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300. Virus-Specific Cytotoxic T Lymphocytes (CTLs) Redirected with a Chimeric Antigen Receptor (CAR) and Engineered To Respond to IL7 Resist Regulatory T Cell (Treg) Inhibition
CANCER-TARGETED GENE & CELL THERAPY 300. Virus-Specific Cytotoxic T Lymphocytes (CTLs) Redirected with a Chimeric Antigen Receptor (CAR) and Engineered To Respond to IL7 Resist Regulatory T Cell (Treg) Inhibition
Serena K. Perna,1 Daria Pagliara,1 Aruna Mahendravada,1 Hao Liu,1 Malcolm K. Brenner,1 Barbara Savoldo,1 Gianpietro Dotti.1 1 Center For Cell And Gene Theraphy, Baylor College of Medicine, Houston, TX.
In human studies of CAR-expressing T cells, expansion and/ or persistence are likely critical for effective clinical responses. Persistence may be prolonged by expression of CARs in cytotoxic T cells with native receptor specificity for persistent virus infections, since this allows the CAR-T cells to receive co-stimulation by professional antigen presenting cells expressing viral antigens. We previously demonstrated that it was safe to administer Epstein-Barr virus (EBV)-specific CTLs (EBV-CTLs) expressing a CAR targeting the neuroblastoma (NB) associated antigen GD2, and that the cells produced complete tumor responses in 3/11 patients with refractory tumors. Nonetheless, these CAR-GD2+ EBV-CTLs had limited expansion and persistence in vivo, in part due to host inhibitory factors such as tumor-associated Tregs. To overcome inhibition attributable to Tregs we exploited the lack of IL7R expression in this cell subset and modified the IL7 cytokine-cytokine receptor axis in CAR-redirected EBV-CTLs. We generated a bicistronic retroviral vector encoding the IL7R and CAR-GD2 (II generation) and genetically modified EBV-CTLs to express these genes. Gene transfer resulted in a stable expression of both IL7R and CAR-GD2 (34%±9% and 64%±3%, respectively). To determine if IL7 supported the antitumor activity of IL7R.CAR-GD2+ EBV-CTLs even in the presence of Tregs, CTLs were co-cultured with NB cells and Tregs. CTL antitumor activity was impaired in the presence of Tregs when IL2 was used to support CTL growth (residual NB cells 6±1% without Tregs vs 14±3% with Tregs). By contrast, antitumor activity remained unaffected in the presence of Tregs when IL7 was added to the culture (residual NB cells 4±1% without Tregs vs 7±2% with Tregs). To ensure that IL7 sustained the proliferation of IL7R.CAR-GD2+ EBV-CTLs in the presence of Tregs, CFSE-labeled CTLs were cultured with LCL and Tregs with IL2 or IL7. CTL proliferation was significantly impaired in the presence of IL2 and Tregs (68±4% without Tregs vs 34±6% with Tregs) but not in the presence of IL7 and Tregs (63%±3% without Tregs vs 56%±2% with Tregs). Finally, we assessed in vivo if IL7 supported the antitumor activity of IL7R.CAR-GD2+ EBV-CTLs in the presence of Tregs. NSG mice were engrafted with the FFLuc+ NB cell line CHLA-255 and then infused with IL7R. CAR-GD2+ EBV-CTLs ± IL2 or IL7 ± Tregs. We found that mice infused with IL-7R.CAR-GD2+ EBV-CTLs and IL2 controlled tumor growth (tumor luminescence 1.6x108±2x107 photons at day 34), but this effect was abrogated when Tregs were co-infused (tumor luminescence 2.4 x 108±4 x 107 photons at day 34, p<0.05). In contrast, mice infused with IL7R.CAR-GD2+ EBV-CTLs and IL7 controlled tumor growth equally well in the absence of Tregs (tumor luminescence 1.2x108±3x107 photons) or in the presence of Tregs (tumor luminescence 1.3x108±6x106 photons, at day 34). Our study suggests that restoring the responsiveness of CAR-redirected EBV-CTLs to IL7 overcomes Treg-mediated inhibition and may further increase the benefits of this validated therapeutic approach.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
301. Engineering Hematopoiesis for TumorTargeted Delivery of IFNa by Tumor-Infiltrating Macrophages Achieves Safe and Effective Inhibition of Breast Cancer and Its Metastases in Both Murine and Human Hematochimeric Models
Giulia Escobar,1,2 Davide Moi,2 Anna Ranghetti,2 Mario L. Squadrito,2 Michele De Palma,2 Roberta Mazzieri,2 Luigi Naldini.1,2 1 Vita-Salute San Raffaele University, Milan, Italy; 2HSR-TIGET, Milan, Italy.
