CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

these limitations to improve CD4CAR T cell therapy. We hypothesize that preventing exhaustion and infection in CAR expressing T cells will enhance antiviral response and efficacy against HIV infection in vivo. Methods: To address these problems and attempt to improve CAR-driven T cell responses and prevent cellular infection, we constructed a truncated CD4 CAR molecule that contains solely the D1 and D2 domains of the CD4 molecule. In addition, we incorporated a PD-1 specific shRNA into the vector to knock down check-point inhibitor PD-1 expression on CAR modified cells to attempt to enhance their function. Humanized mice were constructed and infected with HIV and received autologous T cell transplants of modified CD4 and CD8 cells with either a control non-specific shRNA containing D1D2CD4CAR lentiviral vector or a PD1shRNA containing D1D2CD4CAR lentiviral vector. Results: We found that the D1D2CD4CAR maintains the HIV Env binding site but does not allow HIV infection when introduced into CD8 T cells. The PD-1 shRNA effectively knocked down PD-1 expression on modified T cells both in vitro and in vivo. Further, we found that the PD-1shRNA containing D1D2CD4CAR cells have improved function and proliferation. Mice that received PD1shRNA D1D2CD4CAR cells showed improved viral control as compared to mice that received control CAR modified T cells. Conclusion: Specific knockdown of PD-1 expression on CAR modified T cells can enhance anti-HIV efficacy and highlights the potential of targeting immune check point inhibitors to enhance T cell gene therapy. 332 HEMATOPOIETIC STEM-CELL–BASED CHIMERIC ANTIGEN RECEPTOR THERAPY FOR HIV INFECTION Anjie Zhen 1 , Christopher Peterson 2 , Mayra A. Carrillo 1 , Valerie Rezek 1 , Masakazu Kamata 1 , Jerome Zack 1 , Hans-Peter Kiem 2 , Scott G. Kitchen 1 1 Univ of California Los Angeles, Los Angeles, CA, USA, 2 Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA Background: HIV-1 specific cytotoxic T lymphocytes are key immune response to HIV and are crucial for the elimination of HIV infected cells. Previous studies showed that a chimeric antigen receptor containing the CD4 molecule linked to the signaling domain of the T cell receptor ζ chain (CD4CAR) can be used to redirect peripheral T cells to target HIV infected cells. CD4 CAR modified T cells can kill HIV infected cells in vitro, but showed limited efficacy in clinical trials. Hematopoietic Stem Cells (HSCs) based gene therapy has several advantages over T cell adoptive therapy as it provides long-term engraftment; and, modified stem cells undergo normal T cell differentiation and selection. Our previous studies using humanized mice demonstrated that HSCs modified with a protective CD4 CAR resulted in successful differentiation of CD4CAR expressing T cells and significant suppression of HIV replication, suggesting feasibility of redirecting immunity with a HSCs based approach. Methods: We tested the safety and feasibility of engineering T cell immunity via HSCs in a non-human primate (NHP) model of SHIV infection. We utilized CD4 CAR vectors that also carry an anti-HIV protective gene (C46) that would inhibit infection. 2 pigtailed macaques (Macaca nemestrina) were transplanted with C46CD4CAR modified autologous HSCs and 2 were transplanted with control vector C46CD4CARdeltaZeta that lacks the signaling Zeta chain. After hematopoietic recovery, the animals were challenged with SHIV and monitored for viral load and CAR cell detection for over a year. Results: We determined that engraftment of pigtailed macaques with C46CD4CAR-modified HSCs is safe and the animals have normal transplant recovery. We observed long-term engraftment and stable production of C46CD4CAR expressing cells without any significant toxicities and found that C46CD4CAR modified HSCs could differentiate into multiple hematopoietic lineages, including T cells, NK cells, granulocytes, and B cells. Following challenge of the animals with SHIV, we observed significant expansion of C46CD4CAR expressing cells after infection and found that C46CD4CAR expressing cells were capable of killing infected cells. Conclusion: This demonstrates the safety and feasibility of a HSCs based therapy utilizing an HIV-specific chimeric antigen receptor for chronic HIV infection in NHPs. These results set the stage for future investigational development in an attempt to eradicate HIV infection and provide more effective immune surveillance of HIV. 333 LENTIVIRAL DENDRITIC CELL VACCINE RAISES PROTECTIVE CTLS AND TARGETS LATENT HIV Background: Dendritic cells (DCs) orchestrate immune responses to viral pathogens and have been used in vaccine strategies against several diseases. To further develop this approach we have established lentiviral vectors (LVs) that package the