Chronic and Acute Markers of

Interferon Stimulation

Thomas B. Lavoie

Product R&D and Assay Services (PRaDAS)

The Yin-Yang of the Interferon System Lorne, Victoria, Australia 2014

In

Acute and Chronic Markers of IFN/TLR

Stimulation

CXCL-10 ISG15 Others?

In

Why look for markers of IFN stimulation?

• IFN is implicated in a growing number of conditions.

– Lupus, Scleroderma, etc

• IFN can be difficult to measure in vivo.

– Not systemic

– Fast clearance

– Tools limited

• Markers allow inference of IFN activity and may offer

some discrimination.

• Our goal is to create a panel of IFN and Marker

assay(s) to perhaps differentiate diseases.

In

Why look for markers of IFN stimulation?

• Acute Markers

– Allow measurement of current IFN activity

– Disappear quickly when activity not present

• Intermediate markers

– Reveal recent IFN activity

– Fade slowly when activity not present

• Chronic Markers

– Reveal long term IFN activity

– Maintain levels in absence of IFN

In

Measurement of IFN

• Activity

– Cell based PCR assays (WISH assay)

• Indirect stimulation?

– Microarray (IFN signature)

• When, where, protein?

– Reporter Gene

• Specificity issues, indirect stimulation?

• Mass Based

– Immunoassays

• ELISAs prone to artifacts

• Sensitivity issues

In

Measurement of IFN

90% of Patients on IFN

Beta therapy have

quantifiable IFN Beta

2% of Healthy Donors have

1-2 pg/ml IFN-Beta

No elevation of IFN Beta

on other therapies (p=0.4)

IFN Beta in Normal and MS Patient Sera

Detected by ELISA

IFN

Bet

a Tre

ated

n=29

No

IFN

Tre

atm

ent n

-25

NH

S n=

193

0102030405060708090

100110120

IFN

-Bet

a (

pg

/m

l)

In

Measurement of IFN

Activity on average 30% of

Mass

Literature suggests that

there are inhibitors of

IFN-β in serum/plasma

Not correlated is sIFNAR2

Comparison of Reporter Gene

and ELISA Results

0 25 50 75 100 12501

23

4

56

7

89

r2=0.48

IFN- (pg/ml)

IFN

Act

ivit

y (

IU)

IFNAR2 vs Activity Observed

0 2000 4000 6000 80000.00.10.20.30.40.50.60.70.80.91.01.1

sIFNAR2 (pg/ml)

Act

ivit

y/P

red

icte

d

In

Measurement of IFN

41105 41110 New

DMEM Normal Hu Serum Normal Hu Serum

Subtype LLOQ LLOD LLOQ LLOD LLOQ LLOD

a1 (aD) 59.21 15.07 79.43 17.00 2.31 0.76

a2a (aA) 7.13 2.00 7.38 1.87 1.36 0.42

a4a (aM1) 123.68 39.52 220.99 64.91 1.38 0.43

a5 (aG) 40.63 11.39 83.55 20.79 1.70 0.51

a6 (aK) 10.02 3.14 8.04 1.89 1.13 0.33

a7 (aJ1) 8.57 2.96 10.56 2.96 2.09 0.59

a8 (aB2) 6.84 2.95 9.87 3.74 2.81 0.82

a10 (aC) 10.03 3.60 16.23 5.45 1.77 0.65

a14 (aH) 16.18 5.73 32.94 10.09 1.76 0.51

a16 (aWA) 162.98 49.17 519.16 192.07 2.01 0.68

a17 (aI) 6.06 2.07 11.96 4.32 1.36 0.50

a21 (aF) - - - - 6.18 1.67

500-12.5pg/mL 1000-12.5 pg/mL 250-3.9 pg/mL

New Assay sees all 12 subtypes well, more sensitive

In

Measurement of IFN

Most LLOQ’s in the 1-2 pg/ml range ( y axis reciprocal)

a1 (aD

)

a2a (a

A)

a4a (a

M1)

a5 (aG

)

a6 (aK

)

a7 (aJ

1)

a8 (aB

2)

a10 (a

C)

a14 (a

H)

a16 (a

WA)

a17 (a

I)

a21 (a

F)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.941105

41110

41120

1/

LL

OQ

a1 (aD

)

a2a (a

A)

a4a (a

M1)

a5 (aG

)

a6 (aK

)

a7 (aJ

1)

a8 (aB

2)

a10 (a

C)

a14 (a

H)

a16 (a

WA)

a17 (a

I)

a21 (a

F)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.541105

41110

41120

1/

LL

OD

In

Measurement of IFN

IFN-α Detected in 61% of Lupus samples 30% of samples have >1 pg/ml

Again activity by reporter gene assay ~30% of expected.

