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782. Efficacy of the dAd5GNE Anti-Cocaine Vaccine in Models of “Binge” Cocaine Use
IMMUNE RESPONSE TO GENE TRANSFER AND CELL THERAPY 781. Ex Vivo Expanded Multi-Specific Cytotoxic T Lymphocytes Derived From HIV+ Patients and HIV Negative Donors Using GMP Compliant Methodologies Suppress HIV Replication
Sharon Lam,1 Julia Marsh Sung,3 Conrad Cruz,2 Paul CastilloCaro,1 Minh Ngo,1 Caroline Garrido,3 Joanne Kuruc,3 Cliona Rooney,1 David Margolis,3 Catherine Bollard.2 1 Baylor College of Medicine, Houston, TX; 2Children’s National Medical Center, Washington, DC; 3University of North CarolinaChapel Hill, Chapel Hill, NC.
Antiretroviral therapy (ART) is not curative, is a life-long regimen, and has long-term toxicities. Therefore there is a need for alternative therapies. Previous studies have demonstrated the safety and feasibility of infusing single-epitope specific T cells to HIV patients. However, these T cells were restricted to a single HLA restricted epitope and had limited persistence in vivo. We hypothesize that broadly HIV-specific T cells could be expanded from ART patients and HIV negative donors to effectively target HIV infection using a non-HLA restricted approach. PBMCs are stimulated with antigen presenting cells pulsed with gag, pol, and nef pepmixes and cocultured with co-stimulatory K562 cells. From 100mL of blood,T cells expanded to clinically relevant numbers (Mean=1.62e8 cells, Range (3.72e7, 2.87e8 cells), n=7) in the presence of ART to prevent possible viral spread during expansion. Post-expansion, 5 of 7 patient sample lines showed specific activity to all 3 HIV antigens in IFNγ ELISPOT assays. The T cell lines were broadly specific to gag (mean=99.33 SFC/10e5 cells), pol (mean=131.11 SFC/10e5 cells) and nef (mean=337.26 SFC/10e5 cells), and polyclonal as shown by Vb usage analysis (mean usage= 14.67 of the 24 Vb analyzed). Due to the association of gag-specific T cell responses with viral control in long-term nonprogressors we also determined whether gag-specific T cells could be derived from HIV negative donors (HIVneg) for 3rd party use. Gag-specific T cells from HIVneg donors released IFNγ in response to gag pepmix (163.79 SFC/1e5 cells, n=9) but not an irrelevant antigen (mean=7.0 SFC/1e5 cells). Importantly, T cells expanded from both ART patients and HIVneg were cytotoxic, as expanded T cells lysed antigen loaded autologous targets (mean=67.55% specific lysis compared to no antigen: mean=0.46% at 10:1 E:T ratio) in 51Cr release assays. Expanded T cells from ART patients also showed a greater ability to suppress HIV outgrowth in vitro compared to unexpanded CD8 T cells when co-cultured with reactivated resting CD4+ T cells from ART patients, the authentic latently infected cells that define viral reservoirs.. In 5 patients, a statistically lower recovery of virus from resting CD4+ cells was seen in the presence of CTLs as compared to no effectors (p<0.006), while the unexpanded CD8 cells showed only a modest trend towards decreased recovery (p>0.9). Similarly, HIV-specific T cells derived from HIVneg individuals were able to suppress HIV replication more than unexpanded CD8 T cells when co-cultured with infected, autologous CD4 T cells (HIV only condition p24=681.95 pg/ mL, nonspecific CD8 T cells=448.80 pg/mL, expanded CTL=145.82 pg/mL). We are now translating our approach to the clinical setting where we will test HIV-multispecific T cells as part of a strategy to fully eradicate HIV infection.
782. Efficacy of the dAd5GNE Anti-Cocaine Vaccine in Models of “Binge” Cocaine Use
David F. Havlicek,1 Martin J. Hicks,1 Bishnu P. De,1 Jonathan B.
