Elaborating ELAD Mechanism of Action and Linking Cell-Based Models to the Clinic

Transcription

1 Elaborating ELAD Mechanism of Action and Linking Cell-Based Models to the Clinic September 9, 2017 Patricia W Bedard, Jason Lapetoda, Jagadeesha Dammanahalli, Jessica Van Allen, Susan Lin, Lee Landeen

2 Cellular Therapies Supply Many Potentially Therapeutic Factors. List of VTL C3A Cells Secreted Proteins: 148 proteins identified to date via immunoassay; 62 proteins via LC/MS 39 proteins secreted above 1 mg/day/cartridge 15 proteins secreted 10x above normal serum levels Inflammation: Alpha-1-Antitrypsin Complement C3 Ferritin Gelsolin Haptoglobin Intercellular Adhesion Molecule 1 Interleukin-1 Receptor Antagonist Interleukin-8 Tumor Necrosis Factor Alpha Angiogenesis: Angiopoietin-2 Placental Growth Factor Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor-C Regeneration: Amphiregulin Growth/differentiation factor 15 Heat-Shock protein 70 Heparin-Binding EGF-like Growth Factor Hepatocyte Growth Factor Platelet-Derived Growth Factor-BB Tissue Inhibitor of Metalloproteinases 1 Tissue Inhibitor of Metalloproteinases 2 Tissue Inhibitor of Metalloproteinases 3 Transforming Growth Factor Alpha Hematopoiesis: Erythropoietin Transport: Albumin Alpha-Fetoprotein Apolipoprotein A-I Apoliproprotein A-II Apolipoprotein A-IV Apolipoprotein B Apoliproprotein C-I Apolipoprotein C-II Apoliproprotein C-III Apolipoprotein E Apoliproprotein H Fatty Acid-Binding Protein, liver Serotransferrin Oxidative Stress: Peroxiredoxin-4 Fatty Acid-Binding Protein, liver *Proteins may have multiple roles depending on concentration and targeted cell type 2

3 Working Hypothesis: VTL C3A Cell Secreted Factors Could Impact Multiple Cell Types in the Liver Hepatocyte Stellate ELAD C3A Cell Cartridge Goal: Identify what factors are affecting what cells and in what ways Kupffer Endothelial Neutrophils 3

4 THP-1 Cell Models to Evaluate Impact of ELAD on Inflammation Anti- Inflammatory Factors IL-1β ELAD CM s Effects on Measured IL-1β from THP-1 Kupffer Cells M1, Proinflammatory THP-1 Macrophages SAB DEC

5 Inflammation Involves More than Just Leukocytes Inflammation Bacterial LPS Kupffer Cells Neutrophils IL-1β IL-6 Activated Platelets White Blood Cells Inflamed Endothelium Systemic Inflammation Blood Clots ORGAN FAILURE & DEATH Monocytes 5

6 Endothelial Cell Model of LPS-Induced Cell Death Endothelial Cells + LPS +/- ELAD CM 24hr. Cell Death Adapted from Bucki R. Biochemistry Cell death measured by cytoskeleton disruption FITC-labeled phalloidin binds to F-actin Untreated 100 μg/ml LPS 6

7 ELAD CM Inhibits LPS-induced Cell Death in Human Aortic Endothelial Cells (HAEC) & Human Microvascular Endothelial Cells (HMVEC) HAEC *Magnification: 40x HMVEC *Magnification: 10x Untreated LPS only LPS +CM ELAD CM was equally protective of HMVEC and HAEC challenged with LPS 7

8 Vascular Endothelial Growth Factor (VEGF) Partially Protects HAEC and HMVEC Treated with LPS HAEC *Magnification: 40x HMVEC *Magnification: 10x Untreated LPS only LPS + CM LPS + VEGF VEGF at levels found in ELAD CM, reduces LPS damage, but not as well as ELAD CM, suggesting multiple mechanisms 8

9 If ELAD CM Inhibition of Endothelial Cell Apoptosis is due in Part to VEGF, Blockade Should Reverse the Effect LPS VEGF in ELAD CM ELAD CM ELAD CM PAMP-R Pathogen- Associated Molecular Pattern Receptor VEGFR1 VEGFR2 X DEATH SURVIVAL 9

10 If ELAD CM Inhibition of Endothelial Cell Apoptosis is due in Part to VEGF, Blockade should Reverse the Effect LPS VEGF in ELAD CM ELAD CM ELAD CM svegfr1 PAMP-R Pathogen- Associated Molecular Pattern Receptor VEGFR1 VEGFR2 DEATH XSURVIVAL 10

11 svegfr-1 Blocks the Protective Effects of ELAD CM, VEGF, and PlGF Supporting this Mechanism LPS+CM svegfr-1 1 µg/ml 3 µg/ml LPS Only LPS+VEGF 3.25 µg/ml 9.75 µg/ml LPS Only 11

12 Other Mechanisms for ELAD CM s Inhibition of Endothelial Cell Apoptosis? LPS VEGF in ELAD CM 1 ELAD CM ELAD CM PAMP-R Pathogen- Associated Molecular Pattern Receptor VEGFR1 VEGFR2 2 Other Mechanism??? ELAD CM X DEATH SURVIVAL X 12

13 Endothelial Cell Model - H 2 O 2 -induced Oxidative Stress as Measured by Reduced:Oxidized Glutathione Ratio (GSH:GSSG) HAEC and HMVEC + H 2 O 2 4hr. Decreased GSH Cell stress measured by GSH:GSSG Ratio Promega GSH/GSSG-Glo Assay GSH Luciferin-NT GSH-NT Luciferin luciferase ATP Light GSH:GSSG Ratio = (total glutathione RLU GSSG RLU) [GSSG RLU/2] 13

