CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

351 TRIFUNCTIONAL T-CELL ENGAGERS TARGETING PD-1 AND TIGIT IN CHRONIC HIV/SHIV INFECTIONS Wanwisa Promsote 1 , David R. Ambrozak 1 , Catherine Liu 1 , Megan Demouth 1 , Cassandra Almasri 1 , Michelle Cully 2 , Jason M. Hataye 2 , Amarendra Pegu 2 , John R. Mascola 2 , Richard A. Koup 2 1 National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA, 2 Vaccine Research Center, NIAID, Bethesda, MD, USA Background: Immune checkpoint inhibitors, such as PD-1 and TIGIT, are crucial dysregulators of CD8 T cell function during chronic HIV-1/SIV infections. Importantly, these checkpoint inhibitors are highly expressed on the surface of CD4 T cells that harbor latent HIV. We have previously demonstrated that an anti-HIV/anti-CD3 bispecific T-cell engager (BiTE) can be used to redirect functionally compromised follicular CD8 (fCD8) T cells to kill HIV infected cells in vitro. Here, we hypothesize that adding an extra specificity to target PD-1 and TIGIT to the BiTE will further enhance the functional activities of CD8 T cells and simultaneously lower the threshold for reactivation of latently infected cells in HIV infection. Methods: We generated trifunctional anti-HIV/anti-CD3/anti-PD-1 and anti- TIGIT T-cell engagers by linking the scFvs from either anti-TIGIT and anti-PD-1 antibodies to the BiTE molecule. HIV-infected cell lines and primary cells isolated from lymph nodes (LN) of chronically SHIV-infected rhesus macaques were used as target cells in the in vitro and ex vivo killing assays to test whether trifunctional T-cell engagers could enhance the cytolytic activities of functionally compromised CD8 T cells. We also examined whether trifunctional T-cell engagers could stimulate CD8 T-cell lysis of HIV-infected CD4 T cells using a primary HIV latency model. Furthermore, multiparameter flow cytometry was used to investigate the effects of trifunctional T-cell engagers on CD8 and CD4 T cell polyfunctionality. Results: We found that trifunctional anti-HIV/anti-CD3/anti-TIGIT and anti-PD1 increased the killing capability of CD8 T cells compared to the bifunctional anti-HIV/anti-CD3 BiTE in the in vitro and ex vivo killing assays. We also demonstrated that trifunctional anti-HIV-1/anti-CD3/anti-TIGIT and anti-PD1 enhanced the CD8 T cell lysis of latently infected cells. Furthermore, trifunctional anti-HIV-1/anti-CD3/anti-TIGIT and anti-PD1 were shown to increase antigen- specific CD107a degranulation, levels of granzyme B, cytokine and chemokine release by CD8 T cells, which could potentially underlie the observed increase in the killing capability of CD8 T cells. Conclusion: Our results indicate that the use of trifunctional T cell engagers targeting immune checkpoints, PD1 and TIGIT, may serve as novel immunotherapeutic strategies to eliminate infected cells in HIV infected individuals. 352 IMPACT OF RAPAMYCIN ON SIV PERSISTENCE IN RHESUS MACAQUES ON ANTIRETROVIRAL THERAPY Benjamin Varco-Merth 1 , William B. Brantley 1 , Richard Lum 1 , Alejandra Marenco 1 , Derick M. Duell 1 , Walter Flores 2 , Kathleen Engelman 3 , Haesun M. Park 1 , Jeremy Smedley 1 , Michael K. Axthelm 1 , Jeffrey D. Lifson 4 , Louis J. Picker 1 , Cheryl Cameron 5 , Timothy J. Henrich 6 , Afam Okoye 1 1 Oregon Health and Sciences University, Portland, OR, USA, 2 University of Massachusetts, Worcester, MA, USA, 3 MassBiologics, Boston, MA, USA, 4 Leidos Biomedical Research, Inc, Frederick, MD, USA, 5 Case Western Reserve University, Cleveland, OH, USA, 6 University of California San Francisco, San Francisco, CA, USA Background: The mammalian target of rapamycin (mTOR) is a key regulatory kinase that controls glucose metabolism and cell growth. Inhibition of mTOR has been linked with several immune regulatory functions that may limit HIV persistence, including: 1) reducing CCR5 expression, 2) limiting CD4+ T cell homeostatic proliferation, 3) reducing PD-1 expression and 4) increasing anti-viral CD8+ effector T cell responses. Here we evaluated the impact of long- termmTOR inhibition on SIV DNA and RNA in blood and tissues of SIV-infected rhesus macaques (RM) on combination antiretroviral therapy (cART). We also evaluated whether potent T cell activation can induce latent SIV reactivation in the presence of rapamycin. Methods: A total of 14 adult male RM were intravenously infected with SIVmac239 followed by cART (tenofovir, emtricitabine and dolutegravir) 12 days later. After 219 days of cART, RM were randomized into 2 groups that received twice daily IM injections of rapamycin at 0.02mg/kg (n=7) or vehicle control for 312 days. After 464 days of cART, RM on rapamycin received 2 doses of a non-depleting anti-CD3LALA monoclonal antibody at 0.5mg/kg IV at 21-day intervals. Plasma viral loads and cell-associated SIV RNA and DNA were