S302
Rosenberg,1 Stephen M. Kaminsky,1 Kim D. Janda,2 George F. Koob,2 Ronald G. Crystal.1 1 Weill Cornell Medical College, New York, NY; 2The Scripps Research Institute, La Jolla, CA. dAd5GNE, an anti-cocaine vaccine comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE, evokes a high titer of high affinity, circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system (CNS). In prior studies, we have demonstrated efficacy of the dAd5GNE vaccine in models of moderate and intermittent (weekly to monthly) cocaine use. In the present study, we asked: are the anti-cocaine antibody titers induced by dAd5GNE sufficient to protect against a cocaine “binge”, i.e., more frequent and/or higher doses of cocaine aimed at overriding the vaccine? To assess this question, we modeled 2 “binge” scenarios in vaccinated BALB/C mice [dAd5GNE (4 μg) + Adjuplex adjuvant; administered at 4 and 8 wk (±1 wk)]: (1) daily cocaine challenge with 2.5 μg cocaine; and (2) cocaine overdose with 100 μg cocaine. In the model of daily cocaine use, vaccine efficacy was assessed at wk 12 by 3H-cocaine distribution 1 min after intravenous cocaine challenge. In the vaccinated animals receiving a single cocaine administration, cocaine levels in the brain were reduced by 55% compared to nonvaccinated mice receiving a single cocaine dose (p<0.05). Importantly, daily dosing of cocaine for 3 consecutive days did not override the vaccine, as vaccinated mice continued to show abrogation of cocaine access to the brain (64% reduction, p<0.005), a reduction similar to that of vaccinated mice receiving a single cocaine dose (p>0.5). In the cocaine overdose model, locomotor behavior was assessed using an infrared activity chamber following 100 μg intravenous administration of cocaine at 20 wk. Vaccination not only reduced the cocaine-induced hyperactive ambulatory distance traveled by 52% (p<0.012) but strikingly, while 80% (4/5) of the non-vaccinated mice experienced lengthy seizures (>1 min), only 10% (1/10) of the vaccinated mice demonstrated epileptic activity. We conclude from these studies that dAd5GNE vaccination not only protects against intermittent, recreational cocaine use, but also protects the CNS from “binge” use, whether this is repetitive daily use, or high-dose use at levels known to induce seizure. Based on this data, and our prior data in rodents and nonhuman primates, we are moving the dAd5GNE anti-cocaine vaccine to a clinical trial for cocaine addicts who wish to stop cocaine use.
783. Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or CCR5 Fused to the C34 Peptide from gp41 HR2
Jianbin Wang,1 George J. Leslie,2 Josh DeClercq,1 Max W. Richardson,2 Andrea P. O. Jordon,2 Philip D. Gregory,1 James L. Riley,2 James A. Hoxie,2 Michael C. Holmes.1 1 Sangamo Bioscience Inc., Richmond, CA; 2Univ. of Penn., Philadelphia, PA.
HIV-1 entry into CD4+ T cells requires binding to CD4 and either the CCR5 (R5) or CXCR4 (X4) co-receptor. Thus, strategies that disable productive co-receptor (CoR) engagement should provide potent protection from HIV infection. Previously we described a 34 amino acid peptide from the C-terminal heptad repeat-2 domain of gp41 (C34) which, when fused to the amino terminus (NT) of either R5 or X4, inhibits HIV-1 infection in transformed cells in vitro. Moreover, our initial studies suggested that C34-R5 or C34-X4 fusions provided trans-dominant resistance to infection irrespective of viral tropism (i.e. either C34-R5 or C34-X4 could inhibit entry of R5, X4 or dual-tropic isolates). Here we demonstrate that C34-R5 or C34-X4 expression by lentiviral transduction in primary CD4 T-cells from multiple donors results in almost complete inhibition (>98%) of HIV-1 infection based on intracellular p24 levels and RT activities. GFP-only Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Sharon Lam,1 Julia Marsh Sung,3 Conrad Cruz,2 Paul CastilloCaro,1 Minh Ngo,1 Caroline Garrido,3 Joanne Kuruc,3 Cliona Rooney,1 David Margolis,3 Catherine Bollard.2 1 Baylor College of Medicine, Houston, TX; 2Children’s National Medical Center, Washington, DC; 3University of North CarolinaChapel Hill, Chapel Hill, NC.
Antiretroviral therapy (ART) is not curative, is a life-long regimen, and has long-term toxicities. Therefore there is a need for alternative therapies. Previous studies have demonstrated the safety and feasibility of infusing single-epitope specific T cells to HIV patients. However, these T cells were restricted to a single HLA restricted epitope and had limited persistence in vivo. We hypothesize that broadly HIV-specific T cells could be expanded from ART patients and HIV negative donors to effectively target HIV infection using a non-HLA restricted approach. PBMCs are stimulated with antigen presenting cells pulsed with gag, pol, and nef pepmixes and cocultured with co-stimulatory K562 cells. From 100mL of blood,T cells expanded to clinically relevant numbers (Mean=1.62e8 cells, Range (3.72e7, 2.87e8 cells), n=7) in the presence of ART to prevent possible viral spread during expansion. Post-expansion, 5 of 7 patient sample lines showed specific activity to all 3 HIV antigens in IFNγ ELISPOT assays. The T cell lines were broadly specific to gag (mean=99.33 SFC/10e5 cells), pol (mean=131.11 SFC/10e5 cells) and nef (mean=337.26 SFC/10e5 cells), and polyclonal as shown by Vb usage analysis (mean usage= 14.67 of the 24 Vb analyzed). Due to the association of gag-specific T cell responses with viral control in long-term nonprogressors we also determined whether gag-specific T cells could be derived from HIV negative donors (HIVneg) for 3rd party use. Gag-specific T cells from HIVneg donors released IFNγ in response to gag pepmix (163.79 SFC/1e5 cells, n=9) but not an irrelevant antigen (mean=7.0 SFC/1e5 cells). Importantly, T cells expanded from both ART patients and HIVneg were cytotoxic, as expanded T cells lysed antigen loaded autologous targets (mean=67.55% specific lysis compared to no antigen: mean=0.46% at 10:1 E:T ratio) in 51Cr release assays. Expanded T cells from ART patients also showed a greater ability to suppress HIV outgrowth in vitro compared to unexpanded CD8 T cells when co-cultured with reactivated resting CD4+ T cells from ART patients, the authentic latently infected cells that define viral reservoirs.. In 5 patients, a statistically lower recovery of virus from resting CD4+ cells was seen in the presence of CTLs as compared to no effectors (p<0.006), while the unexpanded CD8 cells showed only a modest trend towards decreased recovery (p>0.9). Similarly, HIV-specific T cells derived from HIVneg individuals were able to suppress HIV replication more than unexpanded CD8 T cells when co-cultured with infected, autologous CD4 T cells (HIV only condition p24=681.95 pg/ mL, nonspecific CD8 T cells=448.80 pg/mL, expanded CTL=145.82 pg/mL). We are now translating our approach to the clinical setting where we will test HIV-multispecific T cells as part of a strategy to fully eradicate HIV infection.