14 G S H / G S S G M o l a r R a t i o G S H / G S S G M o l a r R a t i o ELAD CM Blocked H 2 O 2 -Induced Oxidative Stress Similarly in both HMVEC and HAEC H M V E C HMVEC **** **** HAEC G S H / G S S G M o l a r R a t i o U n t r e a C M H 2 O 2 C M + H **** 1 0 **** 0 Untreated ELAD CM H 2 O 2 H 2 O 2 + U n t r e a t e d E L A D C M H 2 O 2 H 2 O 2 + E L A D C M ELAD CM 0 Untreated ELAD CM H 2 O 2 H 2 O 2 + ELAD CM H 2 O 2 reduced the GSH/GSSG ratio below untreated levels in MVEC and HAEC GSH/GSSG ratio was significantly higher in ELAD CM-treated cells exposed to H 2 O 2 (fully protective) ****P< Student s T-test vs. Respective Control 14

15 Current Model of ELAD Inhibition of Endothelial Cell Apoptosis LPS 1 ELAD CM VEGF & PlGF in ELAD CM PAMP-R Pathogen- Associated Molecular Pattern Receptor VEGFR 2 Antioxidants in ELAD CM ELAD CM GSH X DEATH SURVIVAL X Oxidative Stress 15

16 Previously Shown: ELAD C3A Cell Factors Inhibit Fas-Induced Apoptosis in Primary Human Hepatocytes Hepatocytes ELAD C3A Cell Cartridge Amphiregulin, sfas (*** p<0.001 One-way ANOVA with Tukey post-hoc test) 16

17 MitoSoX Red Assay Measures Superoxide ROS in Mitochondria of Primary Human Hepatocytes MitoSoX Red (Fluorescent redox active probe) MitoSoX Red O 2.- Specifically targets superoxide anions (O 2.- ) in mitochondria 17

18 R e l a t i v e F l u o r e s c e n c e ELAD-CM Inhibited Ethanol-Induced ROS Generation in Mitochondria of Primary Human Hepatocytes (PHH) P * * * E t O H W E M E L A D - C M 5-hrs post-plating, whole cells were incubated 30 min with MitoSox Red dye Ethanol (100mM) was added and relative fluorescence was measured as an indication of ethanol-induced ROS production P<0.009 by Student s T-test 18

19 Abstracts to be Presented at EASL/AASLD and AASLD Meetings Ethanol-Induced Oxidative Stress in Primary Human Hepatocytes is Suppressed by VTL C3A Cell-Secreted Factors EASL/AASLD joint meeting on AH, London Initial findings on mitochondrial ROS AASLD The Liver Meeting, Washington DC Additional data added 19

20 Common Methodology for Linking Bench to Bedside Reductionist Biology Complex phenomenon, can be explained by analyzing the simplest, most basic physical mechanisms in operation during the phenomenon Proteins Cells In Vivo Clinical Applied Reductionism While analyzing underlying mechanisms, there must be continuous linking back to the complex system Anchoring findings in current knowledge of the system & clinical biomarkers 20

21 VTL C3A Cells Produce Anti-Inflammatory Proteins in Response to Inflammatory Cytokine Challenge A A T ( n g / m L ) IL-1β + IL-6 C3A 24 hr Increased IL-1Ra No Change AAT AAT p = C o n t r o l I L I L

22 Connecting Bench to Bedside, Biomarkers in Plasma Collected During the VTI-208 Trial Set 1: Four ELAD-Treated Subjects AASLD ET 2016 Set 2: 25 Subjects (n=14 ELAD, n=11 Ctrl) Selected based on: ~Complete sample sets (Screen, EOS DY 3, EOS DY5, EOS DY7, EOS DY14, EOS DY28) Meeting VTL-308 inclusion criteria Set 3: Remaining Subjects not meeting VTL-308 criteria 22

23 Caveats to Proteins measured in Subject Plasma There are a myriad of potentially confounding factors that could be influencing measured changes (e.g. drugs and other administered products) To the extent that we are able we will explore these factors to look for statistical associations There are plans to evaluate additional samples from subjects who did not meet the VTL-308 criteria 23

24 Concentration (pg/ml) IL-1Ra in VTI-208 Subject Plasma Shows a Significant Dose Was Administered 4500 IL-1Ra *** *** CTRL ELAD Time on Treatment *** Study Day Mean SEM ***ELAD vs. CTRL D3 (p=0.0006), D5 (p<0.0001), D7 (p=0.005) ***D0 vs. D3 (p<0.0001) and D5 (p<0.0001) and D7 (p=0.001) 24

25 Cell-based Therapy, Multiple Mechanisms Working in Concert Alcohol Metabolism Oxidative Stress Cell Death Inflammation IL-6 IL-1β In the Liver IL-1Ra ELAD s In Vitro Effects 1. ROS 2. Cell Death 3. Leukocyte activation 4. Potentially less IL-1β to inhibit with IL-1Ra 25

26 For Each Mechanism of Action from Cell-based Models There are Biomarkers Selected to Link to Clinic IL-1Ra C-Reactive Protein Ferritin Alpha-1 Antitrypsin Procalcitonin Amphiregulin Soluble Fas VEGF-A Keratin 18 Erythropoietin Inflammation Cell Survival Red Blood Cell Production Additional Analytes in Development for use with Protein Simple Ella Alpha Fetoprotein Transferrin 26

27 Questions? 27