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599. Clinical Trial Results: Intralesional Injection of a Tumor Selective Oncolytic Vaccinia Virus
CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION but also increased yield titer up to 4-fold. Downstream processing also faced unusual barriers because large plasmids share similar physical/ chemical characteristics contaminating genomic DNA. Alkaline lysis is of particular concern as large plasmids are inefficient at renaturing. Special attention to process shear is required to maintain structural integrity. We have optimized downstream process conditions at every step. For a plasmid of size greater than 12 kb, the product demonstrated high purity suitable for pre-clinical or clinical applications.
Clinical Gene & Cell Therapy Oral Abstract Session 598. A Gene Therapy Approach to HIV: Adoptive Transfer of Zinc Finger Nuclease (ZFN) Modified Autologous CD4 T-Cells to Aviremic HIV-Infected Subjects with Suboptimal CD4 Counts (200-500 Cells/mm3) Shelley Wang,1 Jay Lalezari,2 Ronald Mitsuyasu,3 Steven Deeks,4 Winson Tang,1 Gary Lee,1 Michael Holmes,1 Phillip Gregory,1 Marty Giedlin,1 Dale Ando.1 1 Sangamo Biosciences, Richmond, CA; 2Quest Clin Research, San Francisco, CA; 3UCLA, Los Angeles, CA; 4UCSF, San Francisco, CA.
Background: A significant number of HIV+ patients on HAART are aviremic but continue to have CD4+ T-cells <500 cells/mm3. Previous attempts to use adoptive transfer to protect CD4+ T-cells from HIV infection have shown limited cell persistence. ZFN mediated disruption of CCR5 in CD4+ T-cells and selective expansion has been shown to protect against R5 HIV infection in both in vitro and in vivo T-cell and stem cell models. This study assesses the following effects of a single infusion of autologous CCR5-disrupted CD4+ T-cells administered intravenously to HAART-treated aviremic HIV subjects: 1) safety and tolerability, 2) cell persistence, 3) increases in CD4+ cell count, and 4) homing to gut mucosa. Methods: Nine aviremic HIV infected subjects with CD4+ counts between 200 and 500 cells/mm3 were enrolled, three each at dose levels of 1X1010, 2X1010 and 3X1010 total cells. Autologous CCR5 disrupted CD4+ T-cells were successfully expanded ex vivo with a mean CCR5 disruption efficiency of 26% (range 14-36%). After infusion, subjects were followed weekly for one month and then monthly for 11 months. Results: The median follow-up to date for cohort 1 is 9 months (range 7-11). CCR5-disrupted CD4+ T-cell infusions were safe and well tolerated with only mild and reversible infusion related adverse effects such as fever and chills. CD4 T-cell counts increased at all time points post infusion in all subjects. The average CD4+ T-cell counts at Day 28 increased by 155 cells/mm3 (range 86-211). The number of CCR5-disrupted T-cells in the peripheral blood as measured by PCR was 3.9-, 0.3- and 4.6-fold of the predicted number (2.5% total CD4 in 4.7L of blood) at Day 14 and persisted for the duration of followup (7 to 11 months). The percentage of CCR5 disrupted CD4 cells detected in the peripheral blood was as high as 19% in one subject at Day 28. CCR5 disrupted cells were detected in the rectal mucosa of all cohort 1 subjects at Day 14 and at 3 months. Conclusions: CCR5 disrupted CD4+ T-cells can be consistently manufactured from patients with suboptimal CD4+ counts to generate amounts greater than the intended 10-30 billion cell dose. Reinfusion to HIVinfected subjects is safe and well tolerated with disrupted cells being detected at frequencies up to 4.6-fold higher than the predicted input 14 days after infusion. This level of gene marking is approximately 1-log greater than has been observed previously in adoptive transfer studies with co-stimulated CD4+ T cells. Improvements in CD4+ T-cell counts were seen in all 3 subjects as was homing of these modified cells to the gut mucosa, suggesting that the modified CD4+ T-cells may distribute normally. These preliminary data suggest that Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
the CCR5 disrupted CD4 T cells persist at significant levels post infusion and can bolster CD4 cell counts even in HIV patients with undetectable viral load.
599. Clinical Trial Results: Intralesional Injection of a Tumor Selective Oncolytic Vaccinia Virus
Herbert J. Zeh,1 Mark O’Malley,1 Heather Jones,1 David H. Kirn,2 Moon Anne,2 Hwang H. Tae,3 David L. Bartlett.1 1 Surgery, University of Pittsburgh, Pittsburgh, PA; 2Jennerex Biotherapeutics, Inc., San Francisco, CA; 3Dong-A University, Busan, Korea.
