. It is known that lymphopenia is followed by spontaneous expansion of the remaining T cells in the periphery to restore the original T-cell pool size and maintain homeostasis. Lymphopenia-induced homeostatic proliferation (HP) of T cells following autologous hematopoietic stem cell transplantation (HSCT) is driven by the recognition of self antigens, and there is an opportunity to skew the T-cell repertoire during the T-cell recovery by engaging tumorassociated antigens (TAAs), leading to an induction of antitumor immunity. However, integration of other immunotherapeutic strategies is necessary to successfully suppress pre-existing malignant tumors, because HP-driven antitumor responses decay gradually since they are vulnerable to a development of tolerance. IFNs have important roles in regulating the innate and adaptive arms of the immune system: up-regulation of major histocompatibility complex class I gene, promotion of the priming and survival of T cells, increase of the cytotoxic activity of natural killer cells and CD8+ T cells, and activation of dendritic cells (DCs). Therefore, in this study, we examined whether HP-induced antitumor activity can be enhanced by IFN-α gene transfer during a physiologic immune reconstitution. From the viewpoint of IFN-α immune therapy also, an autologous HSCT following a preconditioning is expected to introduce a fresh immune system, in which tolerance to tumor cells is not yet induced, and may present a unique opportunity for IFN-α to augment efficacy of the immune therapy. Growth of subcutaneous tumors was significantly suppressed in syngeneic hematopoietic stem cell transplantation (synHSCT) recipient mice, and an IFN-α gene transfer into the tumors resulted in synergistic tumor suppression when administered in the early period of synHSCT. The antitumor effect of the combination therapy was evident even in distant tumors such as liver metastases that were not transduced with the IFN-α vector. There was no significant toxicity in the treated animals. To analyze antitumor immune mechanisms induced by the combination therapy, we isolated CD11c+ cells from the IFN plasmid-injected tumors in synHSCT recipients. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c+ cells and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c+ cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including IL-6. The CD11c+ cells rescued effector T-cell proliferation from regulatory T cell-mediated suppression, and IL-6 may play a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy