LA RETE NAZIONALE SOLIDALE E
COLLABORAZIONI INTERNAZIONALI DEL
PROGRAMMA STRAORDINARIO ONCOLOGIA
2006
(ISS PER ACC)
Roma 21 Aprile 2008
“Sviluppo di nuovi farmaci capaci di alterare il
microambiente tumorale e ripristinare la risposta
immune anti-tumorale"
Vincenzo Bronte
Istituto Oncologico Veneto
Venetian Institute for Molecular Medicine,
Padova, Italy.
Tumor escape from immune response
Intrinsic nature:
- Absence of costimulation
- Absence of antigen
- Treg, CD4+CD25+ induction
Genetic mechanisms:
- Antigen loss/mutation
- MHC allele loss, β2-microglobulin
loss
Tumor progression
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
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 (Myeloidderived 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, cellmatrix interactions and interstitial fluid, are
preserved.
TIL FROM PCa PATIENTS DO NOT RESPOND TO STIMULI
MFI fold induction over
unstimulated samples
3.5
3.0
2.5
CD25
2.0
CD69
1.5
*
*
*
1.0
Tumor-free
prostates
PHA
PCa
Tumor-free
prostates
PCa
PMA + iono
CD137
ARG and NOS inhibitors induce spontaneous up-regulation of
activation markers and increase T cell viability
*
2.5
2.0
*
1.5
*
1.0
Control medium
Medium + ARG
and NOS inhibitors
0.5
0
Viability index (ratio)
Fold of induction over
unstimulated samples
3.0
CD25
CD69
4.0
*
3.0
Control medium
Medium + ARG
and NOS inhibitors
2.0
1.0
0
CD137
CD8
APOPTOSIS
TIA-1
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 autologous
tumor. 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.
Development of small molecule inhibitors to revert tumor-induced tolerance
Muller et al. Nature Reviews Cancer 6, 613–625 (August 2006) | doi:10.1038/nrc1929
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
NO-aspirin as adjuvant of anticancer vaccination:
Effect on adoptively transferred CTL
120
C26GM
C26GM + CD8
C26GM + CD8 + IL2
C26GM + NCX4016
C26GM + NCX4016 + CD8
Tumor size (mm2)
100
80
60
40
20
0
2
4
6
8
10
12
20
Days from tumor challenge
Day 0:
tumor
Day 1:
Adoptive transfer of
C26GM tumor-specific
CD8+ T cells
Day +1 to +10:
NCX by gavage
22
Idea/observation/
blind screening
LEAD
4. SAR and structural
optimization
1. Rational design the
lead structure
modification
Emerged lead(s)
Drug-like properties
optimisation
In vivo studies
3. Biochemical,
cellular &
pharmacological
Screenings
2. Synthesis of the
designed compounds
NO-DONOR / DRUG HYBRIDS
DRUG
NO-DONOR moiety
spacer
connection by direct link
connection by a spacer
connection by fusion
NO-DONORS
N-Hydroxyguanidines
Nitrites
Oximes
Diazeniumdiolates
(NONOates)
S-Nitrosothiols
R
Hydroxylamine
containing compounds
R
+
N
N
O
O
-
Furoxans
NO
C-Nitroso and
C-Nitro compounds
Mesoionic oxatriazoles
3,4-Dihydro-1,2-diazete
1,2-dioxides
N-Nitroso and
N-nitro compounds
Organic nitrates
Organic nitrites
NO-Metal complexes
Sydnonimines
O
N
O
O
O
CH3
O
O
O
N
O
N
N
O
O
Glyceryl trinitrate
(GTN)
O
CH3
NO
CN Fe CN
CN
CN
CN
O
2-
N
2 Na+
N
N
O
Amyl nitrite
Sodium nitroprusside
(SNP)
NCOOC2H5
Molsidomine
FUROXAN SYSTEM as NO-donor
thiols
+
N
N
O
Thiol/ascorbic acid
induced
O
-
NO
pH=7.4
cells/tissues
R
NO
+
N
N
O
O
electron withdrawing
groups
-
NO
NO
In vitro drug test
A
Lymphocytes stimulated with
anti-CD3 + anti-CD28 antibody
Myeloid Suppressor Cells
(ONOO- producers)
No Drug
B
D
R
U
G
Immunosuppressed lymphocytes
From tumor-bearing mice
stimulated with
anti-CD3 + anti-CD28 antibody
BALB
350000
300000
300000
250000
200000
250000
200000
150000
100000
100000
50000
50000
0
0
20
10
5
2.5
1.25
Unst
Stim
150000
conc
conc
BALB
C26GM
250000
200000
200000
cpma
250000
150000
150000
100000
100000
50000
50000
**
conc
conc
BALB
C26GM
100000
80000
80000
60000
40000
20000
20000
0
0
20
10
5
2.5
1.25
40000
conc
20
10
5
2.5
1.25
60000
Unst
Stim
cpma
100000
Unst
Stim
cpma
AT49
20
10
5
2.5
1.25
0
Unst
Stim
Unst
Stim
0
20
10
5
2.5
1.25
cpma
AT39
20
10
5
2.5
1.25
cpma
350000
Unst
Stim
cpma
AT38
C26GM
conc
C26GM
100000
80000
80000
60000
40000
20000
20000
0
0
20
10
5
2.5
1.25
Unst
Stim
40000
conc
conc
BALB
120000
100000
100000
80000
60000
80000
60000
40000
20000
20000
0
0
20
10
5
2.5
1.25
Unst
Stim
40000
conc
conc
BALB
C26GM
100000
80000
80000
60000
60000
40000
20000
20000
0
0
20
10
5
2.5
1.25
40000
conc
Unst
Stim
cpma
100000
Unst
Stim
cpma
AT75
20
10
5
2.5
1.25
cpma
120000
Unst
Stim
cpma
AT61
C26GM
20
10
5
2.5
1.25
60000
20
10
5
2.5
1.25
cpma
100000
Unst
Stim
AT58
cpma
BALB
conc
In vitro cytotoxicity assay:
BALB/c + 3% CD11b+ cells
5µg/ml
2,5µg/ml
Ctrl
No drug
Add 1
60
Add 2
40
Add 3
20
140
105
75
35
10µg/ml
Add 3
Add 2
Add 1
Add 3
Add 2
Add 1
0
+3% CD11b
Effector-Target ratio
Ctrl
0
LU 30/106 cells
% of lysis
80
+3% CD11b
10µg/ml
5µg/ml
AT27
In vitro screening of putative drugs
Candidates
#1
#2
#3
#4
#12
#13
#14
#15
#16
CF1500
CF1508
CF1511
CF1513
AT27
AT28
AT30
AT31
AT32
AT33
AT35
AT36
AT37
AT38
AT45
MC24E
MC28B
MC32B
Proliferation assay
Effector function
#12
#13
AT27
AT28
AT38
AT45
CF1508
AT27
AT28
CF1513
AT27
AT28
AT31
AT38
AT45
MC24E
MC28B
MC32B
AT38
AT45
•
•
In vivo tests
Organ cultures
Eliciting in vivo immune responses
Day 0
C26GM s.c. injection
Day 1
Vaccination with
γ-irradiated CT26 cells
Day 8
Day 9
Ex vivo experiments
on splenocytes
In vivo drug administration
Ctrl
No drug
60
IP 100mg/Kg/day
IP 50mg/Kg/day
40
IP 25mg/Kg/day
MLPC+AH1 peptide
(5 days)
20
0
Effector-Target ratio
Gp70+ CD8+ T cell
% of lysis
allo-MLR
C26GM
splenocytes
80
28.9%
Control splenocytes
+ vaccination
1.57%
9.89%
IFN-γ
C26GM splenocytes
+ vaccination
C26GM splenocytes
+ vaccination
+ AT27 I..P. treatment
In vitro drug test: TIL activation in human
prostate cultures
MFI fold induction over
untreated
samples
5
4
3
AT27
AT28
2
1
0
CD25
CD69
CD137
ACC Partners
In vitro and in vivo screening of new molecules to rescue immunity against cancer in murine models
Istituto Oncologico Veneto (IRCCS), Padua - Tumor immunology and Molecular oncology Unit
Group Leader: Vincenzo Bronte, M.D.
Chemokines and chemokines receptor in cancer: RET, CXCR4 and CCR2 as potential targets for antitumor therapy
Istituto Superiore di Oncologia, Naple
Group Leader: Vecchio Giancarlo, Director
Designing and synthesis of new NO-donor drugs
Università di Torino - Department of Drug Science and Technology
Group Leader: Alberto Gasco, Full Professor
Design and development of c-myc and Bcl-XL inhibitors: a way to control tumor cell proliferation
Istituto Superiore di Oncologia - Sperimental Oncology Unit - University of Genoa
Group Leader: Parodi Silvio, Full Professor
Phenotypical and functional characterization of TILs in human and murine prostate cancer specimens
Fondazione Santa Lucia (IRCCS), Rome
Group Leader: Luca Battistini, M.D.
Advance cytofluorimetric analysis of TILs in human and murine prostate cancer specimens
Fondazione Santa Lucia (IRCCS), Rome
Group Leader: Giovanna Borsellino, M.D.
Defining novel molecules to rescue immunity against prostate cancer: molecular and biological bases for new therapies
Istituto Clinico Humanitas (IRCCS), Milan
Group leader: Antonella Viola, Professor
Identification of new networks and molecules involved in the activation of suppressive pathways ARG-NOS-mediated in myeloid
suppressor cells
Istituto Clinico Humanitas (IRCCS), Milan
Group Leader: Antonio Sica, Professor
Identification of new prognostic markers: evaluation of the activity of ARG-NOS drug-based therapy
Istituto Europeo di Oncologia, Milan
Group Leader: Francesco Bertolini, Unit director
Effect of NO-donor molecules on tumor angiogenesis and on tumor blood vessel permeability
Fondazione Istituto FIRC di Oncologia Molecolare, Milan
Group 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 peptidomimetic
molecules and scFv mini-antibodies: Bronte, Viola, Parodi,
Vecchio
Timetable
Scientific activity
Expected timing
Complete in vitro screening of NO-donor compounds to exploit
structure/function relationships
It will continue until summer
2009
Identification of a common chemical structure for the putative
adjuvant activity in order to file a patent
September 2008
Protocol optimization for in vivo drug delivery in tumor-bearing
mice
November 2008
Recostitution of immune competence in tumor-bearing mice
January-February 2009
Analysis of drug activity in human prostate organ cultures
March 2009
In vivo evaluation of NO-donor toxicity
June 2009
Combination therapy protocol
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 scFv
miniantibodies are linked to peptidic motifs that make them capable of efficient cell internalization.
In collaboration with other O.U. of the project, associations with antiinflammatory molecules, for possible synergisms, at the level of cancer
therapy, will be explored.
Thyroid carcinomas
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 samples
and cell lines by IHC and RT-PCR.
- will test the effects of novel NO-donors on thyroid carcinoma cells.