Hepatic Inflammation and Cellular Therapy - Vital...

Hepatic Inflammation and Cellular Therapy

Lee K. Landeen, Jessica Van Allen, Patricia W. Bedard

Vital Therapies, Inc., San Diego, CA, USA

2

Alcoholic Hepatitis: Role of Inflammation

17th International Symposium on Albumin Dialysis

Leaky Gut

Inflammatory Mediators (PAMPs, DAMPs)

Immune

Dysfunction

Cytokine

Storm

Secondary Organ Failure

Activation

Infections

Anti-inflammation Therapies for Alcoholic Hepatitis

• Anti-tumor necrosis factor alpha (TNF) (infliximab)

– Double-blind randomized controlled trial (prednisolone infliximab)

– Naveau S, et al. 2004 Hepatology

– Did not demonstrate clinical benefit (higher infection rates, death rates, lower ex vivo stimulation capacity of neutrophils in treatment group)

• Thalidomide

– Pilot study to reduce TNF

– N0265006466, DH Adams (Queen Elizabeth Hospital, Birmingham, UK), 2006-2007

– Study abandoned

• Steroids (prednisolone, pentaxifylline)

– STOPAH

– Thursz MR, 2015 N Eng J Med

– Did not demonstrate medium- to long-term clinical benefit

• IL-1ra (anakinra)

– NCT01809132, anakinra + pentoxifylline + zinc sulfate vs. methylprednisolone

– On-going


Porcine Hepatocytes Human Hepatoblastoma-derived C3A

Cell Line

Allogeneic Human Hepatocytes Human Hepatocellular Carcinoma-

derived HepaRG Cell Line

The Case for Cell-based Liver Therapies/Treatments


• Cells are capable of producing multiple factors

• Cells can respond dynamically over time

• Cells can potentially respond to individual patients

HepatAssist

(Circe Biomedical

>Arbios Systems)

SRBAL

(Mayo

Clinic)

MELS

(Charite Virchow

Clinic)

ELAD

(Vitagen>Hepatix>

Vital Therapies)

(BALANCE,

Netherlands)

The ELAD® System


VTL C3A

Cells

• Previous emphasis was on factors that ELAD C3A cells could remove:

– Bilirubin, ammonia, toxins, etc.

Mechanism of Action: Soluble Factors


• Current focus is on what ELAD C3A cells can contribute:

− Anti-inflammation, regeneration, coagulation,

transport, anti-oxidation, etc.

VTL C3A Cells Secrete Many Potentially Useful Proteins

7

• 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

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


Tissue Inhibitor of


Transforming Growth Factor Alpha

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 SerotransferrinAngiogenesis:

Angiopoietin-2

Placental Growth Factor

Vascular Endothelial Growth

Factor

Vascular Endothelial Growth

Factor-C

Hematopoiesis:

Erythropoietin


Oxidative Stress:Peroxiredoxin-4

Fatty Acid-Binding Protein,

liver

*Proteins may have multiple roles depending on concentration and targeted cell type

8

Death

Alcohol

Consumption

Liver

Failure

Stabilization

Recovery/

Regeneration

Immune Dysfunction

Cholestasis

Leaky Gut

Toxic Injury

Apoptosis/Necrosis

Alcoholic

Hepatitis (Severe

Inflammation/

Dysfunction)

Coagulopathy

Respiratory

Failure

Kidney Failure

Immune/Sepsis

Re

du

ce

d in

flam

ma

tion

Me

tab

olic

Su

pp

ort

Imm

un

e F

un

ctio

n

Re

ge

ne

ratio

n

Re

du

ce

d A

po

pto

sis

Imp

rov

ed

Bile

Flo

w

ELAD Treatment

C3A Cell Proteins

Multi-organ failure:

Working Model of ELAD Therapeutic Effect


ELAD C3A Cell Cartridge

Inflammatory Factors:

IL-1, IL-6, TNF, LPS

?


Are ELAD C3A Cells Capable of an Acute-Phase Response?

VTL C3A Experimental Model

10

IL-1β,

IL-6,

&/or

TNFα

or

LPS

VTL C3A Cells

?

Supernatants

Collected

24, 48, 54h

Protein

Secretion

(immunoassay)


VTL C3A Cells Exhibit an Acute-Phase Response


C o n t r o l I L - 1 x I L - 6

0

2

4

6

8

Alb

um

in (

g/

mL

)

p = 0 . 0 4 7

Fib

rin

og

en

(n

g/m

L)

C3AIL-1β

+

IL-6

24

hr

Decreased

Albumin

Increased

Fibrinogen

VTL C3A Cells Produce Anti-Inflammatory Proteins

12

AATIL-1Ra

C3AIL-1β

+

IL-6

24

hr

Increased

IL-1Ra

No Change

AAT


C o n t r o l I L - 1 + I L - 6

0

1 0

2 0

3 0

IL-1

Ra

(n

g/m

L)

p = 0 . 0 0 7

C o n t r o l I L - 6 + I L - 1

0

2 0 0

4 0 0

6 0 0

AA

T (

ng

/mL

)

p = 0 . 5 1 4 0

VTL C3A Cells Produce Anti-Inflammatory Proteins


0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

0

5 0

1 0 0

1 5 0

L P S (E U / m L )

IL-1

Ra

(p

g/

mL

)

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

L P S (E U / m L )

AA

T (

ng

/m

L)

C3ALPS

24

hr

Increased

IL-1Ra

Increased

AAT

Will Healthy Neutrophils Respond to ELAD-Treated AH Plasma?


