ADVANCING CANCER BIOTHERAPY WITH PROTEOMICS

Science 291: 1221, 2001

INFLAMMATORY PROTEIN PROFILE DURING INFLAMMATORY PROTEIN PROFILE DURING SYSTEMIC HIGH DOSE INTERLEUKINSYSTEMIC HIGH DOSE INTERLEUKIN--2 2

ADMINISTRATIONADMINISTRATION

Leonardo Rossi, PhDClinical Center Department of Transfusion Medicine

Section of ImmunogeneticsNIH

ALEXANDRIA

iSBTC

10-13 November 2005

Systemic IL-2 can effectively treat metastatic renal cell cancer and melanoma and plays an essential role during active-specific immunization against cancer by increasing the frequency of tumor regressions

Modulatory effects of IL-2 on various pathways of cellular immune responses have been extensively described

The use of high dose IL-2 treatment is limited by the occurrence of several side effects and severe toxicity

The mechanisms of IL-2 mediated cancer regression remain today largely unknown. Recent data suggest that IL-2 acts through the activation of momonuclear phagocytes at the tumor site.

ENIGMATIC ROLE OF INTERLEUKINENIGMATIC ROLE OF INTERLEUKIN--2 IN 2 IN CANCER THERAPYCANCER THERAPY

ILIL--22

PBMCPBMC

MCP-3MIP1-βMIP1-αMCP-1PARCMCP1IL-8GRO-1MIG

φφEnhancement of innateEnhancement of innateeffector mechanisms effector mechanisms

ChemoChemo--attractionattraction

M1M1--immune stimulationimmune stimulation

We characterized the protein profile of sera obtained from ten patients with RCCundergoing high dose (720,000 IU/kg intravenously every 8 hours) IL-2 therapy.

Serumcollection

PRE

3h

I dose

POST 1

IV dose

3h

POST 4

720000 IU/kg IL-2 administration

PROTEOMIC ANALYSIS OF SERUM IN PATIENTS UNDERGOING IL-2 THERAPY

SERUM

Multiprotein array platforms Immuno-nephelometrySELDI

… .… .… .… . H4

SAX2

WCX2

IMAC

MULTIPLEXED PROTEIN ARRAY PLATFORM

serum

… .… .… .… .

UNSUPERVISED HIERARCHICAL CLUSTERING OF SERUM SAMPLES FROM RCC PATIENTS OBTAINED PRE, POST 1 AND POST 4 DOSES OF IL-2 (720,000IU/KG).

IL-2 treatment is able to induce a very complex cytokine storm

1

0

4

1, 4

1

PRE

POST 1 DOSE

POST 4 DOSES

=soluble factor minimally changing from pre to post 4 doses=soluble factor expression enhanced at 4 doses only= soluble factor expression enhanced post 1 dose

= soluble factor expression enhanced at 1 and 4 doses Panelli et al., Journal of Translational Medicine 2004,2:17

Storm of chemokines, cytokines and soluble factors increasing in the circulation in response to IL-2

Many soluble factors that increased during IL-2 administration are also increased in

inflammatory conditions

Soluble forms of adhesion molecules

Chemoattractant factors

Matrix metalloproteinases and their inhibitors

TNF-alpha and soluble TNFR1

FROM PROTEIN ARRAYS TO SELDI ANALYSIS OF IL-2 INDUCED PROTEINS

Multiplexed protein array platforms

sensitive tools which allows the detection of proteins at very low concentration in serum

Discovery limited by the number of capture antibody selected forprotein detection.

Analysis Extended to Surface Enhanced Lased Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS).

SSURFACE URFACE EENHANCED NHANCED LLASED ASED DDESORPTION ESORPTION IIONIZATION ONIZATION TTIME OF IME OF FFLIGHT LIGHT MMASS ASS SSPECTROMETRY (SELDIPECTROMETRY (SELDI--TOFTOF--MS). MS).

Serum samples

matrixDifferent matrix are available to allow the preferential binding of proteins on the basis of their specific chemical or biological characteristics

