Update on HDL Infusions and Atherosclerosis Regression H. Bryan Brewer, Jr. Washington Cardiovascular Associates Medstar Heart and Cardiovascular Institute Washington Hospital Center Washington, DC

Current Model of HDL and Lipoprotein Metabolism Liver B Asztalos Tufts University Boston, MA aHDL Preb-1 Control Plasma B-100 Triglyerides C-III E Intestine Cholesterol B-100 LDLR LPL HL SR-BI Cholesterol A-I CE TG CETP αHDL VLDL LRP LDL Macrophage Arterial Wall LPL HL ABCG1 B-48 Triglyerides C-III E ABCA1 LCAT Preβ-HDL A-I Chylomicron

2 Step Approach to the Synthesis of Preβ-HDL 2 Step Process for Production of Preβ-HDL 1 ApoA-I 243 aa rHDL A-I Phospholipids Step 1 Isolate and Purify ApoA-I Step 2 Combine ApoA-I + Phospholipids 2 Step Process for Production of Preβ-HDL 1 ApoA-I 243 aa rHDL A-I Phospholipids Step 1 Isolate and Purify ApoA-I Step 2 Combine ApoA-I + Phospholipids Clinical Programs ApoA-I Milano Esperion Pfizer Medicines Co Preβ-HDL A-I

ApoA-I Milano ApoA-I Milano Arg Cys 173 In apoAI milano, there is a cysteine substitution for arginine at position 173 allowing for dimerization, formation of large HDL particles that may be particularly active in RCT. It is characterized by the replacement of a single amino acid at R173C R173S apoA-I. Carriers of the apolipoprotein A-IMilano (apoA-IM) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. Discovered by accident, the mutation was found to be present in about 3.5% of the population of Limone sul Garda, a small village in northern Italy. It has been traced to a mutation in a single man, Giovanni Pomarelli[2], who lived in the village in the 18th century and passed it on to his offspring.[3] Figure 1eNOS expression in HDL-treated HUVECs. A, Reverse-transcription polymerase chain reaction analysis for eNOS mRNA levels in untreated cells (medium) and in cells incubated overnight with HDL (1.14 mmol/L cholesterol) isolated from control subjects ...eNOS expression in HDL-treated HUVECs. A, Reverse-transcription polymerase chain reaction analysis for eNOS mRNA levels in untreated cells (medium) and in cells incubated overnight with HDL (1.14 mmol/L cholesterol) isolated from control subjects (control HDL) or apoA-IM carriers (A-IM HDL). eNOS mRNA band intensities were normalized by GADPH values. B, Western blotting analysis of eNOS protein levels in untreated cells and in cells incubated overnight with control HDL or A-IM HDL (1.14 mmol/L cholesterol). eNOS protein band intensities were normalized by β-actin values. Results are expressed as mean±SEM of 3 separate experiments with 3 different preparations of control HDL or A-IM HDL and 2 batches of cells. *P<0.05 vs medium, #P<0.05 vs control HDL. ApoA-I Milano Arg Cys 173 Gomaraschi M et al. Circulation 2007;116:2165-2172

Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 46 Acute Coronary Patients: 5 Weekly Infusions 33 ApoA-I Milano Infusion; 11 Saline Infusions A-I Milano* -14.10 2 4 6 8 10 14 16 - - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

HDL Infusion Therapy Update Medicines Co. – ApoA-I Milano Pilot Study: Safety and Efficacy IVUS Trial

ApoA-I Milano Clinical Trial : Milano Pilot Study Primary Endpoint: Percent Atheroma Volume (PAV) Secondary Endpoint: Total Atheroma Volume -6.90 (-17.5,2.2) P<0.01 -4.7 (-13.,1.7) P<0.01 Placebo MCO-216 (Entire Vessel Length) - 2 - 4 - 6 - 8 - 10 - 12 - .20 P =0.10 P = .77 - .40 Median Change in Percent Atheroma Volume (%) -0.5 (-1.8.,0.5) P=0.11 - .60 Median Change Volume (mm3) - .80 -0.8 (-2.3,0.2) P<0.001 - 1 Placebo MCO-216 - .20 Nicholls S and Nissen S AHA 2017 Presentation

Clinical Programs 2 Step Approach to the Synthesis of Preβ-HDL 2 Step Process for Production of Preβ-HDL Clinical Programs Step 1 Isolate and Purify ApoA-I Step 2 Combine ApoA-I + Phospholipids Medicines Co: ApoA-I Milano 1 ApoA-I 243 aa rHDL A-I Phospholipids CSL: CSL 111/112

Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 183 Acute Coronary Patients: 4 Weekly Infusions , 123 Patients Received CSL111 rHDL Infusion; 60 Patients Control Infusions 2 4 6 8 10 14 16 - - A-I Milano* -14.10 -5.34 ERASE** **Tardif JC et al. JAMA 2007;297:1675-82 - - Change in Total Atheroma Volume (mm)3 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