IFN- Specific Signal in Lupus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 192021 22232425260

2

4

6

8

Lupus Sera Lupus Sera plus blocking PAb

Sample

IFN

- (

pg

/m

l)

In

Acute Markers

Good relationship between IFN

levels after injection,

temperature change, and 11-OH

corticosteriods.

Other Acute Markers.

IL-6/Fever with Consensus

IFN (Colter et al JICR 2001)

From Scott et al Antimicrob Agents Ther. 1983

Effect of IFN-Alpha 2b in Patients

0.0 2.5 5.0 7.5 10.0 12.50

5

10

15

20

25

30

35

40

45

IFN 11-OH Corticosteroids

Temp (delta)

0.0

0.5

1.0

1.5

2.0

20 30

Time (hrs)

IFN

U/

ml

uM

OL

/L

or D

eg C

In

Acute Markers

Good relationship between

IFN levels and CXCL-10

levels after injection.

2’5’OAS has prolonged

elevation.

Other Intermediate markers

might include β2-

Microglobulin and

Neopterin.

From McHutchinson et al Hepatology, 2007

IFN, CXCL-10 and 2'5'OAS induction by CPG

0 10 20 30 40 50 600

50

100

IFN- CXCL10

2'5' OAS

0

1000

2000

3000

4000

5000

6000

7000

100

200

300

Day

IFN

-(p

g/

ml)

or

2'5

'OA

S

fold

in

du

cti

on

CX

CL

-10(p

g/

ml)

In

Acute Markers

CXCL-10 is elevated in MS

patients on IFN therapy and

it’s levels correlate with

IFN-Beta levels

MS

No

IFN

MS

wIF

NN

HS

0

100

200

300

400 *p=0.019 *p=0.01

IP-1

0 (

pg

/m

l)

CXCL-10 vs IFN Beta in MS

0 20 40 60 80 100 1200

100

200

300

400

r = 0.7271

IFN-beta (pg/ml)

CX

CL

-10 (

pg

/m

l)

In

Acute Markers

C24 C48 T24 T48 Bl C20 T20 CSup TSup

A549 OvCAR

ISG-15 Western Blot

I

Acute Markers

A549 Daudi

OvCar U937

IFN-Beta 48 hours ISG-15 Western Blot

I

Acute Markers

ISG-15 is secreted from Daudi Cells stimulated with IFN-Beta

0 25 50 75 100

10

100

1000

100000.02 ng/ml

0.2 ng/ml

20 ng/ml

2 ng/ml

Hours

ISG

15 (

pg

/m

l)

In

Acute Markers

ISG15 is a good candidate

Acute/Intermediate marker

Serum ISG15 after 8 MU Betaseron Injection

1 2 30

500

1000

1500

2000

Time days

ISG

15 p

g/

ml

From D’Cunha et al, J.Immunol 1996

In

Acute Markers

IFN- mediated IFN- and IL-10

release from NK cell model

0 1 2 3 4 5 60

100

200

300

IFN-IL-10

Log [IFNa] pg/ml

An

aly

te (

pg

/m

l)

Previously IL-15 has been shown to induce IL-10 from NK cells

In

Acute Markers

CXCL-10 Induction by IFN- and IFN-

1 10 100 1000100

1000

10000

Beta 24 Beta 48 Beta 72

Gamma 48 Gamma 72Gamma 24

IFN U/ml

CX

CL

-10 (

pg

/m

l)

IL-8 Induction by IFN- and IFN-

1 10 100 1000100

1000

10000

Beta 24 Beta 48 Beta 72

Gamma 48 Gamma 72Gamma 24

IFN U/ml

CX

CL

-10 (

pg

/m

l)