Tumor cells critically depend on a permissive microenvironment for their maintenance and growth. Among other stromal cell types, we have described a population of TIE2-expressing monocytes (TEMs) that are specifically recruited from the bone marrow to the tumors and are required to promote angiogenesis and tumor growth. We thus hypothesized that genetic engineering of hematopoietic stem cells (HSC) with anti-tumor transgenes transcriptionally targeted to TEMs could provide a novel cell and gene-based therapy to treat tumors. We demonstrated striking inhibition of tumor growth in mice engrafted with HSC transduced with a lentiviral vector carrying an interferon alpha (IFNa) transgene driven by the Tie2 promoter (Tie2p). TIE2, however, is also expressed by HSC and chronic exposure to IFNa can activate HSC and lead to their exhaustion. We thus incorporated into the vector target sequences for miR-126 and miR-130a (miRT), which we previously showed to be specifically expressed in HSC and not in mature hematopoietic cells. By this strategy, we made the vector responsive to post-transcriptional regulation by these miRNAs and de-targeted its expression from HSC. Upon transplant of HSC engineered with the new Tie2p-IFNa-miRT vector, we abrogated IFNa effects on HSC cell cycle while achieving a durable inhibition of spontaneous breast cancer and its metastases, which was accompanied and likely mediated by strong infiltration in the regressing tumors of activated CD4 and CD8+ T cells. These encouraging results prompted us to move towards clinical translation. We first generated corresponding vectors with the human TIE2 promoter/enhancer and miRTs sequences, and expressing either a GFP reporter or the human IFNa2b cDNA. We then transduced human cord blood CD34+ cells and transplanted them into NOD-scid-Il2rg-/- (NSG) mice. By using the GFP transgene, we demonstrated sharply demarcated vector expression in the differentiated myeloid/monocytic lineage of the reconstituted human hematopoiesis, with effective miRT-mediated suppression in the HSC/progenitor compartments. By comparing the GFP and IFNa vectors, we demonstrated that the IFNa transgene did not impair engraftment, self-renewal, long-term repopulation and multilineage reconstitution by the transplanted human cells in NSG mice. We then engineered the mice to express the human IL-7, IL-15 and GM-CSF cytokines to improve reconstitution and support functional activity of human T, NK and myeloid cells in the mice. By this strategy, we could stringently assess the potential anti-tumor effects of our strategy. Upon challenging the mice with human breast carcinoma cells, we observed tumor-targeted activation of a type I IFN response and sharp tumor regression in the IFNa human hematochimeras, demonstrating the selectivity and efficacy of our strategy in an immunocompetent model of human hematopoiesis in vivo. Overall, our studies illustrate the safety, feasibility and therapeutic potential of a new gene- and cell-based strategy to treat established tumors.
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Serena K. Perna,1 Daria Pagliara,1 Aruna Mahendravada,1 Hao Liu,1 Malcolm K. Brenner,1 Barbara Savoldo,1 Gianpietro Dotti.1 1 Center For Cell And Gene Theraphy, Baylor College of Medicine, Houston, TX.
In human studies of CAR-expressing T cells, expansion and/ or persistence are likely critical for effective clinical responses. Persistence may be prolonged by expression of CARs in cytotoxic T cells with native receptor specificity for persistent virus infections, since this allows the CAR-T cells to receive co-stimulation by professional antigen presenting cells expressing viral antigens. We previously demonstrated that it was safe to administer Epstein-Barr virus (EBV)-specific CTLs (EBV-CTLs) expressing a CAR targeting the neuroblastoma (NB) associated antigen GD2, and that the cells produced complete tumor responses in 3/11 patients with refractory tumors. Nonetheless, these CAR-GD2+ EBV-CTLs had limited expansion and persistence in vivo, in part due to host inhibitory factors such as tumor-associated Tregs. To overcome inhibition attributable to Tregs we exploited the lack of IL7R expression in this cell subset and modified the IL7 cytokine-cytokine receptor axis in CAR-redirected EBV-CTLs. We generated a bicistronic retroviral vector encoding the IL7R and CAR-GD2 (II generation) and genetically modified EBV-CTLs to express these genes. Gene transfer resulted in a stable expression of both IL7R and CAR-GD2 (34%±9% and 64%±3%, respectively). To determine if IL7 supported the antitumor activity of IL7R.CAR-GD2+ EBV-CTLs even in the presence of Tregs, CTLs were co-cultured with NB cells and Tregs. CTL antitumor activity was impaired in the presence of Tregs when IL2 was used to support CTL growth (residual NB cells 6±1% without Tregs vs 14±3% with Tregs). By contrast, antitumor activity remained unaffected in the presence of Tregs when IL7 was added to the culture (residual NB cells 4±1% without Tregs vs 7±2% with Tregs). To ensure that IL7 sustained the proliferation of IL7R.CAR-GD2+ EBV-CTLs in the presence of Tregs, CFSE-labeled CTLs were cultured with LCL and Tregs with IL2 or IL7. CTL proliferation was significantly impaired in the presence of IL2 and Tregs (68±4% without Tregs vs 34±6% with Tregs) but not in the presence of IL7 and Tregs (63%±3% without Tregs vs 56%±2% with Tregs). Finally, we assessed in vivo if IL7 supported the antitumor activity of IL7R.CAR-GD2+ EBV-CTLs in the presence of Tregs. NSG mice were engrafted with the FFLuc+ NB cell line CHLA-255 and then infused with IL7R. CAR-GD2+ EBV-CTLs ± IL2 or IL7 ± Tregs. We found that mice infused with IL-7R.CAR-GD2+ EBV-CTLs and IL2 controlled tumor growth (tumor luminescence 1.6x108±2x107 photons at day 34), but this effect was abrogated when Tregs were co-infused (tumor luminescence 2.4 x 108±4 x 107 photons at day 34, p<0.05). In contrast, mice infused with IL7R.CAR-GD2+ EBV-CTLs and IL7 controlled tumor growth equally well in the absence of Tregs (tumor luminescence 1.2x108±3x107 photons) or in the presence of Tregs (tumor luminescence 1.3x108±6x106 photons, at day 34). Our study suggests that restoring the responsiveness of CAR-redirected EBV-CTLs to IL7 overcomes Treg-mediated inhibition and may further increase the benefits of this validated therapeutic approach.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
301. Engineering Hematopoiesis for TumorTargeted Delivery of IFNa by Tumor-Infiltrating Macrophages Achieves Safe and Effective Inhibition of Breast Cancer and Its Metastases in Both Murine and Human Hematochimeric Models
Giulia Escobar,1,2 Davide Moi,2 Anna Ranghetti,2 Mario L. Squadrito,2 Michele De Palma,2 Roberta Mazzieri,2 Luigi Naldini.1,2 1 Vita-Salute San Raffaele University, Milan, Italy; 2HSR-TIGET, Milan, Italy.
Tumor cells critically depend on a permissive microenvironment for their maintenance and growth. Among other stromal cell types, we have described a population of TIE2-expressing monocytes (TEMs) that are specifically recruited from the bone marrow to the tumors and are required to promote angiogenesis and tumor growth. We thus hypothesized that genetic engineering of hematopoietic stem cells (HSC) with anti-tumor transgenes transcriptionally targeted to TEMs could provide a novel cell and gene-based therapy to treat tumors. We demonstrated striking inhibition of tumor growth in mice engrafted with HSC transduced with a lentiviral vector carrying an interferon alpha (IFNa) transgene driven by the Tie2 promoter (Tie2p). TIE2, however, is also expressed by HSC and chronic exposure to IFNa can activate HSC and lead to their exhaustion. We thus incorporated into the vector target sequences for miR-126 and miR-130a (miRT), which we previously showed to be specifically expressed in HSC and not in mature hematopoietic cells. By this strategy, we made the vector responsive to post-transcriptional regulation by these miRNAs and de-targeted its expression from HSC. Upon transplant of HSC engineered with the new Tie2p-IFNa-miRT vector, we abrogated IFNa effects on HSC cell cycle while achieving a durable inhibition of spontaneous breast cancer and its metastases, which was accompanied and likely mediated by strong infiltration in the regressing tumors of activated CD4 and CD8+ T cells. These encouraging results prompted us to move towards clinical translation. We first generated corresponding vectors with the human TIE2 promoter/enhancer and miRTs sequences, and expressing either a GFP reporter or the human IFNa2b cDNA. We then transduced human cord blood CD34+ cells and transplanted them into NOD-scid-Il2rg-/- (NSG) mice. By using the GFP transgene, we demonstrated sharply demarcated vector expression in the differentiated myeloid/monocytic lineage of the reconstituted human hematopoiesis, with effective miRT-mediated suppression in the HSC/progenitor compartments. By comparing the GFP and IFNa vectors, we demonstrated that the IFNa transgene did not impair engraftment, self-renewal, long-term repopulation and multilineage reconstitution by the transplanted human cells in NSG mice. We then engineered the mice to express the human IL-7, IL-15 and GM-CSF cytokines to improve reconstitution and support functional activity of human T, NK and myeloid cells in the mice. By this strategy, we could stringently assess the potential anti-tumor effects of our strategy. Upon challenging the mice with human breast carcinoma cells, we observed tumor-targeted activation of a type I IFN response and sharp tumor regression in the IFNa human hematochimeras, demonstrating the selectivity and efficacy of our strategy in an immunocompetent model of human hematopoiesis in vivo. Overall, our studies illustrate the safety, feasibility and therapeutic potential of a new gene- and cell-based strategy to treat established tumors.
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