The HDAC inhibitor romidepsin is safe and effectively reverses HIV-1 latency in vivo as measured by standard clinical assays Thank the organizers Clinical with romidepsin to activate viral transcription and release from latently infected cells. Ole Schmeltz Søgaard, MD PhD Department of Infectious Diseases, Aarhus University Hospital, Denmark

The “kick and kill” approach to cure HIV Reactivate latent viral expression HDAC inhibitors, PKC activators, BET bromodomain inhibitors, etc. Kick cART Kill Put the trial in an hiv cure context Follows the kick and kill strategy Deeks S. Nature 2012

HDAC inhibitors Induce HIV mRNA tran-scription in latently infected resting CD4 cells in vivo Considerable variability in potency between HDACis Ultimately, HDACis should release viral particles in to plasma to allow for immune-mediated killing of infected cells Acetylation of the lysine residues on the histone tails relaxes the chromatin structure and allows for gene expression, whereas deacetylation of the histone tails leads to condensation of the chromatin structure and silencing of gene expression. In accordance with this mechanism, HDACis have been shown to induce HIV transcription in latently infected cells Many HDACis, considerable difference in their effect on HIV gene expression Archin Nature 2012; Lewin CROI 2013; Rasmussen HVIT 2013

Romidepsin Licensed in the US for treatment of PTCL and CTCL Increase extracellular RNA release from memory and resting CD4+ T cells in patients on cART ex vivo EC50 for RMD approx. 4.5 nM compared with 3,950 nM for SAHA in a primary T cell model RMD (IV) T½~4 hrs However, ex vivo viral outgrowth data question the ability of HDACi (incl. RMD) to reverse latency in vivo In the present study, we have used the HDACi, romidepsin Much credit for the advancement of this drug in HIV latency should be given to Romas Geleziunas, who together with his team at Gilead and external collaborators have done a tremendous amount of in vitro/ex vivo work with RMD Wei PLoS Path 2014, Bullen Nature Med 2014

Trial design Non-randomized interventional trial Romidepsin (5 mg/m2) IV day 0, 7, and 14 Primary endpoints: Safety as well as activation of HIV-1 from latency as determined by plasma HIV-1 RNA and cell-associated unspliced HIV-1 RNA in total CD4+ cells Secondary endpoints: H3 acetylation, HIV-1 DNA, T cell activation HIV-1 patients on cART Age >18 years CD4 >500 cells/µL VL <50 copies/mL for >1 year No HBV/HCV infection No significant cardiac disease - Emphasize 3 doses of romidepsin at a dosing which equals 1/3 the standard dosing used in cancer treatment

Patient characteristics N=6, 5 males and 1 female (caucasian) Median CD4+ cell count: 760 (range 510-1000) age: 54 years (range 37-60) duration of cART: 9.5 years (range 4.2-14.5) None started cART during PHI ART regimens: PI+2NRTIs (n=3), NNRTI+2NRTIs (n=2), INT+2NRTIs (n=1) - Just read

Self-reported AEs 40 adverse events (AE) were registered ̶ 36 AEs were considered to be related to the study drug Most AEs were mild (grade 1, n=38) and resolved spontaneously within a few days Two AEs were grade 2 (fatigue and fever in one individual after the 2nd infusion) The number of AEs reported by each study participant during follow-up ranged from 1 to 13 (up to day 21) The most common AEs were abdominal symptoms such as nausea (n=12), borborygmia (n=4), abdominal pain (n=2), diarrhea (n=1), and vomiting (n=1) and fatigue (n=5). - first: the self-reported safety data

Biochemistry Romidepsin and other HDACis are known to affect the white blood cell compartment, often resulting in thrombocytopenia and neutropenia Initial minor reductions in most leucocyte compartments most pronounced in neutrophils. 7 days following the last romidepsin infusion the levels of the various populations of leucocytes had more or less returned to pre-therapy levels

Lymphocyte histone H3 acetylation The degree of histone acetylation is a direct measure of the pharmacodynamic effect of an HDACi on cells. Thus, to closely describe the chain of events leading from RMD administration to activation of HIV-1 transcription, we determined lymphocyte histone H3 acetylation at each study visit. This graft shows the mean H3 acetylation for the 6 patients as a group Emphasize dosing and sampling schedule - The lasting effect of RMD compared to other HDACi may be related to its unique intracellular pharmacology and interaction with HDAC enzymes. RMD acts as an intracellular prodrug that undergoes reduction of its intramolecular disulfide bond upon entering cells. The released free sulfhydryl groups tightly interact with the Zn2+ ion in the active site of various target HDAC isoforms, a mechanism of inhibition that does not apply to VOR or PNB.

