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Current Clinical Therapies for HIV Remission David Margolis MD UNC HIV Cure Center Aiming for sustained remission off ART Cohen J. Science 2014 Luzuriaga et al. NEJM 372;8: 786 Effect of vorinostat, hydroxychloroquine and maraviroc combination therapy on viremia following treatment interruption in individuals treated during acute HIV infection (Fiebig III-IV) Eugène Kroon, Jintanat Ananworanich, Keith Eubanks, Jintana Intasan, Suteeraporn Pinyakorn, Nicolas Chomont, Sharon R Lewin, Sarah Palmer, Lydie Trautmann, Hua Yang, Nitiya Chomchey, Nittaya Phanuphak, Ken Cooper, Praphan Phanuphak, Mark de Souza on behalf of the SEARCH 019 study group HIV Rebound after ART Interruption Median time to first VL detection: 22 days (range 14 to 77 days) ART resume at VL > 1000 c/ml Host cell modification Stochastic Reversal of Latency Chronically producing cell KO: CCR5 (Sangamo) KI: sh5/C46 (Calimmune) • • • CCR5-modified CD4 T cells at 1 week post infusion constituted 13.9% of • Challenges moving forward: circulating CD4 T cells – an Is cytoreductive Modified cells had estimated meantherapy half-life needed? of 48 weeks Acceptable? After ART interruption, decline in circulating CCR5-modified cells (−1.81 cells – Is there X4 escape? per day) was significantly less than the decline in unmodified cells (−7.25 cells – Scalability? Cost? per day) (P = 0.02) • HIV RNA became undetectable in one of four patients who could be evaluated AAsecond first step step to to eliminate eliminate latent latentHIV HIVinfection infection Latency Reversal Latency Reversal and Clearance Candidates • Latency Reversing Agents (LRAs) – HDAC inhibitors 800 * * ! ! * * ! * * ! • “lack potency and killing as single agents” (Mellors) * * * ! • “HDAC inhibitors do not kill non-transformed cells at clinical exposures” and “Appropriate serial dosing regimens of HDAC inhibitors have not yet been performed” (Margolis) 600 Relative HIV-1 gag RNA copies 14 daily doses of vorinostat! Elliot& PLoS& Path& 2014& Lewin CROI 2013! Baseline ART VOR 400 mg 400 200 100 60 40 20 Days! 0 Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Thrice weekly cycles of Panobinostat 2012! Pt. 8 Weekly Romidepsin ! Total CD4 cell-associated unspliced HIV-1 RNA! Sogaard IAS 2014 Rasmussen Lancet ID 2014 PLoS&Path& 2015& Apologies, too manySogaard& references to cite! Latency Reversal and Clearance Candidates • Latency Reversing Agents (LRAs) – HDAC inhibitors • “lack potency and killing as single agents” (Mellors) • “HDAC inhibitors do not kill non-transformed cells at clinical exposures” and “Appropriate serial dosing regimens of HDAC inhibitors have not yet been performed” (Margolis) – PKC agonists • most potent activators but toxicity of concern • Bryostatin clinical trial did not achieve effective drug exposures – TLR agonists: • activate HIV expression and immune control in SIV/macaques Apologies, too many references to cite! TLR7 Agonists Induce Transient Plasma Viremia Plasma SIV (log10 RNA copies/mL) 103 GS-986 (0.1 Plasma SIV RNA copies/ml Placebo mg/kg) 102 GS-9620 (0.05 mg/kg) mg/kg) 103 GS-9620 (0.15 Vehicle V1 V2 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 V1 V2 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 0 24 48 72 168 102 Vehicle Time after TLR7 agonist dose (Hours) • Reduced frequency of blips 38-75% (doses 3-10) • No more blips from animals dosed after 3 month pause (doses # 11-19) Whitney et al., CROI 2016 Phase I/II trial of GS-9620 in HIVinfection Design: • 3 escalating dose cohorts – 1 mg, 2 mg, 4 mg every 2 weeks for 6 doses • Placebo-controlled, randomized, double-blinded (6 active, 2 placebo