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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.