quantified by qRT-PCR and qPCR, respectively. Lymphocyte populations were evaluated by flow cytometry. Results: After 24 weeks, there were significant decreases in the frequencies of Ki67+ (p = 0.0006), HLA-DR+ (p = 0.0262) and PD-1+ (p = 0.04) CD4+ memory T cells in blood of rapamycin-treated RM versus controls. In addition, surface expression of CCR5 (p=0.007) and the glucose transporter Glut1 (p=0.007) were also significantly reduced in rapamycin-treated RM. Despite these perturbations in CD4+ T cell homeostasis, cell-associated SIV DNA and RNA in blood and peripheral lymph nodes remained stable over time with no significant difference observed between treatment groups. However, 4 of 7 rapamycin-treated RM had blips in plasma viral loads >2 logs above threshold (1 RNA copy/ml) in response to anti-CD3LALA, suggesting T cell activation in the presence of rapamycin can induce SIV reactivation in vivo. Conclusion: Despite profound changes in markers of immune activation, proliferation and T cell exhaustion, rapamycin had minimal effect the stability of the SIV reservoir. However, these data indicate that rapamycin used in synergy with potent T cell activation may be an effective strategy to induce viral reactivation while inhibiting global immune activation and T cell proliferation. 353 COMBINATION OF CRISPR AND LASER ART PREVENTS HIV REBOUND IN HUMANIZED MICE Kamel Khalili 1 , Howard E. Gendelman 2 1 Temple University, Philadelphia, PA, USA, 2 University of Nebraska Medical Center, Omaha, NE, USA Background: Advances in CRISPR-Cas9gene editing technology and its in vivo delivery by AAV9 vectors together with cell based nanotechnology for long- acting slow effective release antiretroviral therapy (LASER-ART), were used in NSG-CD34 humanized mice to facilitate eradication of HIV-1 in vivo. Methods: CRISPR-Cas9 proviral DNA excision followed two months of treatment with long-acting slow effective release antiretroviral therapy (LASER-ART), rilpivirine, myristolyated dolutegravir, lamivudine, and abacavir in HIV-1 infected humanized mice. A series of virological, histological, and DNA and RNA assays were used to detect HIV-1 expression and replication in the animal tissues. Ultra deep, whole genome sequencing was employed to assess in vivo off-target effects. Results: Results from three independent sets of studies showed restorations of CD4+ T cells due to ART treatment and complete eradication of replication competent virus by CRISPR in 39% of animals. Ultrasensitive nested and digital droplet PCR and RNA scope assays failed to detect HIV-1 in blood, spleen, lung, kidney, liver, gut-associated lymphoid tissue and brain. Excision of proviral DNA fragments spanning the LTRs and the Gag gene from the integrated proviral DNA was identified, while no off target effects were observed. The absence of viral rebound following cessation of ART with no progeny virus recovery after in vivo adoptive transfer of human immunocytes from dual-treated virus- free animals to uninfected humanized mice verified HIV-1 eradication by the combined treatment strategy. In contrast, HIV-1 was readily detected in all infected animals treated with LASER ART or CRISPR-Cas9 alone. Conclusion: CONCLUSIONS: The sequential application of LASER ART and CRISPR-Cas9 therapies administered to HIV-1 infected humanized mice provides the first proof-of-concept that viral sterilization is possible. 354 A HUMANIZED MOUSE MODEL FOR EVALUATION OF AUTOLOGOUS HIV- SPECIFIC T-CELL THERAPIES Chase McCann 1 , Elizabeth A. Zale 1 , Adam R. Ward 2 , Shabnum Patel 3 , Catherine M. Bollard 3 , R. Brad Jones 1 1 Weill Cornell Medicine, New York, NY, USA, 2 George Washington University, Washington, DC, USA, 3 Children’s Research Institute, Children’s National Health System, Washington, DC, USA Background: Ex vivo expanded HIV-Specific CD8+ T-cell (HST) immunotherapy offers great promise toward achieving an HIV cure. While plans to test HST therapies in humans are currently underway, a small animal model would enable the rapid and cost-effective pre-clinical evaluation of multiple approaches. We have developed a humanized mouse model reconstituted with only the memory subset of human CD4+ T-cells, which has greatly mitigated the effects of GvHD and allows for the in vivo analysis of autologous HST therapies. Methods: NSG mice were engrafted with 5-10x106 memory CD4+ T-cells isolated from HIV- or HIV+ donor leukapheresis samples. Autologous HSTs were generated by stimulating T-cells with pools of overlapping Clade B consensus

Poster Abstracts

CROI 2019 128

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