CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

525

Nanoparticle Delivery of Tat Synergizes With Latency Reversal Agents to Express HIV Antigen Shane D Falcinelli 1 , Samuel L. Raines 1 , Jackson Peterson 1 , Ellen Van Gulck 2 , Jennifer Kirchherr 1 , Isabel Najera 2 , Jerel Vega 3 , Daniel Boden 2 , Nancie M. Archin 1 , David M. Margolis 1 1 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2 Janssen, Beerse, Belgium, 3 Arcturus Therapeutics, San Diego, CA, USA Background: Latency reversal and clearance of persistently infected CD4 T cells during antiretroviral therapy (ART) is a major strategy for HIV cure. Minimal induction of HIV protein antigens by latency reversal agents (LRAs) is at least partially due to limiting intracellular levels of HIV Tat. Here, lipid nanoparticles containing tat mRNA (Tat LNPs) were evaluated for latency reversal activity alone and in combination with well-characterized LRAs. Methods: Tat LNPs, inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi) were assessed for latency reversal activity in cell line models of HIV latency (J89 ♂ , E4 ♂ , 2D10 ♂ , J-Lat 6.3 ♂ , 2B2D ♂ and ACH-2 ♀ ) via quantification of proviral reporters. Findings in cell lines were confirmed in CD4 T cells from ART-suppressed people with HIV (PWH) via digital PCR measurement of HIV cell-associated transcripts and ultrasensitive digital ELISA quantification of p24 protein in the culture supernatant. Results: Tat LNPs alone demonstrated dose-responsive latency reversal, with variable maximal activity across cell line models of HIV latency. Control experiments demonstrated that Tat LNPs induced latency reversal in 2B2D cells (Tat ΔC22G) and were inactive in ACH-2 cells (TAR ΔC37T), confirming the specificity of the reagent. Tat LNPs in combination with IAPi, BETi, and HDACi demonstrated synergistic latency reversal activity that approached 100% proviral GFP reporter expression in J89, E4, 2D10 and 2B2D cell lines, with more limited activity in the J-Lat 6.3 line. In ex vivo studies of CD4+ T cells from aviremic PWH, Tat LNPs alone and in combination with IAPi, HDACi, and BETi resulted in tens to hundreds-fold inductions of unspliced, multiply-spliced, and polyadenylated HIV transcripts. Tat LNPs alone, but not other LRAs, induced detectable HIV p24 protein in the culture supernatants in a fraction of PWH. Remarkably, without toxicity or T cell activation, Tat LNPs combined with LRAs induced p24 protein at a frequency and magnitude similar to T cell mitogens. Conclusion: Tat LNPs induced HIV RNA and p24 protein expression in CD4 T cells from ART-suppressed PWH ex vivo, and synergized with IAPi, HDACi, and BETi LRAs. Further characterization and optimization of Tat LNPs and combinations for in vivo testing is warranted. Non-Neutralizing Antibody Therapeutics to Eliminate HIV-Infected Cells In Vitro and In Vivo via ADCC Jonathan Richard 1 , Dung N. Nguyen 2 , William D. Tolbert 2 , Derek Yang 3 , Seung Tae Kim 3 , Marco A. Diaz-Salinas 4 , Jyothi K. Rajashekar 5 , Li Zhu 5 , Catherine Bourassa 1 , Halima Medjahed 1 , Priti Kumar 5 , James B. Munro 4 , Amos B. Smith 3 , Andrés Finzi 1 , Marzena Pazgier 2 1 Centre de Recherche du CHUM, Montreal, Canada, 2 Uniformed Services University of the Health Sciences, Bethesda, MD, USA, 3 University of Pennsylvania, Philadelphia, PA, USA, 4 University of Massachusetts, Worcester, MA, USA, 5 Yale University, New Haven, CT, USA Background: In HIV-1 infection, a significant fraction of antibodies (Abs) is induced to epitopes occluded in the Env trimer at the surface of infected cells and viral particles that are exposed only upon Env interaction with CD4 (CD4 induced or CD4i). Most CD4i Abs are weakly or non-neutralizing (nnAbs) but their FcR activities, including antibody dependent cytoxicity (ADCC), are usually potent, but strictly dependent on epitope exposure. Studies have shown that HIV-1 evolved to restrict Env's contact with CD4 to avoid ADCC induced by CD4i nnAbs. Interestingly, some CD4i epitopes, including the co-receptor binding site (CoRBS) or gp120 Cluster A, represent the most conserved Env regions, suggesting their great potential as targets for Ab-based approaches. Methods: We developed CoRBS and Cluster A Abs into Ab-CD4 hybrids or Ab-CD4 mimetic (mc) conjugates in which an Ab (e.g. 17b or A32) is linked via a (G4S)6(G4T)2-linker to d1d2 of CD4, or small compound CD4 antagonist, CJF-III-288, via a (PEG)23-linker, respectively. Ab therapeutics were evaluated for binding and ADCC-mediated elimination of HIV-1-infected primary CD4+ T cells, and in vivo for their ability to eliminate infected cells in HIV-1JRFL-infected humanized mice supporting NK cell functions. Results: In in vitro and ex vivo settings, the Ab-CD4 hybrids of both specificities efficiently eliminated cells infected with HIV-1JRFL with potencies outcompeting the best-in-class bnAbs. In addition, these Ab-CD4s were also