per cohort) Study Population: • HIV-infected adults (n=24) • Virologically suppressed ≥12 months on ART Study Monitoring: • Close follow-up – VL 2-3x/w • Repeat dosing only if VL <50 copies/mL • Safety review prior to initiation of each cohort Current status: Enrolling Cohort 3 Latency Reversal and Clearance Candidates • Latency Reversing Agents (LRAs) – HDAC inhibitors • “lack potency and killing as single agents” (Mellors) • “HDAC inhibitors do not kill non-transformed cells at clinical exposures” (Margolis) – PKC agonists • most potent activators but toxicity of concern • Bryostatin clinical trial did not achieve effective drug exposures – TLR agonists: • activate HIV expression and immune control in SIV/macaques – Other Epigenetic targets • Bromodomain inhibitors • Histone methyltransferase inhibitors (eg. EZH2 inhibitors) • Others in development – High-throughput library screening efforts Apologies, too many references to cite! Merck HIV Latency HDAC Inhibitor Synergy Screen Objective: To identify compounds that potentially act synergistically with HDACis to induce HIV transcription 2,900,000 Compounds HIV LTR Induction With 250nM SAHA Initial Screen, n=3 HIV LTR Induction With 250nM SAHA Confirmatory Assays, n=3 Dose Response Assays, n=2 HIV LTR Induction With 250nM SAHA Toxicity @ 48 Hours CTG HIV LTR Induction Without SAHA NFkB BLA Reporter Counter Screen ~4,500 Compounds Known HDAC Inhibitors Known Farnesyl-Transferase Inhibitors Compounds with unknown mechanism 17.4% 16.1% 66.5% Data Analysis/ Hit Selection Follow-up Analysis Testing interventions in vivo LRAs Immunodulators HIV vaccines Novel approaches Reduction in: ART Baseline • Leukapheresis for QVOA and ca-HIV RNA • SCA • Immune assays • Host cell assays & biomarkers • Novel assays, eg. Quanterix Simoa After * intervention • Leukapheresis for QVOA and ca-HIV RNA • SCA • Immune assays • Host cell assays & biomarkers • Novel assays, eg. Quanterix Simoa • Resting CD4 cell infection • Low-level viremia HIV DNA, RNA, antigen & viruses HIV DNA HIV RNA in cells or culture QVOA Replicationcompetent virus Ho, Cell 2013 Ericksson, PLoS Path 2013 HIV Antigen (protein) detector The “Real” Reservoir Persistent HIV infection despite ART (per 106 cells) Frequency Time to eradication > 73.4 years Viral Activation & Clearance or Cell Death 100 10 1 0.1 0.01 - Residual Replication or Cell Proliferation 0.001 •0.0001 Given assay variance, a more than 6-fold RCI decrease would have likelihood 0.023 (2.3%) 0.00001 • Therefore a measurable goal is therapy that can 0 1 2 3 4 5 reduce the latent reservoir log Timeby onhalf ART a(years) 6 7 al. JID 2016 2015 Margolis, Garcia, Hazuda,Crooks HaynesetScience Challenges to clearing persistent infection after latency reversal • Recent absence of antigen – low frequency of HIV-specific antiviral responses • Immune dysfunction, deletion, or exhaustion • Archived viral diversity, including immune escape • Viral antigen is rare, dispersed, compartmentalized, and may be transient • Latency Reversing Agents (LRAs) are hosttargeted, and alone or in combination may alter antiviral immune response Latency Reversal Agent Discovery • New metric of viral antigen production or presentation - Antigen required to allow clearance • Assessment of LRA effect on immune function as part of development path - Immune function required for clearance Quanterix Simoa™ Technology 1. Single-protein molecules are captured and labeled on individual beads using standard ELISA reagents Quanterix Simoa Platform 2. Beads + substrate are loaded in individual femtoliter wells. Oil added to seal well 3. Digital or analog fluorescence readout of individual beads • Ultrasensitive platform (sub-pg/mL sensitivity) • Full automation (samples in – data out) – Rapid readout (~1 hr), >500 data points per day http://www.quanterix.com/ • Broad dynamic range (>4 logs) • Commercial p24 assay applied to plasma and serum (Chang L, et al. J Virol Methods. 