SELDI ADVANTAGES

fast and easy

very reduced amount of samples are necessary to perform the analysis

accurate information about the molecular weight

possibility to discriminate between isoforms(phosphorilation, glycosilation, truncated forms)

possible capture of co-precipitating proteins

SELDI ANALYSIS OF RCC SERUMSELDI ANALYSIS OF RCC SERUMSAX2

Apo

C-I 6

629

Apo

A-II

8679

Apo

C-II

8909

TTR 13

859

Apo

C-I 6

632

Apo

A-II

8688

Apo

C-II

8911

TTR 13

860

Alph

a-glo

bin15

113

Beta-

globin

1585

9

RBP 2

1007

Apo A

-I 28

036

RBP 21

0254

Apo A

-I 28

071

SAA-

1-R 11

508

SAA

1166

8

Alph

a-glo

bin15

125

Beta-

globin

1586

3

Apo

C-I 6

630

Apo

C-I 6

648

SAA-

1-R 11

510

SAA

1166

0

TTR 13

730

TTR 13

812

RBP 21

0114

CRP 23

108

Apo A

-I 28

066

Apo A

-I 28

164

RBP 2

1011

Apo A

-II b

1723

3

Apo A

-II b 1

7250

Apo A

-II b 1

7304

Apo A

-II b 1

7383

C3dg 2

5504

C3dg 2

5464

C3dg 2

5382

C3dg

2538

0

PRE

POST 4DOSESIL-2

PRE

POST 4DOSESIL-2

Non fractionated serumFractionated serum

Inte

nsity

m/z

A

B

C

D

Rossi L. et al., Proteomics 2005 in press

SAA

CRP

7.5

8

8.5

9

9.5

10 11682.6+H

23114.0+H 28154.1+H

Prior IL-2

After IL-2

SELDI IMMUNOAFFINITY CAPTURE OF SAA AND CRP SELDI IMMUNOAFFINITY CAPTURE OF SAA AND CRP

A

B

Inte

nsity

m/z

Rossi L. et al., Proteomics 2005 in press

III

Immunochip/SELDISELDI literatureNephelometry

39,624 DaSAASAA1-RAlpha1 antitrypsin25,476 DaSAA-1-RSAlbuminApoA-IIApo A-IApo C-IApo-A IIbTruncated form of TTRBeta globinAlpha globinTTRRBPApo CIIBeta2 GPIHemopexin

No change in SELDI m/z

Increase in SELDI m/z

Decrease in SELDI m/z

UNSUPERVISED HIERARCHICAL CLUSTERING OF RCC SERUM SAMPLES UNSUPERVISED HIERARCHICAL CLUSTERING OF RCC SERUM SAMPLES OBTAINED BEFORE AND AFTER 1 AND 4 DOSES OF ILOBTAINED BEFORE AND AFTER 1 AND 4 DOSES OF IL--2 (SELDI M/Z AREA).2 (SELDI M/Z AREA).

I

IINegative

acute phase

reactants

Rossi L. et al., Proteomics 2005 in press

QUANTIFICATION OF SERUM FACTORS CONCENTRATION BY IMMUNONEPHELOMETRY

-10000 0 10000 20000 30000 40000 50000 60000 70000 80000

Cys C

PRB

TTR

HDL chol

Apo A-II

chol

a1 AT

Alb

mg/l

Total prot

TFN

HPT

SAA

Apo A-II

CRP

cr

2x1010 4x10103x10101x1010 5x1010 6x1010 7x1010

0 500 1000 1500 2000 2500 3000 3500 4000

chol

HPT

a1 AT

TFN

mg/l

-200 0 200 400 600 800 1000 1200 1400

CRP

TTR

Apo A-II

HDL chol

SAA

Apo A-II

Apo B

mg/l

0 10 20 30 40 50 60 70 80

Cys C

cr

PRB

mg/l

TFN

a1 AT

HPTchol

Apo BApo A-ISAAHDL chol

TTRCRP

RBPcrCys C

Albumin

a1 AT

chol

Apo B

Apo A-I

SAAHDL chol

TTR

RBP

Cys C

Apo A-II

1x107

10x1086x1082x108

25x10815x1085x108 40x108

3x107 5x107 7x107

pg/ml

PREPOST 1 DOSEPOST 4 DOSES

Rossi L. et al., Proteomics, 2005 in press

SEQUENTIAL DILUTION OF ONE POST 4 SAMPLE WITH THE CORRESPONDING PRE SAMPLE.