Increasing phagocytic capacity with Days on ELAD Treatment suggests restored function

FIT

C

Phagocytosis

Normal T0 T1 T2 T3Plasma

14

Normal D0 24h EOT 30d

• Healthy peripheral blood neutrophils

• Exposed to plasma from Normal individuals or ELAD-Treated subjects

– D0=before treatment

– 24 h after treatment initiated

– EOT=end of treatment

– 30 d after treatment initiated

• Measure fluorescent signal of neutrophils actively phagocytizing FITC-labeled E. coli

R Jalan, RP Mookerjee, NA Davies (University College London)

Will Macrophages Respond to ELAD C3A Factors?

15

24-well plate

1x106 cells/well

0 1 6 7

THP-1

monocytic cells

PMA

24-hrs

Polarize

24-hrs

Cytokine

Immunoassays

Collect

Supernatants

Time (Days)

Rest

5-Days

M1

Adherent

macrophages

IFN-γ

+ LPS

+/- ELAD C3A Cell

Conditioned

Medium

8

Pro-Inflammatory

Phenotype


M1 Macrophages Reduce Pro-Inflammatory IL-1 When Treated with ELAD Conditioned Medium

M 1 M 1 + C M

0

1 0

2 0

3 0

4 0

I L - 1

IL

-1

(p

g/m

L)

M 1

M 1 + C M

***

*** <0.001, Student’s T-Test, n=6 biological replicates tested in duplicate


Pro-Inflammatory Cytokines Decrease During ELAD Treatment of AH Subjects


TNFa

day 0

day 1

0

50

100

150

200

pg

/ml

IL-8

Day

0

Day

1

0

200

400

600

800

1000

pg

/ml

IL-6

Day

0

Day

1

0

500

1000

1500

2000

2500

pg

/ml

IL1b

Day

0

Day

1

0

10

20

30

40

pg

/ml

R Jalan, RP Mookerjee, NA Davies (University College London)

TNF IL-8

IL-6 IL-1

Day 0 Day 1 Day 0 Day 1

Day 0 Day 1 Day 0 Day 1

VTL C3A Cells Produce IL-8 In Response to IL-1

• But not in response to IL-6 or LPS

• IL-8 is elevated in patients with chronic alcoholic liver disease

– Swiatkowska-Stodulska 2006 Med Sci Monit

• IL-8 can rescue human hepatocytes from TNF-induced apoptosis, and induce DNA synthesis

– Osawa 2002 Infect Immun


LPS

Is There Evidence of an Anti-Inflammatory Response in ELAD-Treated AH Subjects?

• Case Study

19

• Plasma collected prior to, during, and after ELAD Treatment

• Immunoassayed for IL-1Ra


IL-1Ra Levels Increased During ELAD Treatment of AH Subject


0.0

0.5

1.0

1.5

2.0

2.5

0 5 10 15 20 25 30

Co

ncen

trati

on

(n

g/m

L)

Study Day

IL-1Ra

Translational Biomarkers for Survey of VTI-208 Samples

Inflammation:

• IL-1Ra - anti-inflammatory protein expressed by VTL C3A cells

• AAT - anti-inflammatory protein expressed by VTL C3A cells

• IL-1β - Inflammatory protein in AH

• IL-6 - Inflammatory protein in AH

• TNFα - Inflammatory protein in AH

• C-reactive protein - increased in AH with Systemic Inflammatory Response Syndrome (SIRS)

• Pro-calcitonin – marker of SIRS

Anti-Apoptosis:

• Cytokeratin 18 (CK18) - a marker of caspase-mediated apoptosis

• Amphiregulin - EGFR ligand most highly expressed by VTL C3A cells

• Soluble Fas - blocks Fas-ligand, expressed by VTL C3A cells

• VEGF- reduces HAEC apoptosis, expressed by VTL C3A cells


Conclusions

• Single-drug therapies have not proved successful in treating AH

• ELAD cell-based treatment may provide improved clinical benefit by reducing inflammation, among other mechanisms of action

22

Death

Alcohol

Consumption

Liver

Failure

Stabilization

Recovery/

Regeneration

Immune Dysfunction

Cholestasis

Leaky Gut

Toxic Injury

Apoptosis/Necrosis

Alcoholic Hepatitis

(Severe

Inflammation/

Dysfunction)

Coagulopathy

Respiratory Failure

Kidney Failure

Immune/SepsisR

ed

uced

infla

mm

atio

n

Meta

bo

lic S

up

po

rt

Imm

un

e F

un

ctio

n

Reg

en

era

tion

Red

uced

Ap

op

tosis

Imp

roved

Bile

Flo

w

ELAD Treatment

C3A Cell Proteins

Multi-organ failure:


Acknowledgements

• Neutrophil studies were completed by the Liver Failure Group, University College London Institute for Liver and Digestive Health, Royal Free Hospital (London, UK)

– Rajiv Jalan, MD

– Rajeshwar P Mookerjee, MBBS, PhD

– Nathan A Davies, PhD

• VTL Scientific Advisory Board

– Cliff Steer, M.D. (University of Minnesota)

– Charles Dinarello, M.D. (University Colorado Denver)

– George Michalopoulos, M.D. (University of Pittsburgh School of Medicine)

– Alan Hofmann, M.D. (emeritus) (University of California, San Diego )

– Fernando Camargo, Ph.D. (Harvard and Boston Children’s Hospital)

– Nikolaos Pyrsopoulos, M.D., M.B.A. (Rutgers New Jersey Medical School)

– Mike Millis, M.D. (University of Chicago Medicine)



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Hepatic Inflammation and Cellular Therapy

Lee K. Landeen, PhD, Jessica Van Allen, BS,

and Patricia W. Bedard, PhD

Date post:	28-May-2018
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