2013;188(1-2):153-160.) • In-house Merck assay optimized to reduce nonspecific binding and enhance sensitivity, thus enabling p24 quantitation in cell lysates Howell et al. submitted Latency Clearance Assay CD8+ by negative selection PBMCs CD8+ Resting CD4+ cells by negative selection Culture Resting CD4+ cells Latency Reversing Agent or CD8+, HXTCs , or DARTs or No Effectors Limiting Dilution Co-culture Add Effectors Remove Effectors Measure HIV Production at 2 weeks PHA Number of positive wells (out of 12 total) HXTCs Reduce Recovery of Virus from autologous resting CD4+ T cells stimulated with: No Effectors CD8 HXTC 8 7 6 5 † 4 †† 3 2 †† † 1 0 Patient 423 Patient 250 Patient 231 Patient 492 Patient 532 Patient 425 VOR % Viral recovery Patient 425 120 †p<0.05 compared to No Effector 100 ††p<0.05 compared to BOTH 80 60 †† † 40 †† 20 0 Patient 425 Patient 532 Patient 250 Sung et al. JID 2015 Latency Reversal and Clearance Candidates • Natural and Engineered Antibodies – Broadly-neutralizing monoclonal antibodies (bnMAbs) • Can delay rebound and promote cell clearance in humans (3BNC117) • Resistance can rapidly develop (VRC01, 3BNC117) • Effect in individuals on ART? (VRC01) – Engineered bnMAb • Can prolong half-life and enhance Fc effector functions (e.g. PGT-151) – Bispecific Ab (anti-HIV/anti-host, e.g. CD3 or CD16) • Enhance effector function ex vivo and in animal models Apologies, too many references to cite! A5342/VRC01 Study • • • Double-blind, randomized, placebo-controlled, Phase I study 40 participants (20 per arm) VRC01 40 mg/kg IV at Day 0 & 21 (Arm A) or Day 42 & 63 (Arm B) Trial Completed and Analyses Underway! • • • • SIV-infected monkeys were treated with a 90-day course of ART initiated 5 weeks post infection 9 weeks post infection infused with primatized monoclonal antibody against the α4β7 integrin every 3 weeks until week 32 All animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts for more than 9 months, after all treatment was withdrawn. Human trial underway at NIH Dual Affinity ReTargeting (DARTs) Molecules for HIV • Do not require pre-existing HIV specificity • Not impacted by archived CTL escape variants • Anti-Env arm based on well characterized mAbs that have: • Are broad neutralizing antibodies -- bind virions and infected cells: DH542 (V3 glycan bnAb), CH557 (CD4bs bnAb), DH511-K3 (gp41 MPER bnAb) • Are ADCC mediating antibodies -- bind only infected cells: (7B2, gp41 immunodominant), A32 (C1) Dual Affinity Re-Targeting proteins direct T cell – mediated cytolysis of latently HIV-infected cells. Sung, JA, Pickeral, J, Liu, L, Stanfield-Oakley, SA, Lam, CY, Garrido, C, Pollara, J, LaBranche C. Bonsignori, M. Moody, MA, …..Haynes, BF, Nordstrom JL, Margolis, DM, Ferrari, G. JCI 2015 Targeting HIV Reservoir in Infected CD4 T cells by DARTs that bind Envelope and Recruit CTLs. Sloan DD et al. PLoS Pathogens, 2015 Sung et al.: • Screened ADCC, non-neutralizing Abs that bound HIV-infected CD4 T cells for optimal ADCC • Constructed DARTs with nonNeutralizing mAbs A32XCD3 and 7B2XCD3 • Showed DARTs + CD8 CTL eliminated HIV-infected CD4 T cells in vitro by CD8 T cell-mediated cytolysis. Sloan et al.: • Constructed bnAb PGT145 (V1V2), PGT121 (V3 glycan), VRC01 (CD4 bs), and 10E8 (distal MPER)--compared to A32 and 7B2 nonneutralizing DARTs • Best were PGT121, A32 and 7B2 DARTs ( n o r m a liz e d t o n o E f f e c t o