able to synergize with nnAbs present in HIV+ plasma, further enhancing their ADCC activity. Our experiments utilizing single molecule FRET (smFRET) imaging confirmed that the activity of the Ab-CD4s directly results from their ability to stabilize State 2A, an Env conformation known to be ADCC vulnerable. Furthermore, results in humanized mice showed that A32-CD4 can control HIV-1 replication and substantially reduce the level of integrated HIV DNA in an FcR-dependent manner. Finally, our data also indicates that the CD4 moiety in an Ab-CD4 hybrid can be replaced by a small molecule CD4 mimetic. Our preliminary data confirm in vitro activity to eliminate HIV-1 infected cells with an Ab- CJF-III-288 conjugate utilizing an Ab of the CoRBS specificity. Conclusion: Our data confirm the utility of engineered CD4i Abs as therapeutics capable of overcoming obstacles to eliminate HIV-1 infected cells via ADCC. CD4i Ab therapeutics could be utilized in strategies to reduce or eliminate the viral reservoir in people living with HIV-1. A New CD4mc Enables Fc-Mediated Reservoir Reduction for Durable Post-ART HIV-1 Control in Hu-Mice Li Zhu 1 , Sri Lakshmi T. Boodapati 1 , Jonathan Richard 2 , Christopher J. Fritschi 3 , Derek Yang 3 , Hongil Kim 1 , Yaping Sun 1 , Lorie Marchitto 2 , Guillaume Beaudoin Bussieres 2 , Debashree Chatterjee 2 , Catherine Bourassa 2 , Joseph Sodroski 4 , Amos B. Smith III 3 , Andrés Finzi 2 , Priti Kumar 1 1 Yale University, New Haven, CT, USA, 2 Centre de Recherche du CHUM, Montreal, Canada, 3 University of Pennsylvania, Philadelphia, PA, USA, 4 Dana–Farber Cancer Institute, Boston, MA, USA Background: Persistently-infected HIV-1-positive cells are heterogeneous, rare, and inherently difficult to eliminate; shock-and-kill approaches have thus far proven inefficient in measurably reducing infected cell frequencies in clinical trials. CD4-mimetic compounds (CD4mcs) sensitize HIV-1-infected cells to antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) non-neutralizing antibodies (nnAb) that are frequently found in plasma of people living with HIV (PLWH) (Richard et al., PNAS 2015, PMID: 25941367; Rajashekar, Richard et al., Cell Host Microbe 2021, PMID: 34019804). The development of a new indoline CD4mc, CJF-III-288, with improved potency in neutralization and ADCC (Fritschi et al., PNAS 2023, PMID: 36961924), prompted us to investigate the impact of treatment, administrated at ART initiation or ART cessation, on viral tissue reservoirs and post-ART virus rebound dynamics in humanized mice (hu-mice) that support antibody effector function. Methods: NSG-Tg(IL15) hu-mice were treated with CJF-III-288 in combination with CD4i A32 (anti-cluster A) and 17b (anti-coreceptor binding site) nnAbs either at ART initiation or at ART withdrawal. Post-ART plasma viremia and tissue viral reservoir size were analyzed by longitudinal measurements of HIV-1 RNA and DNA respectively. We also analyzed the contributions of natural killer (NK) and CD8 T cells to the observed effects in in vivo cell-depletion studies. Results: Striking differences in HIV-1 rebound dynamics were observed depending on the timing of CJF-III-288/CD4i nnAb treatment. While treatment at ART cessation resulted in a significant delay of viral rebound (up to 1 month), treatment at ART initiation had an even greater effect, delaying viral rebound by more than twice as long as treatment at ART cessation. Remarkably, durable control of plasma viral loads (in spite of transient viremic blips) with a highly significant reduction of the viral reservoir led to ART-free viral remission in a subset of mice treated during the viremic phase of infection, concomitant with initiation of ART. While the outcome was primarily mediated by NK cells supporting the major contribution of ADCC effects, a role for CD8+ T cells in the continued suppression of viral loads and control of viral rebound was suggested from the results of in in vivo cell-depletion experiments. Conclusion: The overall results indicate that CD4mc have therapeutic potential in the presence of anti-Env CD4i antibodies, especially when administered at early stage. Combination CAR T-Cell Therapy Restricts HIV Escape and Durably Suppresses HIV Replication In Vivo Federica Severi , Alexandria Criswell, Dalia Bercow, Tyler Yang, Francesco Pennino, Reyes Acosta, Daniel T. Claiborne Wistar Institute, Philadelphia, PA, USA Background: In most cases, the cellular immune response is unable to durably control HIV replication. Chimeric antigen receptor (CAR) T cell therapy provides an avenue to engineer a more potent T cell response. Broadly neutralizing antibodies (bNAbs) targeting conserved regions of HIV Envelope may further facilitate the creation of a CAR T cell therapy effective against the majority of HIV isolates.

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Poster Abstracts

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CROI 2024 140