- Email: [email protected]
33. Genetic Modification of Antigen-Specific Cytotoxic T Lymphocytes (CTLs) Restores Their Ability To Respond to Interleukin-7 (IL-7)
Cancer - Immunotherapy: Designer Effector Cells eyes treated with GRP78. Conclusions: The over-expression of GRP78 in photoreceptors decreases the rate of retinal degeneration in ADRP animal models caused by misfolded P23H rhodopsin protein. The mechanism by which the GRP78 provide the therapy remains to be elucidated. Optimization of the GRP78 gene delivery might be necessarily to elevate the therapeutic effect.
32. Long Term Expression of an Anti-VEGF Molecule for Inhibition of Ocular Angiogenesis by Intra-Ocular Gene Delivery in Murine and Primate Models
Abraham Scaria,1 Peter Pechan,1 Michael Lukason,1 Hillard Rubin,1 Elizabeth DuFresne,1 Tim Maclachlan,1 Margaret Wills,2 Christina Flaxel,3 Ivana Kim,4 Szilard Kiss,4 Joan Miller,4 Gabor Veres,5 William Hauswirth,6 Samuel Wadsworth.1 1 Molecular Biology, Genzyme Corporation, Framingham, MA; 2 Preclinical Services, Charles River Labs, Sparks, NV; 3Casey Eye Institute, Portland, OR; 4Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA; 5Applied Genetic Therapies, Corp., Alachua, FL; 6Ophthalmology, Univ of Florida, Gainesville, FL. Vascular endothelial growth factor (VEGF) plays a critical role in pathological neovascularization which is a key component of ocular diseases like neovascular age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). There are numerous preclinical and clinical studies that demonstrate that antagonizing VEGF is a useful strategy for treating such disorders, however current treatments require monthly intravitreal injections. We have designed and constructed a soluble hybrid anti-VEGF molecule (sFLT01) and delivered it by intravitreal injection of an adeno-associated viral (AAV2) vector. The AAV2-sFLT01 vector delivered intravitreally transduces predominantly ganglion cells in the murine retina and results in persistent expression of sFLT01 protein for at least one year. We have shown that AAV2-sFLT01 inhibits ocular neovascularization in the murine oxygen induced retinopathy (OIR) model in neonatal mice and the laser-CNV model performed at different time points post vector administration in adult mice. When injected into the eyes of cynomolgus monkeys, we find that AAV2-sFLT01 gives expression levels persistent for at least one year following administration of the AAV vector. We performed laser-CNV experiments several months after vector administration in non-human primates and show that sFLT01 was very effective at inhibiting neovascularization in this NHP model. These results suggest an alternate method of long term treatment for diseases of ocular neovascularization without the burden of repeated injections.
Cancer – Immunotherapy: Designer Effector Cells 33. Genetic Modification of Antigen-Specific Cytotoxic T Lymphocytes (CTLs) Restores Their Ability To Respond to Interleukin-7 (IL-7)
Juan F. Vera,1 Valentina Hoyos,1 Barbara Savoldo,1 Concetta Quintarelli,1 Greta Giordano Attianese,1 Aaron E. Foster,1 Helen M. Heslop H,1 Cliona M. Rooney,1 Malcolm K. Brenner,1 Gianpietro Dotti.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Systemic administration of IL2 may improve the expansion and persistence of adoptively transferred anti-tumor CTLs, but toxicity and concomitant expansion of regulatory T cells (Tregs) limit the clinical value of this strategy. IL7 plays a crucial role in maintaining T-cell homeostasis, and administration appears well tolerated and not to expand Tregs. Unfortunately, IL7 may be unable to expand tumor-specific CTLs in vivo because they lack expression of the Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
IL7 receptor (IL7Ra): the receptor is, for example, essentially undetectable on EBV-specific CTL lines due to downregulation of IL7Ra transcripts. To determine whether transgenic expression of IL7Ra enables established EBV-CTLs to respond to rhIL7, hIL7Ra was cloned in the SFG retroviral vector (SFG/IL7Ra), which was then used to transduce EBV-CTLs from 5 healthy donors. We compared their growth kinetics and antigen specificity with EBV-CTLs that were transduced with the SFG retroviral vector encoding a truncated CD34 molecule (SFG.DCD34). After transduction with SFG/IL7Ra, IL7Ra was detectable in 58% to 76% of EBV-CTLs. The transgenic IL7Ra was functional since addition of IL7 (2ng/mL) to transgenic cells induced phosphorylation of STAT5 within 10 min. To evaluate whether rhIL7 sustained the expansion of EBV-CTLs/IL7Ra+, transduced CTLs were stimulated weekly with autologous EBVLCLs in the presence of IL2 (50U/mL) or IL7 (2ng/mL). Control EBV-CTLs and EBV-CTLs/IL7Ra+ expanded equally well with IL2. However, only EBV-CTL/IL7Ra+ significantly proliferated in the presence of IL7 [from 1x106 cells to 1.03x108 cells (range, 0.38-2.9x108)] over a period of 5 weeks. CTL expansion remained antigen dependent since antigen withdrawal halted CTL growth. The EBV-CTLs/IL7Ra+ showed the expected selective growth advantage in the presence of IL7, increasing from 55%±15% to 79%±5% of total cells within 5 weeks. In contrast, the proportion of IL7Ra+ cells marginally declined when the cells were expanded with IL2 (from 64%±14% to 40%±11%). Importantly, EBV-CTLs/IL7Ra+ expanded with IL7 retained their ability to respond to other common-g-chain cytokines such as IL2 and IL15. Control CTLs grown with IL2 and EBV-CTLs/IL7Ra+ grown with rhIL7 both remained polyclonal and were mostly CD3+/CD8+ (> 90±8%) with an effector-memory profile. EBV-CTLs/IL7Ra+ also retained antigen specificity measured by tetramer staining, by IFNg release in response to EBV-peptides and by MHC-restricted killing of autologous LCLs (52%±18% at a ratio 20:1 vs. 9±17% of allogeneic LCL). These in vitro characteristics are replicated in vivo. We used a SCID mouse model, in which EBVCTLs/IL7Ra+ labelled with Firefly luciferase (10x106) were injected i.v. in mice engrafted subcutaneously with EBV-LCLs (10x106). We found a significant increase of bioluminescence from CTLs in mice receiving hIL7 (500ng 3 times per week) compared to mice without cytokine (7.5 fold± 3.6 vs. 1.2 fold ± 0.6). Response to IL2 was conserved. This approach may improve the clinical efficacy of CTL therapies.