782. Efficacy of the dAd5GNE Anti-Cocaine Vaccine in Models of “Binge” Cocaine Use
David F. Havlicek,1 Martin J. Hicks,1 Bishnu P. De,1 Jonathan B.
S302
Rosenberg,1 Stephen M. Kaminsky,1 Kim D. Janda,2 George F. Koob,2 Ronald G. Crystal.1 1 Weill Cornell Medical College, New York, NY; 2The Scripps Research Institute, La Jolla, CA. dAd5GNE, an anti-cocaine vaccine comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE, evokes a high titer of high affinity, circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system (CNS). In prior studies, we have demonstrated efficacy of the dAd5GNE vaccine in models of moderate and intermittent (weekly to monthly) cocaine use. In the present study, we asked: are the anti-cocaine antibody titers induced by dAd5GNE sufficient to protect against a cocaine “binge”, i.e., more frequent and/or higher doses of cocaine aimed at overriding the vaccine? To assess this question, we modeled 2 “binge” scenarios in vaccinated BALB/C mice [dAd5GNE (4 μg) + Adjuplex adjuvant; administered at 4 and 8 wk (±1 wk)]: (1) daily cocaine challenge with 2.5 μg cocaine; and (2) cocaine overdose with 100 μg cocaine. In the model of daily cocaine use, vaccine efficacy was assessed at wk 12 by 3H-cocaine distribution 1 min after intravenous cocaine challenge. In the vaccinated animals receiving a single cocaine administration, cocaine levels in the brain were reduced by 55% compared to nonvaccinated mice receiving a single cocaine dose (p<0.05). Importantly, daily dosing of cocaine for 3 consecutive days did not override the vaccine, as vaccinated mice continued to show abrogation of cocaine access to the brain (64% reduction, p<0.005), a reduction similar to that of vaccinated mice receiving a single cocaine dose (p>0.5). In the cocaine overdose model, locomotor behavior was assessed using an infrared activity chamber following 100 μg intravenous administration of cocaine at 20 wk. Vaccination not only reduced the cocaine-induced hyperactive ambulatory distance traveled by 52% (p<0.012) but strikingly, while 80% (4/5) of the non-vaccinated mice experienced lengthy seizures (>1 min), only 10% (1/10) of the vaccinated mice demonstrated epileptic activity. We conclude from these studies that dAd5GNE vaccination not only protects against intermittent, recreational cocaine use, but also protects the CNS from “binge” use, whether this is repetitive daily use, or high-dose use at levels known to induce seizure. Based on this data, and our prior data in rodents and nonhuman primates, we are moving the dAd5GNE anti-cocaine vaccine to a clinical trial for cocaine addicts who wish to stop cocaine use.
783. Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or CCR5 Fused to the C34 Peptide from gp41 HR2
Jianbin Wang,1 George J. Leslie,2 Josh DeClercq,1 Max W. Richardson,2 Andrea P. O. Jordon,2 Philip D. Gregory,1 James L. Riley,2 James A. Hoxie,2 Michael C. Holmes.1 1 Sangamo Bioscience Inc., Richmond, CA; 2Univ. of Penn., Philadelphia, PA.
HIV-1 entry into CD4+ T cells requires binding to CD4 and either the CCR5 (R5) or CXCR4 (X4) co-receptor. Thus, strategies that disable productive co-receptor (CoR) engagement should provide potent protection from HIV infection. Previously we described a 34 amino acid peptide from the C-terminal heptad repeat-2 domain of gp41 (C34) which, when fused to the amino terminus (NT) of either R5 or X4, inhibits HIV-1 infection in transformed cells in vitro. Moreover, our initial studies suggested that C34-R5 or C34-X4 fusions provided trans-dominant resistance to infection irrespective of viral tropism (i.e. either C34-R5 or C34-X4 could inhibit entry of R5, X4 or dual-tropic isolates). Here we demonstrate that C34-R5 or C34-X4 expression by lentiviral transduction in primary CD4 T-cells from multiple donors results in almost complete inhibition (>98%) of HIV-1 infection based on intracellular p24 levels and RT activities. GFP-only Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy