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Recent advances in the medical treatment of melanoma
Roma 21/06/2013
Pre-clinical and clinical
aspects of peptide-based
melanoma vaccines
Giorgio Parmiani
San Raffaele Foundation, Milano
Tumor destruction by the immune system
(are T cells the main player?)
NK/NKT
TCD8
Monocytes, DC
c
IL-2, IFN, TNF...
B ly
Tumor-specific
antibodies
TCD4
IL-2
• The Melanoma-Associated
Antigens
Melanoma-associated antigens (MAAs)
recognized by T cells. Which is the best
MAA or combination of?
1. Shared/self/differentiation MAAs (e.g.
Mart1, tyrosinase)
2. Shared/self/cancer testis or germinal
MAAs (e.g.MAGE, NY-ESO1)
3. Universal MAAs (survivin, hTERT)
4. Mutated, unique MAAs
• These different MAAs have been used
as immunogens in clinical trials.
• Which was the outcome in terms of
toxicity, immune response and clinical
response?
• The immunogenicity
1-2. Shared self MAAs
• Normal subjects and cancer patients show some form of
tolerance to “self” MAAs (immune ignorance, peripheral
or central tolerance, low frequency of T cell precursors, T
regs).
• Tolerance needs to be broken (spontaneously or not)
in order to elicit a T cell immune response against “self”
MAAs.
• Thus, these MAAs are considered to be “weak antigens”
though despite being the first to be molecularly
characterized and used in patients
In vivo spontaneous tolerance
break (immunogenicity) of
shared self MAAs.
2 examples
FREQUENCY OF ANTI-MAA CD8 T LYMPHOCYTES
AND PROGRESSION OF THE DISEASE
gp100209-217
3.3
2.8
2.3
1.8
1.3
0.8
0.3
n.s.
***
Tyrosinase368-377
***
3.3
2.8
2.3
1.8
1.3
0.8
0.3
n.s.
***
**
Melan-A/Mart-126-35
3.3
2.8
2.3
1.8
1.3
0.8
0.3
0.3
0.3
0.3
0.2
0.2
0.2
0.1
0.1
0.1
0.0
0.0
0.0
Donors
Donors
Patients
**
***
***
Donors
Patients
Patients
CONCLUSIONS
•
A hierarchy exists in the spontaneous
recognition of “self” TAAs.
•
Recognition of “self” TAAs increases with
the increased tumor burden (e.g. MelanA/MART1)
Break of tolerance to shared
self TAAs by vaccination
Peptide-based vaccines
• Advantages:
• Sequence and biochemical features are
known
• Easy to synthesize (large availability)
• Allow a specific immune-monitoring of the
patient response to vaccine
• Allow assessing the expression of targeted
TAA in patients’ tumor cells
Peptide-based vaccines (cont.)
• Disadvantages:
• Easy degradable in absence of adjuvants
• Require appropriate HLA allele to be
recognized by T cells (patient selection)
• Induce T cells that may not recognize
tumor cells
• Costs (40-160’000€/each)
Results of first generation (1998-2006) of self
peptide-based vaccination of metastatic
melanoma patients (Phase I/II studies).
N. of
Clinical response
Immune response
Type of
peptide MAA patients (CR+PR) (mean %) (%)
Lineage related
(e.g. Melan-A)
159
14
20-65
Cancer/Testis
(e.g. MAGE)
92
17
30-50
DC peptides
124
16
56
DC lysates
106
18
46
In a recent study Slingluff et al. (2008) reported 100% immune response
and survival benefit in melanoma patients vaccinated with 12 peptides.
Vaccination with MAGE3.A1
peptide
Multicentric European Study
Ludwig Institute,Brussels; INT and HSR, Milano
Adapted from M.Marchand et al., Int.J. Cancer 1999
14
Use of dendritic cells:
which progress ?
see Ridolfi’s presentation
Disappointment followed by a
drop in the interest in new
studies
• Which are the reasons of such negative
clinical results of active immunotherapy of
cancer?
Reasons for the limited clinical
response
Factors that interfere with the T cellmediated anti-tumor response
Tumor (Immunosubversion)
• Lack of antigen or downregulation of HLA
• Dysfunction of antigen
presentation
• Release of immunesuppressive factors (IL-10,
TGFβ, VEGF)
• Tumour counterattack
(Fas/FasL)
• IDO, SPARC
• Expression of FoxP3, CTLA4
• Tumor ER stress
• NFAT1, Exosomes
Immune system
• Immune anergy or ignorance
• Lack of tissue homing
molecules; defective
adhesion
• T-cell receptor dysfunction
• Inactivation of T-cells within
the tumor environment
(granzyme B)
• T-regulatory cells
• MDSC
• Epithelial/mesenchimal
transition
• Tie+ Monocytes dysfunction
• …but, despite this long list of obstacles,
under some conditions immune cells can
manage to find and sometime destroy
cancer cells.