Introduction: The WR strain of vaccinia virus (VV) has numerous potential advantages as an oncolytic virus. We have created a tumor selective mutant of VV (vvDD) by deleting the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene. These gene products are non-essential in tumor cells with E2F and/or EGFR pathway activation mutations (including ras), but essential for replication in normal, non-dividing cells. After extensive pre-clinical work, we embarked on a phase I trial of vvDD as an intralesional injection in patients with accessible tumors. Methods: Patients with metastatic breast cancer (n=4), colorectal cancer (n=10), and pancreatic cancer (n=2) were treated with a single percutaneous injection of vvDD. A standard phase I dose escalation was performed with a starting dose of 3X107 plaque forming units (pfu), and a maximum feasible dose of 3X109 pfu. Up to 3 lesions were injected. Patients were observed in the hospital for 24 hours, then followed closely with serial samples for viral shedding analysis and analysis of response. Cutaneous tumors allowed the direct observation of the response and biopsies for viral recovery, while CT scans were used for internal lesions. Patients were stratified for prior vaccinia exposure, but only one patient had not been previously vaccinated. Results: There were no dose limiting toxicities. Grade 3 toxicities possibly related to the treatment, included pain after injection. This was predominately abdominal pain after deep injection of hepatic metastases. Grade 1 and 2 adverse events included fever, chills, swelling and erythema at the injection site, diffuse rash (not systemic vaccinia), and thrombocytopenia. We did not see evidence of viral shedding by vaccinia titers. 4 patients underwent biopsies of their tumors on day 8, and 2 patients had recoverable virus. These 2 patients also had evidence of viral spread from the injected lesions to other metastatic deposits. 2 patients had complete resolution of their injected lesions, and 1 patient had evidence of a distant lesion responding. The virus did not spread to normal skin. Conclusions: We have demonstrated the safety of vvDD as a single injection into tumors, with evidence of replication, tumor response, and recoverable virus in distant tumors. The vaccinia replication was specific for tumor, with complete sparing of the normal skin. Future plans include systemic delivery of this tumor selective virus.
600. Safety and Efficacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations
Jean Bennett,1,2 Albert M. Maguire,1,2 Federico Mingozzi,2 Eric A. Pierce,1,2 Daniel C. Chung,1,2 Jeannette Bennicelli,1 Junwei Sun,2 J. Fraser Wright,1,2 Kathleen Marshall,2 Jennifer M. Wellman,2 Katherine A. High.1,2 1 Ophthalmology, University of Pennsylvania, Philadelphia, PA; 2 Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA.
Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We and others previously demonstrated that adenoassociated virus (AAV)-mediated delivery of RPE65 via subretinal injection results in improved vision/retinal function in animal models of and in humans with Leber Congenital Amaurosis due to RPE65 S229
Clinical Gene & Cell Therapy Oral Abstract Session 598. A Gene Therapy Approach to HIV: Adoptive Transfer of Zinc Finger Nuclease (ZFN) Modified Autologous CD4 T-Cells to Aviremic HIV-Infected Subjects with Suboptimal CD4 Counts (200-500 Cells/mm3) Shelley Wang,1 Jay Lalezari,2 Ronald Mitsuyasu,3 Steven Deeks,4 Winson Tang,1 Gary Lee,1 Michael Holmes,1 Phillip Gregory,1 Marty Giedlin,1 Dale Ando.1 1 Sangamo Biosciences, Richmond, CA; 2Quest Clin Research, San Francisco, CA; 3UCLA, Los Angeles, CA; 4UCSF, San Francisco, CA.