Cell-associated unspliced HIV-1 RNA We determined intracellular HIV RNA levels in total CD4+ cells by ddPCR as a measure of HIV transcriptional activity Top graft shows the actual changes in HIV RNA for each individual and the bottom graft shows fold-changes from pre-therapy level for the whole group We observed significant increases in CA US HIV RNA in all 6 patients Highest levels observed after the 2nd and 3rd dosing (mean 3.5 fold increase)

Plasma HIV-1 RNA Plasma viral RNA was determined using the standard COBAS taqman assay ”Take 1 infusion at a time” 5 of 6 patients reached quantifiable plasma RNA levels during the 3 infusions Using the same qualitative transcription mediated assay that blood bank use for screening of donor blood for early signs of HIV infections, we noted that 50% of the pre-infusion samples were positive for plasma HIV RNA, whereas more than 90% were positive 3 days post-infusion Collectively, these data may be represent convincing evidence of a reactivating agent that ”kicks” the latently infected cells out of latency Viral load: COBAS® TaqMan® HIV-1 Test, v2.0 TMA: Qualitative NAT screening system (PROCLEIX ULTRIO Plus, Genprobe)

T cell subsets, activation and PD-1 expr. - RMD induced immediately changes in the composition of the peripheral CD4+ and CD8+ compartment Shift towards larger proportions of naive cells and fever effector and central memory cells. However, changes had almost reversed by day 10 CD4 T cell activation as measured by CD69 increased primarily in the effector memory and term diff subsets which is consistant with ex vivo findings Expression of the exhaustion marker PD1 decreased initially in both CD4+ and CD8+ Collectively, these changes may impact measurements of the HIV reservoir and the chance of clearing reactivated cells

Total HIV-1 DNA in CD4+ T cells Individual HIV DNA levels in total CD4+ cells measured by qPCR shown left, fold changes from pre-therapy level shown to the right One person had an 80% decrease in HIV DNA but on a group level there was no change from baseline to day 21

Conclusions RMD safely activated latently infected cells and induced transient quantifiable plasma viremia Phenotypic changes occurred in the T cell compartment during RMD treatment The HIV-1 reservoir was not significantly reduced by RMD (as measured by HIV-1 DNA) A clinical trial combining a therapeutic HIV vaccine (Vacc-4x) and RMD is ongoing

Acknowledgments THE STUDY PARTICIPANTS Department of Infectious Diseases, Aarhus University Hospital Martin Tolstrup Thomas Aagaard Rasmussen Lars Østergaard Steffen Leth Mette Graversen Paul Denton Christel Rothe Brinkmann Rikke Olesen Anne Sofie Kjær Sara Konstantin Nissen Lene Svinth Jøhnke Erik Hagen Nielsen Department of Clinical Immunology, Aarhus University Hospital Christian Erikstrup Bionor Pharma (SPONSOR) Maja Sommerfelt Anker Lundemose Potential cART and RMD: Tested in a TZM-bl assay. Using a range of concentrations of PI, NRTIs, NNRTIs and INT, we did not see any see reduced sensitivity to the antiviral effect of these drugs when co-administered with HDACis including RMD and PAN - Potential RMD and PI interaction: Romidepsin is metabolized by CYP3A4. In recently conducted drug interaction clinical trial evaluating the strong CYP3A4 inhibitor, ketoconazole, showed co-administration of 8mg/m2 romidepsin (4-hour infusion) with ketoconazole, resulted in an overall increase in romidepsin exposure by approximately 25% and 10% for AUC0-∞ and Cmax, respectively, compared to romidepsin. As a comparison, ritonavir has been shown to have an inhibitory potency on CYP3A4 slightly less than that of ketoconazole (von Moltke, Greenblatt et al. 1998). Given the lower dosing of romidepsin in the current study and the estimated <25% increase in romidepsin (AUC0-∞) with ritonavir regimens, subjects on ritonavir boosted protease inhibitors will be allowed in the current study but will be monitored closely for toxicity related to increased romidepsin exposure. BUT PHARMACOKINETICS VS PHARMACODYNAMICS (H3 acetylation). Highest VL blip in a NNRTI treated pt, least and most side effects observed in two PI patients.