Istituto Oncologico VenetoVenetian Institute for Molecular Medicine,

Padova, Italy.

“Sviluppo di nuovi farmaci capaci di alterare il microambiente tumorale e ripristinare la risposta

immune anti-tumorale"

Vincenzo Bronte

LA RETE NAZIONALE SOLIDALE E

COLLABORAZIONI INTERNAZIONALI DEL

PROGRAMMA STRAORDINARIO ONCOLOGIA 2006

(ISS PER ACC)

Roma 21 Aprile 2008

Tumor escape from immune response

Tumo r p rog re ssion

Epigenetic mechanisms:- Cytokines (TGF-β, IL-10, VEGF)- Membrane molecules (↓Fas, ↑Fas-L,Muc-1, B7-H1, TRAIL, HLA-G)

- Reduced antigen presentation (TAP)- Altered myelopiesis

Genetic mechanisms:- Antigen loss/mutation- MHC allele loss, β2-microglobulinloss

Intrinsic nature:- Absence of costimulation- Absence of antigen- Treg, CD4+CD25+ induction

tumor-free tumor-bearer

CD11b+/Gr-1+ cells expanded in tumor bearing mice:

Are heterogeneous and accumulate in blood, bone marrow, spleen, and lymph nodes

Can home to tumor site

Comprise cells inhibiting T cell activation (Myeloid-derived suppressor cells, MDSC)

Can be found also in chronic infections, autoimmune diseases, and following chemotherapy

Comprise cells with pro-angiogenetic program

Sica and Bronte, JCI, 2007

Immune dysfunction

induced by MDSC depends on the

interaction between enzymes

which utilize L-arginine as

substrate

PROSTATE ORGAN CULTURE

The advantage of using PCa organ cultures is that the microenvironment of the tumor remains intact and all the factors that may affect TIL function, such as cell-cell interactions, cell-matrix interactions and interstitial fluid, are preserved.

MFI

fol

din

duct

ion

over

u

nst

imu

late

dsa

mpl

es

TIL FROM PCa PATIENTS DO NOT RESPOND TO STIMULI

CD25

CD69

CD137

1.0

1.5

2.0

2.5

3.0

3.5

Tumor-freeprostates

PCa

* **

Tumor-freeprostates

PCa

PHA PMA + iono

0

1.0

2.0

3.0

4.0

CD8

Via

bilit

y in

dex

(rat

io)

0

0.5

1.0

1.5

2.0

2.5

3.0

CD25 CD69 CD137

**

*

Fold

of

indu

ctio

n o

ver

un

stim

ula

ted

sam

ples

*

Control mediumControl medium

Medium + ARGMedium + ARGand NOS inhibitorsand NOS inhibitors

Control mediumControl medium

Medium + ARGMedium + ARGand NOS inhibitorsand NOS inhibitors

ARG and NOS inhibitors induce spontaneous up-regulation of

activation markers and increase T cell viability

Bronte, V., Kasic, T., Gri, G., Gallana, K., Borsellino, G., Marigo, I., Battistini, L., Iafrate, M., Prayer-Galetti, T., Pagano, F., and A. Viola. Boosting anti-tumor responses of T lymphocytes infiltrating human prostate cancers. J. Exp. Med., 2005.

Human prostate cancer cells express ARG and NOS and through these enzymes they induce a functional paralysis of tumor-infiltrating lymphocytes (TILs). ARG and NOS inhibitors added to prostate cancer organ cultures are sufficient to restore the immune reactivity of TILs, which thus become able to recognize the autologoustumor. These findings identify the L-arginine metabolism in tumors as a dominant mechanism to restrain immune attack and open the possibility to design novel immunotherapeutic approaches.

APOPTOSIS TIA-1

Muller et al. Nature Reviews Cancer 6, 613–625 (August 2006) | doi:10.1038/nrc1929

Development of small molecule inhibitors to revert tumor-induced tolerance

Actions of NSAID-NO drugs:

1. Down regulation of iNOS/NOS2 (NO donor group)2. Down regulation of ARG (salicylate portion)3. Inhibition of leukocyte adherence to endothelial cell in tumor vessels

(guanylate cyclase-dependent)4. Inhibition of inflammatory cytokine synthesis

2 4 6 8 10 12 20 22

Tum

or s

ize

(mm

2 )

0

20

40

60

80

100

120C26GMC26GM + CD8C26GM + CD8 + IL2C26GM + NCX4016C26GM + NCX4016 + CD8

Days from tumor challenge

Day +1 to +10: NCX by gavage

Day 0: tumor

Day 1: Adoptive transfer of C26GM tumor-specificCD8+ T cells

NO-aspirin as adjuvant of anticancer vaccination:Effect on adoptively transferred CTL