lentiviral accessory protein Vpx. Vpx increases DC transduction by two-logs allowing for long-term antigen expression, a significant improvement over peptide-pulsing. To test this approach, we generated vectors that expressed the lymphocytic choriomeningitis virus (LCMV) GP33 epitope or the major HLA-A2-restricted HIV Gag epitope SL9. The vectors co-expressed CD40L, increasing their ability to stimulate antigen-specific T cells. Cytokines released by transduced DCs reactivated latent HIV-1 provirus in latency models, supporting their use as a two-pronged approach for activating and targeting the latent reservoir in HIV+ patients. We have now tested the lentiviral DC vaccine strategy in HIV-infected humanized mouse and LCMV mouse models. Methods: Bone marrow, liver, thymus humanized mice expressing transduced SL9-specific T cell receptor (TCR) CD8 T cells were injected with CD40L-SL9 expressing LV-transduced antigen presenting cells and then challenged with HIV-1. Expansion of SL9 TCR+ cells was quantified by flow cytometry and peripheral viral loads were quantified by RT-PCR. Wild- type mice were injected with CD40L-GP33 LV-transduced bone marrow-derived from SAMHD1-/- mice and challenged with LCMV. CD8 T cells expressing GP33-specific TCRs cells were quantified by flow cytometry and viral loads were quantified by plaque assay. Survival was monitored following lethal challenge with LCMV. Results: In mice injected with transduced DCs, the number of SL9 TCR+ and GP33 TCR+ CD8 T cells expanded 30-fold and 10-fold, respectively. Mice injected with SL9 expressing LV-transduced DCs and then infected with HIV-1 dramatically suppressed virus loads 2-6 weeks post-infection. Mice injected with GP33 expressing LV-transduced DCs were protected against LCMV challenge. No infectious virus was detected in 4/5 immunized mice and the survival rate was close to 100%, while in mice injected with control LV- transduced DCs, virus loads reached > 2 X 10E6 PFU/mL and resulted in death. Conclusion: This DC vaccine approach induces antigen-specific CTLs that protect against LCMV and HIV infection. This is a promising approach to restore CTLs and reduce the latent reservoir in chronically infected individuals. 334 SELF-ACTIVATING VECTORS THAT EXPRESS VIF-RESISTANT APOBEC3G FOR HIV-1 GENE THERAPY Krista Delviks-Frankenberry 1 , Daniel Ackerman 1 , Nina Timberlake 2 , Maria Hamscher 1 , Olga A Nikolaitchik 1 , Wei-Shau Hu 1 , Bruce E. Torbett 2 , Vinay K. Pathak 1 1 NCI, Frederick, MD, USA, 2 The Scripps Rsr Inst, La Jolla, CA, USA Background: Strategies to suppress HIV-1 replication in the absence of antiviral therapy are needed to achieve a functional cure. APOBEC3G (A3G) and APOBEC3F (A3F) are host restriction factors that inhibit HIV-1 replication by inducing lethal hypermutation and inhibiting reverse transcription and integration. However, HIV-1 encodes the protein Vif, which induces A3G/A3F degradation, allowing viral replication to proceed. Here we developed novel self-activating lentiviral vectors to express Vif-resistant A3G/A3F mutants that inhibit HIV-1 replication and evaluated their potential to control HIV-1 replication in T cell lines. Methods: Standard lentiviral vectors cannot be used for delivery of A3G/A3F because their expression in the virus producing cells inactivates the therapeutic virus. We designed novel self-activating lentiviral vectors that maintain an inactive Vif-resistant A3G(D128K) in virus producing cells using directly repeated nucleotide sequences. Upon infection, direct repeats are removed during reverse transcription to express functional A3G(D128K) in the target cells. HIV-1 replication kinetics were evaluated in infected T cell lines expressing A3G(D128K) and tested for the emergence of resistant virus. Results: Self-activating vectors allowed for successful virus production and delivery of A3G(D128K) to target cells; direct repeat deletion was >99% efficient. CD4+ T cell lines CEM and PM1 expressing A3G(D128K) successfully restricted NL4-3 infection. Subtype C and intersubtype recombinant subtype AE also failed to replicate in A3G(D128K) expressing cells; however, SIV and HIV-2 whose Vif can neutralize A3G(D128K) were able to replicate. No A3G(D128K)-resistant NL4-3 virus emerged in CEM/A3G(D128K) cells in culture after passaging for 3.5 months. Analysis of proviral DNA showed typical G-to-A hypermutation patterns in the A3G context, consistent with inhibition by A3G(D128K) expression. Infectious titers of >10 8 /ml allowed for efficient delivery of A3G(D128K) to CD34+ hematopoietic stem cells without cytotoxicity. Thomas D. Norton 1 , Anjie Zhen 2 , Takuya Tada 1 , Scott G. Kitchen 2 , Nathaniel R. Landau 1 1 New York Univ, New York, NY, USA, 2 Univ of California Los Angeles, Los Angeles, CA, USA

Poster and Themed Discussion Abstracts

CROI 2017 131