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561. Expansion of FoxP3+ Regulatory T Cells in Mucopolysaccharidosis Type I Mice Prior to Lentiviral Gene Transfer Promotes Tolerance to α-l-Iduronidase
IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY CD34 stem cell enriched, mismatched allogeneic stem cell grafts. All patients had pre-existing viral infections and received chemotherapy conditioning but not serotherapy. The purity of transduced T cells by CliniMacs selection on the basis CD34 expression was between 92-97%, and all products were sensitive to the prodrug Ganciclovir in vitro. All infusions were completed without any evidence of acute toxicity, and resulted in low levels of circulating gene modified T cells detectable by flow cytometry for CD3+CD34+ T cells. No patients developed acute graft versus host disease >Grade I. Adoptive transfer of virus specific cellular immunity against Varicella zoster, Adenovirus and H1N1 influenza virus was confirmed on the basis of gamma interferon secretion by donor T cells following challenge with specific antigen. Conclusion: Preliminary findings confirm the feasibility and safety of donor T cells modified to express a HSVTKCD34 suicide gene, and reconstitution data shows adoptive transfer of T cell immunity against viral pathogens.
560. Autophagy Induced by Oncolytic Adenoviruses Combined with Temozolomide Triggers Antitumor Immune Responses Preclinically and in Cancer Patients
Ilkka Liikanen,1 Mari Hirvinen,1 Simona Bramante,1 Vincenzo Cerullo,1 Iulia Diaconu,1 Sari Pesonen,1 Anniina Koski,1 Lotta Kangasniemi,2 Fang Zhao,3 Timo Joensuu,4 Anna Kanerva,1 Akseli Hemminki.1 1 Cancer Gene Therapy Group, Molecular Cancer Biology Program, Transplantation Laboratory and Haartman Institute, University of Helsinki, Finland; 2Oncos Therapeutics, Ltd., Helsinki, Finland; 3Advanced Microscopy Unit, Haartman Institute, University of Helsinki, Finland; 4International Comprehensive Cancer Center Docrates, Docrates Clinic, Helsinki, Finland. Purpose: Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cell death. Combinations of these, however, have not been studied in humans. We hypothesize that the combination of oncolytic adenovirus with temozolomide is safe, effective and capable of inducing antitumor immune responses. Experimental design: We assessed the combinations of oncolytic adenovirus, temozolomide, and immunomodulatory cyclophosphamide in prostate cancer cells in vitro and in vivo. Cell viability, autophagy, and immunogenicity assays were conducted. 17 patients with solid tumors refractory to conventional therapies were treated in a personalized advanced therapy access program with oncolytic adenovirus together with a low-dose pulse of temozolomide. Concurrent low-dose cyclophosphamide was administered to reduce regulatory T-cells. The primary end-point was safety. In addition, immunological, virological, and efficacy parameters were studied. Results: The combination of oncolytic adenovirus, temozolomide, and cyclophosphamide resulted in superior cell killing in vitro and enhanced tumor growth control in mice. Electron microscopy and immunohistochemistry revealed increased autophagy in combination treated tumors, while elevated extracellular ATP levels suggested immunogenic cell death in vitro. Combination treatments in patients were well-tolerated with mostly grade 1–2 flu-like symptoms, nausea, and transient lymphopenia and anemia. Immunostimulatory cytokine elevations and T-cell activations against tumor-associated antigen were observed. Objective evidence of antitumor activity (imaging or tumor marker response) was seen in 15/22 evaluable treatments. Long-term survival was increased over virus-only treated matched non-randomized controls. Conclusions: Low-dose pulse temozolomide and low-dose cyclophosphamide in combination with oncolytic adenovirus increased autophagy, suggested antitumor immune activation, enhanced efficacy in preclinical tests, and were well-tolerated in patients. Further studies are needed to assess if Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
temozolomide increases the efficacy of oncolytic virotherapy in humans. However, these results support the hypothesis that oncolysis and chemotherapy-mediated autophagy can lead to antitumor immunity.