1 0 0 0 0 1 5 0 0 0 2 0 0 0 0

0

2 . 5

5

7 . 5

1 0

6 4 2 5 . 1 + H

6 6 2 2 . 8 + H

6 9 3 3 . 5 + H 8 6 7 1 . 8 + H8 7 9 8 . 1 + H8 9 0 2 . 4 + H

9 4 0 9 . 1 + H1 1 5 0 5 . 1 + H1 1 6 6 1 . 9 + H

1 3 8 5 0 . 3 + H

1 5 1 0 1 . 2 + H1 5 8 4 6 . 5 + H 1 7 2 2 0 . 8 + H

0

2 . 5

5

7 . 5

1 0

6 4 2 6 . 3 + H

6 6 2 4 . 1 + H

6 9 3 3 . 6 + H8 6 7 4 . 6 + H8 7 9 9 . 9 + H

8 9 0 2 . 8 + H9 4 1 1 . 3 + H

1 1 5 0 3 . 9 + H1 1 6 6 3 . 7 + H

1 3 7 4 0 . 9 + H

1 3 8 5 0 . 0 + H

1 5 1 0 0 . 6 + H1 5 8 4 7 . 8 + H 1 7 2 2 4 . 2 + H 2 2 1 9

0

2 . 5

5

7 . 5

1 0

6 4 2 6 . 8 + H

6 6 2 4 . 6 + H

6 9 3 4 . 6 + H 8 6 7 4 . 7 + H8 8 0 0 . 0 + H

8 9 0 4 . 1 + H

9 1 2 3 . 3 + H

9 4 1 0 . 6 + H

1 1 6 6 4 . 8 + H

1 3 7 4 2 . 8 + H

1 3 8 5 6 . 6 + H

1 5 1 0 4 . 9 + H1 5 8 5 4 . 2 + H 1 7 2 4 1 . 8 + H1 00

0

2 . 5

5

7 . 5

1 0

6 4 2 4 . 9 + H

6 6 2 3 . 2 + H

6 9 3 2 . 7 + H 8 6 7 2 . 6 + H8 7 9 8 . 9 + H

8 9 0 2 . 7 + H9 4 0 8 . 3 + H

1 3 7 4 5 . 5 + H

1 3 8 5 4 . 1 + H

1 5 1 0 3 . 5 + H1 5 8 5 0 . 8 + H 1 7 2 3 4 . 4 + H

1 0

0.3% of tot prot

0.17% of tot protInte

nsity

m/z

SAA

SAA

SAA

SAA

1 POST42 PRE

1 POST44 PRE

1 POST49 PRE

1 POST414 PRE

0.08% of tot prot

0.06% of tot prot

CORRELATION BETWEEN % SERUM SAA IN TOTAL SERUM PROTEIN AND THE RELATIVE M/Z AREA IN PATIENT SERA

R2 = 0.9371

0

500

1000

1500

2000

2500

3000

3500

4000

0 0.2 0.4 0.6 0.8 1 1.2 1.4

% SAA in total serum protein

P3

m/z

area

R2 = 0.971

0

500

1000

1500

2000

2500

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

% SAA in total serum protein

m/z

area

P9

R 2 = 0.90420

500

1000

1500

2000

2500

3000

3500

0 0.2 0.4 0.6 0.8

% SAA in total serum protein

m/z

area P14

R2 = 0.9787

0

500

1000

1500

2000

2500

3000

0 0.2 0.4 0.6 0.8 1 1.2 1.4

% SAA in total serum protein

m/z

area

P2

Rossi L. et al., Proteomics, 2005 in press

The interplay between different platforms could provide a varietThe interplay between different platforms could provide a variety of y of information information

Critical aspect: collection of good quality material

1) Simultaneous detection of the expression levels of several factors

2) Bio-functional suggestions isoforms

Protein to proteininteraction

3) Discovery of new factors

SUMMARYSUMMARY

We observed an outburst of a multitude of proteins increasing/reducing in the circulation in response to IL-2:

Changes became more dramatic with increasing doses

Subclasses of soluble factors displayed different kinetics

All these data well correlate with a systemic inflammatory profile

1

2

3

4

PROTEIN INTERPLAY FOLLOWING HIGH DOSE ILPROTEIN INTERPLAY FOLLOWING HIGH DOSE IL--2 THERAPY 2 THERAPY

IL-2

ECM DEGRADING ENZYMES

Act as an inflammatory stimulus

ICAM, VCAM E-SELECTIN

MCP-1

ACTIVATION OF MONOCYTESAND RELASE OF TNFR

CHEMOATTRACTANT ACTIVE RECRUITMENT

OF MONONUCLEAR CELLSTO INFLAMMATORY SITES

SAACRP After the 4° dose

MMP-2,3,8,9,10,13MMP-1

IL-1 IL-6 TNF αAfter the 1° dose

sTNFR1

Capillary leak syndrome and hypothension

MPs activation

ACKNOWLEDGMENTS

Department of Transfusion Medicine Section of Immunogenetics, Clinical Center /NIHFranco MarincolaMonica PanelliEna WangKatia ZavagliaEleonora AricòMarianna SabatinoSara DeolaSilvia SelleriSonia VoiculescuPing JinKina AmstrongXin LiDavid StroncekHarvey Klein

Perbio/PierceChristine BurnsMoreyLinda LavigneAmy Wilson

North Carolina Health OrganizationRichard WhiteMareva Foster

Clinical Center NIH Department of Clinical Chemistry NIHGlenn Hortin

Unit of Molecular Structure NIMHBrian MartinRamy Moharram