Novel high-throughput ELISAs to measure apoC-III on apoB-100, apoA-I and Lp(a): Reduction in lipoprotein-associated apoC-III

levels with volanesorsen (ISIS 304801 or ISIS-APOC-IIIRx) to apoC-III in a Phase 2 randomized trial Xiaohong Yang,1 Sang-Rok Lee,1 Yun-Seok Choi,1 Veronica J. Alexander,2 Qingqing Yang, 2 Joseph L Witztum,1 Sotirios Tsimikas1,2 1Department of Medicine, University of California San Diego, 2Isis Pharmaceuticals

Abstract Background Results – Baseline Patient Characteristics Summary Volanesorsen Safety Population (N=88) Background Elevated apolipoprotein C-III (APOC-III) • ApoC-III is a 79 amino acid glycoprotein synthesized principally in the Placebo 100 mg 200 mg 300 mg 1) ApoC-III can be detected directly levels are associated with and are liver and associated with apoB-containing lipoproteins and HDL. Characteristic (N=24) (N=13) (N=23) (N=28) on apoB-100, Lp(a) and apoA-I by causally associated with cardiovascular events. ApoC- • ApoC-III plays a key role in determining serum triglyceride levels and Gender, F:M 7:17 5:08 3:20 12:16 immunocapture from plasma. III present on LDL and HDL particles predicts is a potent inhibitor of lipoprotein lipase (LPL)-catalyzed lipolysis of Age, years 52.1 ±14.1 52.5 ±9.7 52.8 ±11.7 52.9 ±12.5 2 2) These ELISA techniques are increased cardiovascular risk. triglyceride rich lipoproteins. BMI, kg/m 31.9 ±3.8 29.4 ±4.1 30.4 ±3.1 31.0 ±4.4 • ApoC-III inhibits LPL activation by apoC-II and also inhibits hepatic , n (%) 8 (33.3) 6 (46.2) 8 (34.8) 10 (35.7) adaptable to high-throughput † lipase that plays an important role in the conversion of dense VLDL Maximal 3 (12.5) 2 (15.4) 6 (26.1) 3 (10.7) methodology. Objectives Traditional methods to detect apoC-III on Triglycerides, mg/dL 500.8 ±322.5 573.2 ±248.5 529.7 ±304.9 624.6 ±491.1 to IDL. LDL or HDL density fractions utilize labor-intensive Median (IQR) 464 (316, 582) 558 (351, 649) 464 (285, 715) 451 (310, 723) 3) Volanesorsen significantly • ApoC-III inhibits receptor-mediated uptake of lipoprotein remnants by the liver. immunoprecipitation of apoC-III and/or HDL-C, mg/dL 33.6 ±7.7 31.5 ±4.7 33.6 ±6.8 31.8 ±11.7 reduces apoC-III on apoB, Lp(a) • Elevated apoC-III levels are a causal, independent risk factor for CVD and is a target ultracentifugation methods. We developed high- LDL-C, mg/dL 101.4 ±59.3 95.6 ±33.3 93.2 ±34.8 75.8 ±36.7 and HDL in hyper-triglyceridemic throughput sandwich chemiluminescent ELISAs to of drug therapy • Currently available drugs have modest effects on apoC-III levels, ranging from -10% to patients, including those on detect apoC-III on individual lipoproteins in plasma. Results – Baseline Patient Characteristics of Subgroups -30%. and with FCS. • Volanesorsen, a second generation antisense oligonucleotide (ASO) potently Volanesorsen Monotherapy Volanesorsen Add-on to * 4) Significant correlations were were Methods Monoclonal antibodies MB47, LPA4 and Placebo 100 mg 200 mg 300 mg Placebo 200 mg 300 mg decrease plasma apoC-III levels and triglyceride levels in animal models, human present between apoCIII-apoB, sheep anti-human apoA-I were plated to capture volunteers, subjects with familial chylomicronemia syndrome and primary Characteristic (N=16) (N=13) (N=15) (N=13) (N=8) (N=8) (N=12) apoB-100, Lp(a) and apoA-I lipoproteins, respectively, hypertriglyceridemia by a mean of approximately 70%. Gender, F:M 3:13 5:08 1:14 5:08 4:04 2:06 5:07 apoCIII-Lp(a) and apoCIII-apoAI and the content of apoC-III on each was detected with Age, years 48.6 ±11.9 52.2 ±9.7 49.7 ±12.8 52.8 ±10.4 58.9 ±16.6 58.3 ±6.7 54.0 ±13.8 complexes with several lipid and an anti-apoC-III monoclonal antibody. ApoC-III-apoB- 2 Methods BMI, kg/m 31.1 ±3.0 29.4 ±4.1 30.8 ±3.0 30.9 ±3.6 33.5 ±5.2 29.6 ±3.5 32.5 ±4.6 lipoprotein measurements. 