r s c o n t r o l) % v ir a l r e c o v e r y HIVxCD3 DART Mediated Clearance of Resting Patient CD4 Cells Exposed to Vorinostat V O R - E x p o s e d L a t e n t ly In f e c t e d C e ll s 120 N o E ffe c to r s 100 C D 8 o n ly C D 3x4420 80 C om bo D AR Ts 60 40 20 0 0 0 674 408 407 795 795 P a t ie n t 2 4 h r c u lt u r e w it h D A R T s 96hr HIVxCD3 DART-mediated virus clearance in 4 of 4 patients (longer time needed for Pt 795) Sung, et al. JCI 2015 Latency Reversal and Clearance Candidates • Immune Checkpoint Blocking Antibodies – Major advance in cancer immunotherapy – Reverse immune exhaustion – Examples: Anti-PD-1/PD-L1, LAG-3, 2B4, CD160, TIM-3, others • Cellular therapies – CD8+T-cells with chimeric antigen receptors – Ex-vivo Effector cell expansion/re-infusion – Activated NK cells • Therapeutic Vaccines – Multiple approaches – Chimp Adeno vector, CMV vector, VSV vector, Ad26/MVA vectors, Dendritic cells – Can induce broad CTL responses Covering immune escape variants is critical! Apologies, too many references to cite! Tools to Test Latency Reversal and Clearance Minimal effect of VOR on immune function Clutton Sci Rep 2016 Persistent LRA activity of multiple VOR doses p < 0 .0 0 0 1 H IV - 1 g a g R N A c o p ie s p e r w e ll Autologous DC vaccine 2500 2000 Archin Keystone Symposium on HIV Persistence 2016 1500 1000 500 0 Baseline Multidose Combination Latency Reversal and Clearance Trial I Step 1 Screen Step 2 Enrollment Step 3 Interval Step 4 AGS Approximately ~Week 10 - Week ~Week 15 Week 24 Weeks 0 - 8 ~Week 6 - Week 13 18 Visits 1&2 Visit 3 & 4 Vists 5 - 7 BASELINE Step 5 AGS Dosing Step 6 Multiple VOR Dosing Step 7 AGS Dosing Step 8 Multiple VOR Dosing ~ Week 27 - Week 46 ~Week 39 - Week 54 ~Week 52 - Week 73 ~Week 64 - Week 81 ~Week 73 Week 89 Visit 9 - 13 Visits 14 - 17 Visits 18 - 22 Visits 23 - 26 27 Visit 8 EOS AGS Manufacturing Single Dose Paired Doses Interval dosing X 10 doses Injections X4 LRA x 1 EOS Term Interval dosing X 10 doses Injections X4 LRA x 2 LRA x 10 Vaccine visits - no dosing Leukapheresis & rc-RNA visits - Vorinistat doses Safety labs Vaccine = Argos dendritic cell therapy visits - AGS-004 injections PK Samples measurement determines progress LRA =rc-RNA vorinostat LRA x 10 Vaccine IM and Proviral SCA Exploratory Immunology Viral inhibition and latency clearance assays Acetylation Combination Latency Reversal and Clearance Trial II STEP 3: HXTCs produce d STEP 4: FirstSTEP Combination 1 STEPREST 2: STEPSTEP 3: 5: First STEPCombination 4: First Combination STEP 6: Immune REST Response STEP 5:monitoring First Combination treatment Pretreatm Single PERIO HXTCs treatment treatment PERIO treatment VOR every 72 enthours Phase x 10 VORD produce VOR every VOR 72every hours72 and hours x 10 D VOR every 72 hours and doses and Exhibit Ex- 400mg d 2 HXTC Infusions doses and 2 HXTC Infusions 2 HXTC Infusions vivo dose 2 HXTC Infusions response to VOR ~4 weeks ~8 weeks ~6-8 weeks 4 weeks Visits 1 2 3 6 7 8 9Visits 101 11 2 123 3.1 4 135 14 15 6 16 7 17 8 18 9 19 10 cART VOR every 72 hours x 10 doses ~45 weeks2-4 weeks HXTC ART 5 ~8 2-4 ~6-84 weeks 4 weeks weeks weeks weeks HXTC 4 HXTC 3.1 ~4 weeks HXTC STEP 2: Single VOR 400mg dose HXTC STEP 1 Pretreatm ent Phase Exhibit Exvivo response to VOR 11 2012 21 22 4 weeks 13 23 14 15 24 16 25 17 18 19 cART VOR every 72 VOR hours every 72 hours x 10 doses x 10 doses VOR every 72 hours x 10 doses Cath Bollard Immune Response Assays Immune HXTC Infusion Response Assays Leukapheresis HXTC Infusion Clio Rooney Leu Steps to eliminate HIV infection Latency Reversal Finally: the addition of durable vaccine protection if rebound or reexposure occurs