561. Expansion of FoxP3+ Regulatory T Cells in Mucopolysaccharidosis Type I Mice Prior to Lentiviral Gene Transfer Promotes Tolerance to α-l-Iduronidase
Kevin Goudy,1 Alessio Cantore,1 Andrea Annoni,1 Mahzad Akbarpour,1 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Maria Grazia Roncarolo.1,2 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 vita Salute San Raffaele University, Milan, Italy. The frequency and potency of FoxP3-expressing regulatory T cells (Treg) are critical factors in regulating tolerance to transgenes since their suppressive abilities influence the quantity and function of transgene-specific, pathogenic effector T cells (Teff) after gene therapy. Gene replacement strategies to treat Mucopolysaccharidosis Type I (MPS-I) mice, a lysosomal storage disease resulting from the absence of α-l-Iduronidase (IDUA), requires long-term immunosuppression via systemic co-stimulatory blockade in order to prevent Teff cells from clearing IDUA producing cells. In an effort to induce tolerance by other means than immunosuppressive drugs, we injected MPS-1 mice using a known tolerogenic, Treg promoting lentiviral vector encoding a liver specific promoter, microRNA 142 target sequences, and human IDUA (LV.ET.IDUA.142T). Surprisingly, this approach was insufficient in preventing pathogenic immune responses against IDUA after in vivo administration in mice. Thus, to achieve IDUA tolerance and to evaluate the effectiveness of Treg cell expansion in maintaining tolerance after LV administration, we tested whether the expansion of Treg prior to LV.ET.IDUA.142T can prevent the immune-mediated clearance of IDUA expressing cells. Preconditioning regimens of 1ug or 2ug of an IL-2 complex (recombinant mIL-2 coupled with the IL-2 antibody (JES61)) were injected on day -5 before LV injection and given daily for three days to expand Tregs. After two days of rest, MPS-1 mice were injected with LV.ET.IDUA.142T on day 0 and followed for immunological responses and transgene persistence. Mice from the 1ug and 2ug IL-2 complex treated groups exhibited increased frequencies of Treg and elevated serum levels of IL-10 at the time of LV injection (blood Treg were two-fold higher in 1ug treated mice and two-three fold higher in the 2ug treated group compared to injected only). However, the increased Treg and IL-10 levels were transient since one week after LV injection, the frequency of Treg in blood of the 1ug and 2ug treated groups were comparable to injected only animals and serum IL-10 was undetectable in all groups. Interestingly, all mice in the 2ug treated group had a significantly lower level of anti-IDUA antibodies and significantly reduced number of IDUA specific IFNγ secreting CD8+ T cells suggesting that the tolerogenic effects were dose dependent. Furthermore, we identified an immunogenic epitope of the IDUA protein that can induce IFNγ release from CD8+ T cells. Taken together, this is the first known report to show that the immune response to IDUA can be controlled by a cellular mechanism and to identify a CD8+ T cell epitope encoded by IDUA. This study also puts forth the possibility that expansion of Treg in immuno-stringent gene therapy settings may provide a means to induce tolerance to transgenes while negating the use of immunosuppressive drugs.
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560. Autophagy Induced by Oncolytic Adenoviruses Combined with Temozolomide Triggers Antitumor Immune Responses Preclinically and in Cancer Patients
Ilkka Liikanen,1 Mari Hirvinen,1 Simona Bramante,1 Vincenzo Cerullo,1 Iulia Diaconu,1 Sari Pesonen,1 Anniina Koski,1 Lotta Kangasniemi,2 Fang Zhao,3 Timo Joensuu,4 Anna Kanerva,1 Akseli Hemminki.1 1 Cancer Gene Therapy Group, Molecular Cancer Biology Program, Transplantation Laboratory and Haartman Institute, University of Helsinki, Finland; 2Oncos Therapeutics, Ltd., Helsinki, Finland; 3Advanced Microscopy Unit, Haartman Institute, University of Helsinki, Finland; 4International Comprehensive Cancer Center Docrates, Docrates Clinic, Helsinki, Finland. Purpose: Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cell death. Combinations of these, however, have not been studied in humans. We hypothesize that the combination of oncolytic adenovirus with temozolomide is safe, effective and capable of inducing antitumor immune responses. Experimental design: We assessed the combinations of oncolytic adenovirus, temozolomide, and immunomodulatory cyclophosphamide in prostate cancer cells in vitro and in vivo. Cell viability, autophagy, and immunogenicity assays were conducted. 17 patients with solid tumors refractory to conventional therapies were treated in a personalized advanced therapy access program with oncolytic adenovirus together with a low-dose pulse of temozolomide. Concurrent low-dose cyclophosphamide was administered to reduce regulatory T-cells. The primary end-point was safety. In addition, immunological, virological, and efficacy parameters were studied. Results: The combination of oncolytic adenovirus, temozolomide, and cyclophosphamide resulted in superior cell killing in vitro and enhanced tumor growth control in mice. Electron microscopy and immunohistochemistry revealed increased autophagy in combination treated tumors, while elevated extracellular ATP levels suggested immunogenic cell death in vitro. Combination treatments in patients were well-tolerated with mostly grade 1–2 flu-like symptoms, nausea, and transient lymphopenia and anemia. Immunostimulatory cytokine elevations and T-cell activations against tumor-associated antigen were observed. Objective evidence of antitumor activity (imaging or tumor marker response) was seen in 15/22 evaluable treatments. Long-term survival was increased over virus-only treated matched non-randomized controls. Conclusions: Low-dose pulse temozolomide and low-dose cyclophosphamide in combination with oncolytic adenovirus increased autophagy, suggested antitumor immune activation, enhanced efficacy in preclinical tests, and were well-tolerated in patients. Further studies are needed to assess if Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
temozolomide increases the efficacy of oncolytic virotherapy in humans. However, these results support the hypothesis that oncolysis and chemotherapy-mediated autophagy can lead to antitumor immunity.