100, apoCIII-Lp(a) and apoC-III-apoA-I complexes Statins, n (%) 4 (25.0) 6 (46.2) 4 (26.7) 5 (38.5) 4 (50.0) 4 (50.0) 4 (33.3) 5) These assays may be used to were measured at baseline and day 57, 92 and 176 in Maximal Statin† 2 (12.5) 2 (15.4) 2 (13.3) 2 (15.4) 1 (12.5) 4 (50.0) 0 (0.0) Study subjects - The study subjects were derived from one phase 2 study of volanesorsen in 3 groups Triglycerides, mg/dL 522.7 ±369.7 573.2 ±248.5 662.0 ±299.0 565.6 ±213.7 457.0 ±213.5 281.8 ±74.5 383.7 ±207.4 understand the cardiovascular 88 patients with hypertriglyceridemia treated with 100, of patients with: 1- familial chylomicronemia syndrome (FCS) and primary elevation in triglycerides; 2- Median (IQR) 459 (356, 582) 558 (351, 649) 588 (464, 858) 566 (422, 702) 475 (253, 602) 272 (230, 295) 302 (246, 451) 200, 300mg volanesorsen (previously known as SIS volanesorsen as monotherapy; or 3- as add-on to subjects on fibrate therapy. The protocol was effects of triglyceride lowering HDL-C, mg/dL 33.0 ±7.5 31.5 ±4.7 31.9 ±4.0 33.2 ±8.9 34.9 ±8.4 36.9 ±9.8 35.1 ±11.5 304801 or ISIS-APOC-III ), a generation 2.0+ designed to administer volanesorsen (previously called ISIS 304801 and ISIS-APOC-IIIRx ), a generation therapies. Rx LDL-C, mg/dL 105.1 ±56.3 95.6 ±33.3 79.5 ±25.1 70.9 ±31.0 94.5 ±68.1 118.8 ±37.5 94.5 ±27.7 antisense drug to apoC-III or placebo, for 85 days. 2.0+ antisense drug directed to APOC3 messenger RNA (mRNA). Eligible patients were randomly 6) Whether these complexes predict assigned to receive either volanesorsen, at doses ranging from 100 to 300 mg, or placebo, once weekly range, Q1 and Q3. * 25 of 28 (89%) patients were on stable doses of (median daily dose = 145 mg); for 13 weeks and patients were then followed up to 176 days. FCS patients had triglyceride levels 3 of 28 (11%) patients were on stable doses of (median daily dose = 1200 mg). cardiovascular events waits to be Results The ELISAs detected apoC-III-apoB, apoC-III- ranging from 1406 to 2083 mg per deciliter (15.9 to 23.5 mmol per liter). In the other 2 groups the trial † Maximal statin (total daily dose): 40 mg and above of , , , or determined in future studies. apoA-I and apoCIII-Lp(a) with high signal-to-noise was designed as a randomized, double-blind, placebo-controlled, dose-ranging, phase 2 study in ratios. Compared to placebo, a dose-response effect untreated patients with fasting triglyceride levels between 350 mg per deciliter (4.0 mmol per liter) and was noted with volanesorsen at day 92 with 2000 mg per deciliter (22.6 mmol per liter), as well as in patients receiving stable fibrate therapy who Results – Changes in lipoprotein-associated ApoC-III levels with volanesorsen References ± ± ± had fasting triglyceride levels between 225 mg per deciliter (2.5 mmol per liter) and 2000 mg per 84.2 10.1%, 80.7 16.6%, and 80.7 14.3% deciliter (ISIS 304801-fibrate cohort). The primary outcome was the percentage change in APOC-III All groups Monotherapy Add-on Fibrate Familial Chylomicronemia reductions in ApoC-III-apoB, apoCIII-Lp(a) and apoC- levels from baseline. combined Gaudet D, Alexander VJ, Baker BF et al. Antisense Inhibition III-apoA-I, respectively (300 mg dose; p<0.001 for all), of Apolipoprotein C-III in Patients with Hypertriglyceridemia. and return to baseline by day 176. Strong correlations Determination of lipoprotein-associated apoCIII levels (ApoCIII-apoB, apoCIII-Lp(a), apoCIIII- N Engl J Med 2015;373:438-47. in all assay measures were noted with changes in total apoAI) - A sensitive and quantitative sandwich-based chemiluminescent enzyme-linked immunosorbent assay (ELISA) to measure apoC-III associated with plasma lipoproteins apoB-100, Lp(a) and HDL Gaudet D, Brisson D, Tremblay K et al. Ta rg e tin g APOC3 in plasma apoC-III and triglycerides. (Figure below). Microtiter 96-well plates are coated overnight at 4°C with antibodies MB47 and LPA4 the familial