Background: A significant number of HIV+ patients on HAART are aviremic but continue to have CD4+ T-cells <500 cells/mm3. Previous attempts to use adoptive transfer to protect CD4+ T-cells from HIV infection have shown limited cell persistence. ZFN mediated disruption of CCR5 in CD4+ T-cells and selective expansion has been shown to protect against R5 HIV infection in both in vitro and in vivo T-cell and stem cell models. This study assesses the following effects of a single infusion of autologous CCR5-disrupted CD4+ T-cells administered intravenously to HAART-treated aviremic HIV subjects: 1) safety and tolerability, 2) cell persistence, 3) increases in CD4+ cell count, and 4) homing to gut mucosa. Methods: Nine aviremic HIV infected subjects with CD4+ counts between 200 and 500 cells/mm3 were enrolled, three each at dose levels of 1X1010, 2X1010 and 3X1010 total cells. Autologous CCR5 disrupted CD4+ T-cells were successfully expanded ex vivo with a mean CCR5 disruption efficiency of 26% (range 14-36%). After infusion, subjects were followed weekly for one month and then monthly for 11 months. Results: The median follow-up to date for cohort 1 is 9 months (range 7-11). CCR5-disrupted CD4+ T-cell infusions were safe and well tolerated with only mild and reversible infusion related adverse effects such as fever and chills. CD4 T-cell counts increased at all time points post infusion in all subjects. The average CD4+ T-cell counts at Day 28 increased by 155 cells/mm3 (range 86-211). The number of CCR5-disrupted T-cells in the peripheral blood as measured by PCR was 3.9-, 0.3- and 4.6-fold of the predicted number (2.5% total CD4 in 4.7L of blood) at Day 14 and persisted for the duration of followup (7 to 11 months). The percentage of CCR5 disrupted CD4 cells detected in the peripheral blood was as high as 19% in one subject at Day 28. CCR5 disrupted cells were detected in the rectal mucosa of all cohort 1 subjects at Day 14 and at 3 months. Conclusions: CCR5 disrupted CD4+ T-cells can be consistently manufactured from patients with suboptimal CD4+ counts to generate amounts greater than the intended 10-30 billion cell dose. Reinfusion to HIVinfected subjects is safe and well tolerated with disrupted cells being detected at frequencies up to 4.6-fold higher than the predicted input 14 days after infusion. This level of gene marking is approximately 1-log greater than has been observed previously in adoptive transfer studies with co-stimulated CD4+ T cells. Improvements in CD4+ T-cell counts were seen in all 3 subjects as was homing of these modified cells to the gut mucosa, suggesting that the modified CD4+ T-cells may distribute normally. These preliminary data suggest that Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
the CCR5 disrupted CD4 T cells persist at significant levels post infusion and can bolster CD4 cell counts even in HIV patients with undetectable viral load.
599. Clinical Trial Results: Intralesional Injection of a Tumor Selective Oncolytic Vaccinia Virus
Herbert J. Zeh,1 Mark O’Malley,1 Heather Jones,1 David H. Kirn,2 Moon Anne,2 Hwang H. Tae,3 David L. Bartlett.1 1 Surgery, University of Pittsburgh, Pittsburgh, PA; 2Jennerex Biotherapeutics, Inc., San Francisco, CA; 3Dong-A University, Busan, Korea.
Introduction: The WR strain of vaccinia virus (VV) has numerous potential advantages as an oncolytic virus. We have created a tumor selective mutant of VV (vvDD) by deleting the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene. These gene products are non-essential in tumor cells with E2F and/or EGFR pathway activation mutations (including ras), but essential for replication in normal, non-dividing cells. After extensive pre-clinical work, we embarked on a phase I trial of vvDD as an intralesional injection in patients with accessible tumors. Methods: Patients with metastatic breast cancer (n=4), colorectal cancer (n=10), and pancreatic cancer (n=2) were treated with a single percutaneous injection of vvDD. A standard phase I dose escalation was performed with a starting dose of 3X107 plaque forming units (pfu), and a maximum feasible dose of 3X109 pfu. Up to 3 lesions were injected. Patients were observed in the hospital for 24 hours, then followed closely with serial samples for viral shedding analysis and analysis of response. Cutaneous tumors allowed the direct observation of the response and biopsies for viral recovery, while CT scans were used for internal lesions. Patients were stratified for prior vaccinia exposure, but only one patient had not been previously vaccinated. Results: There were no dose limiting toxicities. Grade 3 toxicities possibly related to the treatment, included pain after injection. This was predominately abdominal pain after deep injection of hepatic metastases. Grade 1 and 2 adverse events included fever, chills, swelling and erythema at the injection site, diffuse rash (not systemic vaccinia), and thrombocytopenia. We did not see evidence of viral shedding by vaccinia titers. 4 patients underwent biopsies of their tumors on day 8, and 2 patients had recoverable virus. These 2 patients also had evidence of viral spread from the injected lesions to other metastatic deposits. 2 patients had complete resolution of their injected lesions, and 1 patient had evidence of a distant lesion responding. The virus did not spread to normal skin. Conclusions: We have demonstrated the safety of vvDD as a single injection into tumors, with evidence of replication, tumor response, and recoverable virus in distant tumors. The vaccinia replication was specific for tumor, with complete sparing of the normal skin. Future plans include systemic delivery of this tumor selective virus.
600. Safety and Efficacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations
Jean Bennett,1,2 Albert M. Maguire,1,2 Federico Mingozzi,2 Eric A. Pierce,1,2 Daniel C. Chung,1,2 Jeannette Bennicelli,1 Junwei Sun,2 J. Fraser Wright,1,2 Kathleen Marshall,2 Jennifer M. Wellman,2 Katherine A. High.1,2 1 Ophthalmology, University of Pennsylvania, Philadelphia, PA; 2 Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA.
Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We and others previously demonstrated that adenoassociated virus (AAV)-mediated delivery of RPE65 via subretinal injection results in improved vision/retinal function in animal models of and in humans with Leber Congenital Amaurosis due to RPE65 S229