LEAD

Idea/observation/blind screening

1. Rational design the lead structure modification

2. Synthesis of the designed compounds

3. Biochemical, cellular &pharmacologicalScreenings

4. SAR and structuraloptimization

Emerged Emerged lead(slead(s) ) DrugDrug--like propertieslike properties

optimisationoptimisationIn vivo studiesIn vivo studies

DRUG NO-DONOR moiety

connection by direct link connection by a spacer connection by fusion

NONO--DONOR / DRUG HYBRIDSDONOR / DRUG HYBRIDS

spacer

3,4-Dihydro-1,2-diazete 1,2-dioxides

Mesoionic oxatriazoles

NONO--DONORS DONORS

SydnoniminesOrganic nitrates

OO

O

O O

ON

O

NO

NO

Glyceryl trinitrate(GTN)

Hydroxylaminecontaining compounds

N-Hydroxyguanidines

Diazeniumdiolates(NONOates)

Furoxans

Oximes S-NitrosothiolsNitrites

NO

N-Nitroso andN-nitro compounds

C-Nitroso andC-Nitro compounds

Organic nitrites

CH3

CH3

ON

O

Amyl nitrite

NO-Metal complexes

2-

Fe

NO

CNCNCN CN

CN

Sodium nitroprusside(SNP)

N

NO

NCOOC2H5

N

O

Molsidomine

2 Na+

ON N

R R

O+

-

FUROXAN SYSTEM as NOFUROXAN SYSTEM as NO--donor donor

ON N

O

+-

thiols

pH=7.4NO

NO

cells/tissuesThiol/ascorbic acidinduced

ON N

O

+-

R

NO

electron withdrawinggroups NO

In vitro In vitro drugdrug testtest

Lymphocytes stimulated withanti-CD3 + anti-CD28 antibody

Myeloid Suppressor Cells(ONOO- producers)

A

B

Immunosuppressed lymphocytesFrom tumor-bearing mice

stimulated withanti-CD3 + anti-CD28 antibody

DRUG

No Drug

AT38

AT49

AT39

0

50000

100000

150000

200000

250000

300000

350000

10 2.5520 1.

25

Uns

tSt

im

cpm

a

conc

BALB

0

50000

100000

150000

200000

250000

300000

350000

10 2.5520 1.

25

cpm

a

Uns

tSt

im

conc

C26GM

0

50000

100000

150000

200000

250000

BALB

10 2.5520 1.

25

Uns

tSt

im

cpm

a

conc

0

50000

100000

150000

200000

250000

C26GM

10 2.5520 1.

25

cpm

a

Uns

tSt

im

conc

0

20000

40000

60000

80000

100000BALB

10 2.5520 1.

25

Uns

tSt

im

cpm

a

conc

0

20000

40000

60000

80000

100000

C26GM

Uns

tSt

im 10 2.5520 1.

25

cpm

a

conc

**

AT58

AT75

AT61

0

20000

40000

60000

80000

100000

10 2.5520 1.

25

Uns

tSt

im

cpm

a

conc

BALB

0

20000

40000

60000

80000

100000

10 2.5520 1.

25

cpm

a

Uns

tSt

im

conc

C26GM

0

20000

40000

60000

80000

100000

120000

BALB

10 2.5520 1.

25

Uns

tSt

im

cpm

a

conc

0

20000

40000

60000

80000

100000

120000

C26GM

10 2.5520 1.

25

cpm

a

Uns

tSt

im

conc

0

20000

40000

60000

80000

100000

BALB10 2.

5520 1.25

Uns

tSt

im

cpm

a

0

20000

40000

60000

80000

100000

C26GM

Uns

tSt

im 10 2.5520 1.

25

cpm

a

concconc

In vitro cytotoxicity assay:BALB/c + 3% CD11b+ cells

10µg/ml 5µg/ml 2,5µg/ml

0

20

40

60

80

% o

f ly

sis

Effector-Target ratio

Ctrl

No drugAdd 1Add 2Add 3 +3

% C

D11

b

LU 3

0/10

6ce

lls

0

35

75

105

140

Ctrl

+3%

CD

11b

Add

1

Add

2

10µg/ml 5µg/ml

Add

3

Add

1

Add

2

Add

3

AT27

In vitro screening of putative drugsCandidates#1#2#3#4#12#13#14#15#16CF1500CF1508CF1511CF1513AT27AT28AT30AT31AT32AT33AT35AT36AT37AT38AT45MC24EMC28BMC32B