chylomicronemia syndrome. N Engl J Med (generated at UCSD) and sheep anti-human apoA-I (The Binding Site, Birmingham, UK) (all at 5µg/ml 2014;371:2200-6. Conclusions Novel high-throughput ELISAs were antigen of 40µl/well). Excess material is washed off and the plates blocked with 1% tris buffered developed to detect lipoprotein-associated apoC-III, saline/bovine serum albumin (TBS/BSA) for 45 minutes. After the plates are washed, EDTA plasma is including for the first time on Lp(a). Volanesorsen added at 1:50 dilution (40 µL/well) for 75 minutes to bind apoB-100, Lp(a) and HDL, respectively. After the plates are washed, rabbit anti-apoC-III polyclonal antibody (Abgent, San Diego, CA) at 1µg/ml, uniformly lowers apoC-III on apoB-100, Lp(a) and 40µ/well, is allowed to incubate with the plates for 60 minutes. After washing ex c ess material off the Disclosures apoA-I. Ongoing studies with cardiovascular endpoints plates, alkaline phosphatase-labeled goat anti-rabbit IgG (Sigma, St Lous, MO) (40 µl/well) is added for will define whether lipoprotein-associated apoC-III 60 min. After a final washing, lumi-phos 530 (Lumigen, Inc., Southfield, Michigan) (25µl/well) is added biomarkers have prognostic utility and/or can be for 75 minutes and luminescence read on a Dynex luminometer (Chantilly, Virginia). The results are ST and JLW are co-inventors and receive royalties from reported as relative light units (RLU) in 100 milliseconds after the background (TBS/BSA) RLU are patents owned by the University of California San Diego on targets of apoC-III-directed therapy. oxidation-specific antibodies. A patent has been applied for subtracted. High and low values are added to each 96-well plates as internal controls. by UCSD on lipoprotein-associated apoC-III assays. ST has a dual appointment at UCSD and Isis Pharmaceuticals. JLW is a consultant for Isis Pharmaceuticals, Intercept, CymaBay Aims ApoCIII-Lp(a) ApoCIII-apoB ApoCIII-ApoAI and Prometheus. VJA and QY are employees of Isis Pharmaceuticals. The other co-authors have no conflicts of The CVD risk of circulating plasma apoC-III is highly interest. dependent on its association with specific lipoproteins. For example, LDL or HDL containing Acknowledgment apoC-III predict CVD, whereas LDL or HDL without apoC-III does not, even after adjustment for The authors thank Tracy Reigle for creating the artwork. triglycerides. Currently established methods to detect apoC-III on LDL or HDL utilize labor-intensive methods that can only be done on small numbers of samples and are not easily translatable to large outcomes studies or clinical applications. Therefore, our goal was to develop high-throughput sandwich chemiluminescent ELISAs to detect apoC- Results – Correlations between lipids, lipoproteins and lipoprotein-associated apoC-III levels III on individual lipoproteins, directly from plasma, as a tool to assess their relationship to CVD risk and Chylomicron Chylomicron - Chylomicron - VLDL-ApoC- LDL-ApoC- effectiveness of apoC-III lowering therapies. These TG TC LDL-C HDL-C VLDL-C non-HDL ApoCIII -Apoc-III TG Cholesterol III HDL-ApoC-III III agents include antisense oligonucleotides (ASO) and ApoCIII-apoB 0.32 (0.016) 0.17 (0.215) -0.35 (0.009) -0.24 (0.080) 0.38 (0.004) 0.20 (0.144) 0.60 (<.001) 0.49 (<.001) 0.36 (0.007) 0.39 (0.003) 0.61 (<.001) 0.13 (0.360) 0.25 (0.061) ApoCIII-apoA-I 0.21 (0.121) 0.36 (0.006) 0.06 (0.671) -0.27 (0.050) 0.32 (0.017) 0.39 (0.003) 0.59 (<.001) 0.33 (0.014) 0.15 (0.273) 0.23 (0.095) 0.66 (<.001) 0.11 (0.409) 0.36 (0.006) drugs that Indirectly affect apoC-III, such as fibrates, ApoCIII-apo(a) 0.46 (<.001) 0.35 (0.008) -0.24 (0.072) -0.44 (<.001) 0.51 (<.001) 0.41 (0.002) 0.67 (<.001) 0.44 (<.001) 0.41 (0.002) 0.45 (<.001) 0.68 (<.001) 0.07 (0.598) 0.32 (0.016) fish-oil or other triglyceride lowering agents. R-value (p-value)