34. Zinc Finger Nucleases Targeting the Glucocorticoid Receptor Allow IL-13 Zetakine Transgenic CTLs To Kill Glioblastoma Cells In Vivo in the Presence of Immunosuppressing Glucocorticoids
Andreas Reik,1 Yuanyue Zhou,1 Araceli Hamlett,2 Jamie Wagner,2 Matthew C. Mendel,1 Colin W. Flinders,1 Pei-Qi Liu,1 Gary Lee,1 David E. Paschon,1 Edward J. Rebar,1 Dale Ando,1 David L. DiGiusto,2 Philip D. Gregory,1 Michael C. Holmes,1 Michael C. Jensen.2 1 Sangamo BioSciences, Richmond, CA; 2Division of Cancer Immunotherapeutics & Tumor Immunology, City of Hope National Medical Center, Duarte, CA. Glioblastoma-specific cytolytic T-lymphocytes (CTLs) can be generated by introducing a chimeric T-cell receptor consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain (IL13zetakine) into CD8+ T-cells. Both in vitro and in animal models these CTLs effectively kill malignant glioma cells which are characterized by high expression of the IL13 receptor a2. Indeed, patient-specific IL13-zetakine expressing CD8+ T-cell products have entered early stage clinical trials. However, these CTLs targeting recurrent glioblastoma multiforme are rendered ineffective if, as is often the case, patients require anti-inflammatory glucocorticoids postS13
32. Long Term Expression of an Anti-VEGF Molecule for Inhibition of Ocular Angiogenesis by Intra-Ocular Gene Delivery in Murine and Primate Models
Abraham Scaria,1 Peter Pechan,1 Michael Lukason,1 Hillard Rubin,1 Elizabeth DuFresne,1 Tim Maclachlan,1 Margaret Wills,2 Christina Flaxel,3 Ivana Kim,4 Szilard Kiss,4 Joan Miller,4 Gabor Veres,5 William Hauswirth,6 Samuel Wadsworth.1 1 Molecular Biology, Genzyme Corporation, Framingham, MA; 2 Preclinical Services, Charles River Labs, Sparks, NV; 3Casey Eye Institute, Portland, OR; 4Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA; 5Applied Genetic Therapies, Corp., Alachua, FL; 6Ophthalmology, Univ of Florida, Gainesville, FL. Vascular endothelial growth factor (VEGF) plays a critical role in pathological neovascularization which is a key component of ocular diseases like neovascular age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). There are numerous preclinical and clinical studies that demonstrate that antagonizing VEGF is a useful strategy for treating such disorders, however current treatments require monthly intravitreal injections. We have designed and constructed a soluble hybrid anti-VEGF molecule (sFLT01) and delivered it by intravitreal injection of an adeno-associated viral (AAV2) vector. The AAV2-sFLT01 vector delivered intravitreally transduces predominantly ganglion cells in the murine retina and results in persistent expression of sFLT01 protein for at least one year. We have shown that AAV2-sFLT01 inhibits ocular neovascularization in the murine oxygen induced retinopathy (OIR) model in neonatal mice and the laser-CNV model performed at different time points post vector administration in adult mice. When injected into the eyes of cynomolgus monkeys, we find that AAV2-sFLT01 gives expression levels persistent for at least one year following administration of the AAV vector. We performed laser-CNV experiments several months after vector administration in non-human primates and show that sFLT01 was very effective at inhibiting neovascularization in this NHP model. These results suggest an alternate method of long term treatment for diseases of ocular neovascularization without the burden of repeated injections.