561. Expansion of FoxP3+ Regulatory T Cells in Mucopolysaccharidosis Type I Mice Prior to Lentiviral Gene Transfer Promotes Tolerance to α-l-Iduronidase
Kevin Goudy,1 Alessio Cantore,1 Andrea Annoni,1 Mahzad Akbarpour,1 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Maria Grazia Roncarolo.1,2 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 vita Salute San Raffaele University, Milan, Italy. The frequency and potency of FoxP3-expressing regulatory T cells (Treg) are critical factors in regulating tolerance to transgenes since their suppressive abilities influence the quantity and function of transgene-specific, pathogenic effector T cells (Teff) after gene therapy. Gene replacement strategies to treat Mucopolysaccharidosis Type I (MPS-I) mice, a lysosomal storage disease resulting from the absence of α-l-Iduronidase (IDUA), requires long-term immunosuppression via systemic co-stimulatory blockade in order to prevent Teff cells from clearing IDUA producing cells. In an effort to induce tolerance by other means than immunosuppressive drugs, we injected MPS-1 mice using a known tolerogenic, Treg promoting lentiviral vector encoding a liver specific promoter, microRNA 142 target sequences, and human IDUA (LV.ET.IDUA.142T). Surprisingly, this approach was insufficient in preventing pathogenic immune responses against IDUA after in vivo administration in mice. Thus, to achieve IDUA tolerance and to evaluate the effectiveness of Treg cell expansion in maintaining tolerance after LV administration, we tested whether the expansion of Treg prior to LV.ET.IDUA.142T can prevent the immune-mediated clearance of IDUA expressing cells. Preconditioning regimens of 1ug or 2ug of an IL-2 complex (recombinant mIL-2 coupled with the IL-2 antibody (JES61)) were injected on day -5 before LV injection and given daily for three days to expand Tregs. After two days of rest, MPS-1 mice were injected with LV.ET.IDUA.142T on day 0 and followed for immunological responses and transgene persistence. Mice from the 1ug and 2ug IL-2 complex treated groups exhibited increased frequencies of Treg and elevated serum levels of IL-10 at the time of LV injection (blood Treg were two-fold higher in 1ug treated mice and two-three fold higher in the 2ug treated group compared to injected only). However, the increased Treg and IL-10 levels were transient since one week after LV injection, the frequency of Treg in blood of the 1ug and 2ug treated groups were comparable to injected only animals and serum IL-10 was undetectable in all groups. Interestingly, all mice in the 2ug treated group had a significantly lower level of anti-IDUA antibodies and significantly reduced number of IDUA specific IFNγ secreting CD8+ T cells suggesting that the tolerogenic effects were dose dependent. Furthermore, we identified an immunogenic epitope of the IDUA protein that can induce IFNγ release from CD8+ T cells. Taken together, this is the first known report to show that the immune response to IDUA can be controlled by a cellular mechanism and to identify a CD8+ T cell epitope encoded by IDUA. This study also puts forth the possibility that expansion of Treg in immuno-stringent gene therapy settings may provide a means to induce tolerance to transgenes while negating the use of immunosuppressive drugs.
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