HDL Infusion Therapy Update Medicines Co. – ApoA-I Milano Pilot Study: Safety and Efficacy IVUS Trial CSL Behring – AEGIS-I Trial CSL112: Safety and Efficacy Trial

CSL Behring AEGIS-I Trial: Clinical Infusion Therapy With CSL112 Subjects:1258 Acute Coronary Syndrome Patients Randomized 1:1:1 2 g/A-I, 6g A-I, placebo Stratified renal function Trial: 4 weekly infusions of CSL 112 End Points Primary Endpoint : Clinical Assessment of Renal and Liver Function Secondary Endpoint : 1. Composite CV Death, non-fatal MI, stroke hospitalization for unstable angina 2. Cholesterol efflux Results No Significant Alteration in Liver and Kidney Function Dose Dependent Elevated Cholesterol Efflux Gibson CM Et al Circulation 2016;134:1918-1930

Clinical Programs 2 Step Approach to the Synthesis of Preβ-HDL 2 Step Process for Production of Preβ-HDL Clinical Programs Step 1 Isolate and Purify ApoA-I Step 2 Combine ApoA-I + Phospholipids Medicines Co, ApoA-I Milano 1 ApoA-I 243 aa rHDL A-I Phospholipids CSL, CSL 111/112 Cerenis, CER-001

Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 570 Acute Coronary Patients: 6 Weekly Infusions, Infusion Dose CER-001 3 mg/kg, 6 mg/kg and 12 mg/kg 2 4 6 8 10 14 16 - + CHI-SQUARE 3 mg/kg*** ***Tardif JC et al Eur Heart J 2014;35:3277-3286 -3.13 A-I Milano* -14.10 -5.34 ERASE** **Tardif JC et al. JAMA 2007;297:1675-82 - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

HDL Infusion Therapy Update Medicines Co. – ApoA-I Milano Pilot Study: Safety and Efficacy IVUS Trial CSL Behring – AEGIS-I Trial CSL112: Safety and Efficacy Trial Cerenis – CER-001 CARAT: Safety and Efficacy IVUS Trial

Cerenis CARAT Trial: Clinical HDL Infusion Therapy With CER-001 Subjects: 292 Acute Coronary Syndrome Patients Trial: 9 weekly infusions of 3 mg/kg CER-001 End Points Primary Endpoint : Change in Percent Athero Volume (PAV) Results To Be Presented ACC Anderson J et Cardiovasc Diagn Ther 2017;7:45-51

HDL Therapeutics Therapy Employs a 1 Step Process To Produce Preβ-HDL 2 Step Process for Production of rHDL 1 Step Process for Synthesis of Preβ-HDL αHDL Preβ-HDL A-I Advantages of Selective Delipidation 1. Safe 2. High Yield 4. Low Cost 3. Selective increase in preβ-HDL for Specificity for ABCA1 Transport 5. Low Regulatory Hurdles Isolated ApoA-I Phospholipids 1 243 aa rHDL A-I Convert αHDL to Preβ-HDL by Selective HDL Delipidation CSL, CSL111/CSL112

a HDL a HDL a HDL Preb-1 Preb-1 Preb-1 2-D GEL ELECTROPHORESIS OF CONTROL PLASMA, SHAM TREATED PLASMA AND SELECTIVE HDL DELIPIDATED PLASMA DEMONSTRATING A SHIFT FROM aHDL TO Preb-HDL With SELECTIVE HDL DELIPIDATION a HDL a HDL a HDL Preb-1 Preb-1 Preb-1 Sham Treated Plasma Selective HDL Delipidation Control Plasma Analysis performed by Dr. B. Asztalos, Tufts University Boston, MA

Acute Preβ-HDL Infusions: IVUS Clinical Trial De-risking and Feasibility Trials In ACS Patients

HDL Selective Delipidation**** Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 2 4 6 8 10 14 16 - + 28 Acute Coronary Patients: 7 Weekly Infusions, 14 Received Delipidated HDL and 14 Control Plasma Infusions HDL Selective Delipidation**** ****Waksman R et al J. Am. Coll Card 2010;55:2727-2735C -12.10 A-I Milano* -14.10 -5.34 ERASE** **Tardif JC et al. JAMA 2007;297:1675-82 CHI-SQUARE 3 mg/kg*** ***Tardif JC et al Eur Heart J 2014;35:3277-3286 -3.13 - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

HDL Infusion Therapy Update New Approaches to the Quantitation of Vascular Disease Following HDL Infusions

TVCTM: New FDA Approved Technical For Vulnerable Plaque Analysis Coronary Artery Lesions Distinguishes Cholesterol Filled Plaques From Cholesterol Free Lesions Measures Cholesterol Content of Vulnerable Plaques Before and After Therapy NIR Scan Cholesteryl Oleate Cholesterol Cholesteryl Linoleate Collagen

Take Home Messages √ Selective HDL infusions in ACS patients were associated with marked regression of atherosclerosis and have a high probability of decreasing clinical events. √ Clinical trials will be required to definitively establish if increasing Preβ-HDL will decrease cardiovascular events.