IFN-β and IFN-γ differentially up-regulate CXCL-10 and IL-8 in OvCAR cells

In

Acute Markers

IFN- induction by poly I:C in Hepatocytes and Monocytes

0 2 4 6 80.9

9

90

900

Hepatocytes Monocytes

24

Lymphocytes

Hours

IFN

- (

pg

/m

l)

IFN- induction by poly I:C in Hepatocytes and Monocytes

0 2 4 6 810

100

1000

Hepatocytes Monocytes

24

Lymphocytes

Hours

IFN

- (

pg

/m

l)

IFN- induction by poly I:C in Hepatocytes and Monocytes

0 2 4 6 80.01

0.1

1

10

100

1000

10000

Hepatocytes Monocytes

24

Lymphocytes

Hours

IFN

- (

pg

/m

l)

Tissue dependent regulation of IFN expression

May give indications of tissue involved in activation

In

Acute Markers

Tissue dependent regulation of marker expression

May give indications of tissue of activation

CXCL-10 induction by poly I:C in Hepatocytes and Monocytes

0 2 4 6 81

10

100

1000

10000

100000

Hepatocytes Monocytes

24

Lymphocytes

Hours

CX

CL

-10 (

pg

/m

l)

IL-6 induction by poly I:C in Hepatocytes and Monocytes

0 2 4 6 810

100

1000

10000

100000

Hepatocytes Monocytes

24

Lymphocytes

Hours

IL-6

(p

g/

ml)

TNF induction by poly I:C in Hepatocytes and Monocytes

0 2 4 6 81

10

100

1000

10000

Hepatocytes Monocytes

24

Lymphocytes

Hours

TN

F

(p

g/

ml)

In

Chronic Markers

In

Chronic Markers

Soluble Type I and Type II Receptors are elevated in Males

In

Chronic Markers

Soluble Type I and Type II Receptors are not

coordinately controlled

sIFNGR1 may be elevated in RA (Bello et al Biotherapy 1998)

In

Chronic Markers

From Gilli et al Neurology 2008

In

Chronic Markers

sIFNAR2 Levels in Autoimmunity

Normal n

=59

SLE n=111

RA n=16

Sjogren's n=11

Scleroderma n=10

0

2500

5000

7500

100001000035000

p=0.008

sIF

NA

R2 p

g/

ml

sIFNAR2 Elevated in Lupus

Not in RA, Sjogren’s, Scleroderma

Percent IFN positive?

Strength of signal?

Duration of signal?

In

Chronic Markers

sIFNAR2 does not correlate with IFN detected in samples

sIFNAR2 vs IFN- in MS

0 20 40 60 80 100 1200

2000

4000

6000

r = 0.05367

IFN- pg/mL

sIF

NA

R2 p

g/

mL

In

Chronic Markers

From Miyake et al Cytokine 2009

Chronic IFN stimulation in Cancer

Stage dependent?

In

Chronic Markers

From Muzugoshi et al Hepatology 1999

and Hep C

In

Why look for markers of IFN stimulation?

• Summary and Conclusions

– Acute Markers

• 11-OH Steroids CXCL-10 (9, 11?)

• IL-6 Fever

• ISG15?

– Intermediate Markers

• Neopterin 2’5’OAS

• β2-Microgloblulin

• ISG15?

– Chronic Markers

• sIFNAR2

• sIFNGR1?

In

Why look for markers of IFN stimulation?

• Summary and Conclusions

– Good panel of Acute and Intermediate markers

• May provide Flare/Exacerbation Information

– Limited panel of Chronic Markers

• May be suitable for patient stratification/prognosis

– Most useful panel will be a mixture of both

• Provides real time and long term information.

In

Acute and Chronic Markers of IFN-Stimulation

PRaDAS Quality Control

Taher Fatakdawala* Sehrish Ajmal*

Robert Niemczyk* Karen Zipf*

Xiao-Hong Lin*

Tara Stauffer* Production Team

Michael Skawinski*

Lara Izotova *

Eric Zhu*

Barbara Schwartz Ray Donnelly (FDA/NIH)*

Nirupama Putcha

Sanjna Sanghvi

Katherine Carrino-Bednarski

Jonathon Ferreira