Cancer – Immunotherapy: Designer Effector Cells 33. Genetic Modification of Antigen-Specific Cytotoxic T Lymphocytes (CTLs) Restores Their Ability To Respond to Interleukin-7 (IL-7)
Juan F. Vera,1 Valentina Hoyos,1 Barbara Savoldo,1 Concetta Quintarelli,1 Greta Giordano Attianese,1 Aaron E. Foster,1 Helen M. Heslop H,1 Cliona M. Rooney,1 Malcolm K. Brenner,1 Gianpietro Dotti.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Systemic administration of IL2 may improve the expansion and persistence of adoptively transferred anti-tumor CTLs, but toxicity and concomitant expansion of regulatory T cells (Tregs) limit the clinical value of this strategy. IL7 plays a crucial role in maintaining T-cell homeostasis, and administration appears well tolerated and not to expand Tregs. Unfortunately, IL7 may be unable to expand tumor-specific CTLs in vivo because they lack expression of the Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
IL7 receptor (IL7Ra): the receptor is, for example, essentially undetectable on EBV-specific CTL lines due to downregulation of IL7Ra transcripts. To determine whether transgenic expression of IL7Ra enables established EBV-CTLs to respond to rhIL7, hIL7Ra was cloned in the SFG retroviral vector (SFG/IL7Ra), which was then used to transduce EBV-CTLs from 5 healthy donors. We compared their growth kinetics and antigen specificity with EBV-CTLs that were transduced with the SFG retroviral vector encoding a truncated CD34 molecule (SFG.DCD34). After transduction with SFG/IL7Ra, IL7Ra was detectable in 58% to 76% of EBV-CTLs. The transgenic IL7Ra was functional since addition of IL7 (2ng/mL) to transgenic cells induced phosphorylation of STAT5 within 10 min. To evaluate whether rhIL7 sustained the expansion of EBV-CTLs/IL7Ra+, transduced CTLs were stimulated weekly with autologous EBVLCLs in the presence of IL2 (50U/mL) or IL7 (2ng/mL). Control EBV-CTLs and EBV-CTLs/IL7Ra+ expanded equally well with IL2. However, only EBV-CTL/IL7Ra+ significantly proliferated in the presence of IL7 [from 1x106 cells to 1.03x108 cells (range, 0.38-2.9x108)] over a period of 5 weeks. CTL expansion remained antigen dependent since antigen withdrawal halted CTL growth. The EBV-CTLs/IL7Ra+ showed the expected selective growth advantage in the presence of IL7, increasing from 55%±15% to 79%±5% of total cells within 5 weeks. In contrast, the proportion of IL7Ra+ cells marginally declined when the cells were expanded with IL2 (from 64%±14% to 40%±11%). Importantly, EBV-CTLs/IL7Ra+ expanded with IL7 retained their ability to respond to other common-g-chain cytokines such as IL2 and IL15. Control CTLs grown with IL2 and EBV-CTLs/IL7Ra+ grown with rhIL7 both remained polyclonal and were mostly CD3+/CD8+ (> 90±8%) with an effector-memory profile. EBV-CTLs/IL7Ra+ also retained antigen specificity measured by tetramer staining, by IFNg release in response to EBV-peptides and by MHC-restricted killing of autologous LCLs (52%±18% at a ratio 20:1 vs. 9±17% of allogeneic LCL). These in vitro characteristics are replicated in vivo. We used a SCID mouse model, in which EBVCTLs/IL7Ra+ labelled with Firefly luciferase (10x106) were injected i.v. in mice engrafted subcutaneously with EBV-LCLs (10x106). We found a significant increase of bioluminescence from CTLs in mice receiving hIL7 (500ng 3 times per week) compared to mice without cytokine (7.5 fold± 3.6 vs. 1.2 fold ± 0.6). Response to IL2 was conserved. This approach may improve the clinical efficacy of CTL therapies.
34. Zinc Finger Nucleases Targeting the Glucocorticoid Receptor Allow IL-13 Zetakine Transgenic CTLs To Kill Glioblastoma Cells In Vivo in the Presence of Immunosuppressing Glucocorticoids
Andreas Reik,1 Yuanyue Zhou,1 Araceli Hamlett,2 Jamie Wagner,2 Matthew C. Mendel,1 Colin W. Flinders,1 Pei-Qi Liu,1 Gary Lee,1 David E. Paschon,1 Edward J. Rebar,1 Dale Ando,1 David L. DiGiusto,2 Philip D. Gregory,1 Michael C. Holmes,1 Michael C. Jensen.2 1 Sangamo BioSciences, Richmond, CA; 2Division of Cancer Immunotherapeutics & Tumor Immunology, City of Hope National Medical Center, Duarte, CA. Glioblastoma-specific cytolytic T-lymphocytes (CTLs) can be generated by introducing a chimeric T-cell receptor consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain (IL13zetakine) into CD8+ T-cells. Both in vitro and in animal models these CTLs effectively kill malignant glioma cells which are characterized by high expression of the IL13 receptor a2. Indeed, patient-specific IL13-zetakine expressing CD8+ T-cell products have entered early stage clinical trials. However, these CTLs targeting recurrent glioblastoma multiforme are rendered ineffective if, as is often the case, patients require anti-inflammatory glucocorticoids postS13