Proliferation assay

#12#13

CF1508

AT27AT28CF1513

AT31

AT38 AT45MC24EMC28BMC32B

Effector function

AT27AT28

AT38 AT45

AT27AT28AT38AT45

• In vivo tests• Organ cultures

Eliciting in vivo immune responses

Day 0C26GM s.c. injection

Day 1Vaccination with

γ-irradiated CT26 cells

Day 8 Day 9Ex vivo experiments

on splenocytes

In vivo drug administration

0

20

40

60

80

% o

f ly

sis

Effector-Target ratio

Ctrl

IP 100mg/Kg/day

C26G

Msp

leno

cyte

sNo drug

IP 50mg/Kg/dayIP 25mg/Kg/day

Control splenocytes+ vaccination

C26GM splenocytes+ vaccination

C26GM splenocytes+ vaccination+ AT27 I..P. treatment

IFN-γGp70

+ CD

8+ T

cel

l

MLPC+AH1 peptide(5 days)

28.9% 1.57% 9.89%

allo-MLR

In vitro drug test: TIL activation in humanprostate cultures

CD25 CD69 CD137

MFI

fold

indu

ction

over

un

trea

ted

samples

0

1

2

3

4

5

AT27AT28

ACC PartnersIn vitro and in vivo screening of new molecules to rescue immunity against cancer in murine modelsIstituto Oncologico Veneto (IRCCS), Padua - Tumor immunology and Molecular oncology UnitGroup Leader: Vincenzo Bronte, M.D.

Chemokines and chemokines receptor in cancer: RET, CXCR4 and CCR2 as potential targets for antitumor therapyIstituto Superiore di Oncologia, NapleGroup Leader: Vecchio Giancarlo, Director

Designing and synthesis of new NO-donor drugsUniversità di Torino - Department of Drug Science and TechnologyGroup Leader: Alberto Gasco, Full Professor

Design and development of c-myc and Bcl-XL inhibitors: a way to control tumor cell proliferationIstituto Superiore di Oncologia - Sperimental Oncology Unit - University of GenoaGroup Leader: Parodi Silvio, Full Professor

Phenotypical and functional characterization of TILs in human and murine prostate cancer specimensFondazione Santa Lucia (IRCCS), RomeGroup Leader: Luca Battistini, M.D.

Advance cytofluorimetric analysis of TILs in human and murine prostate cancer specimensFondazione Santa Lucia (IRCCS), RomeGroup Leader: Giovanna Borsellino, M.D.

Defining novel molecules to rescue immunity against prostate cancer: molecular and biological bases for new therapiesIstituto Clinico Humanitas (IRCCS), MilanGroup leader: Antonella Viola, Professor

Identification of new networks and molecules involved in the activation of suppressive pathways ARG-NOS-mediated in myeloidsuppressor cellsIstituto Clinico Humanitas (IRCCS), MilanGroup Leader: Antonio Sica, Professor

Identification of new prognostic markers: evaluation of the activity of ARG-NOS drug-based therapyIstituto Europeo di Oncologia, MilanGroup Leader: Francesco Bertolini, Unit director

Effect of NO-donor molecules on tumor angiogenesis and on tumor blood vessel permeabilityFondazione Istituto FIRC di Oncologia Molecolare, MilanGroup Leader: Elisabetta Dejana, Full Professor

Integrated Tasks

Evaluate the effect of new molecules on tumor microenvironment and immune response: Gasco, Bonte, Viola, Battistini, Borsellino, Sica, Vecchio

Evaluate the effect of new molecules on NFΚB pathways in MDSC and TAMS: Sica, Bronte, Viola

Evaluate the effect of new molecules on tumor angiogenesis: Sica, Dejana, Bertolini

Synergy between new NO-donos with peptidomimeticmolecules and scFv mini-antibodies: Bronte, Viola, Parodi, Vecchio

Timetable

Complete in vitro screening of NO-donor compounds to exploit structure/function relationships

Identification of a common chemical structure for the putative adjuvant activity in order to file a patent

Protocol optimization for in vivo drug delivery in tumor-bearingmice

Recostitution of immune competence in tumor-bearing mice

Analysis of drug activity in human prostate organ cultures

In vivo evaluation of NO-donor toxicity

Combination therapy protocol

Expected timingScientific activity

It will continue until summer2009

September 2008

November 2008

January-February 2009

March 2009

June 2009

October 2009

Internalization of Anti-Myc-Int(+) scFv Antibody

The goal of Parodi O.U. will be the production of inhibitors of protein-protein interactions at the level of the higher-order-structures around c-Myc and Bcl-XL. Both peptidomimetic molecules and scFvminiantibodies are linked to peptidic motifs that make them capable of efficient cell internalization.

In collaboration with other O.U. of the project, associations with anti-inflammatory molecules, for possible synergisms, at the level of cancer therapy, will be explored.

ThyroidThyroid carcinomascarcinomas

In the framework of our project, the groups of Viola (Padua, Milan) and Melillo (ISO, Naples):- will characterize thyroid cancer inflammatory infiltrate.- will evaluate the expression levels of arginase and NOS in thyroid cancer samplesand cell lines by IHC and RT-PCR.- will test the effects of novel NO-donors on thyroid carcinoma cells.