• The understanding of the escape
mechanism helps to inhibit or downregulate them in the clinic thus improving
efficacy.
• Possible examples
% CD14+CD11b+HLA-DR-/lo
Myeloid suppressor cells are increased
in peripheral blood of stage II-III melanoma patients
20
N=30
p<0.01
15
p<0-01
N=40
10
5
N=40
0
HD
STAGE IIBIIIC
STAGE IV
MELANOMA
p= 0.0002
15
n.s.
10
p= 0.002
p=0.04
5
e
ag
II
IA
e
ag
St
St
-I
II
C
II
C
II
Be
ag
St
IV
0
H
D
+
%CD4 CD25
high
Foxp3
+
Increased frequency of CD4+CD25Foxp3+ Treg
is a late event in melanoma patients
10
10
(gated on lymphocytes)
% CD4+CD25highFoxP3+ cells
Monitoring T regs : unpredictable effect of low-dose
cyclophosphamide on the frequency of CD4+CD25highFoxp3+
IIB-IIIC HIGH RISK MELANOMA PATIENTS
88
300mg/m2 3-5 days
before vaccination
cyclophosphamide
6
6
4
4
2
2
0
0
HD
P0
P1
VACCINATION ARM
P4
cyclophosphamide
• Cyclophosphamide is effective in a
different trial with RCC patients (Walter et
al., Nature Med 2013)
Multipeptide
vaccination
In RCC patients
de010203040020406080100Time
(months)Percentage survival3+Cy 1+Cy 0–Cy 0–
Cy 1210010203040020406080Percentage
survival100Time
(months)bc0246810020406080100Time
(months)Percentageprogression-free survival+
Conclusions
1. Myeloid-derived suppressor cells are
increased
in the peripheral blood of stage II-III
melanoma patients
2. Increased frequency of CD4+CD25FoxP3+
Tregs is a late event in melanoma patients
Not clear the effect of low-dose
cyclophosphamide on the frequency of
CD4+CD25+Foxp3+
3.
Recent progress in cancer vaccines.
The Renaissance of cancer
immunotherapy
: Phase II-III positive randomized trials:
• Melanoma (gp100)
• B cell lymphoma
• Prostate cancer (Sipuleucel, Provenge)
• NSCLC (MAGE)
• Renal Cell Carcinoma (IMA901)
Phase III study of gp100 peptide
vaccine in melanoma
• A phase III multi-institutional randomized
study of immunization with the gp100
(210M) peptide followed by high-dose of
IL-2 compared with high dose IL-2 alone in
patients with metastatic melanoma.
• Schwartzentruber et al. NEJM 2011
Phase III study of gp100 peptide
vaccine (cont.)
• 21 centers; total of 185 patients
• Stage IV or locally advanced stage III,
HLA-A*0201
• Therapy. IL-2: 720’000IU/kg/dose+/Gp100 (210M) peptide+Montanide
• Results. High toxicity (IL-2); RR 22.1% vs.
9.7% (P=0.0223); PFS: 2.9 vs. 1.6 mos
(P=0.010); Median OS: 17.6 vs. 12.9 mos
(P=0.096)
EVIDENCE FOR CLINICAL ACTIVITY OF
CANCER VACCINES
Vaccine
Tumor
Phase
N.
patients
Stage
Statistics
MAGE-3
NSCLC
II R
182
IB-II
Trend
IDM-2101
NSCLC
II
63
IIIB.IV
NA
IL2+/gp100
Melanoma
III
185
IV
P<0.02
Provenge
DC
Prostate
Cancer
III
341/171
HR
P<0.03
E75/Her2/n Breast
eu
cancer
IIR
101/75
IV
P<0.04
DC/NHL
NHL
II
18
3CR,3PR,8
SD
BiovaxID
Follicular
Lymphoma
III
76/41
P<0.04
IMA901
RCC
II R
96
advanced
P<0.02
Ongoing Phase II trials of active cancer
immunotherapy: number of trials by immunotherapy
and cancer type
Breast
Breast
Gastrointestinal
Colorectal
Type of active
immunotherapy
Other
Genitourinary
Autologous T-cell/
lymphocyte infusion
Prostate
Renal
Gynecologic
Dendritic cell
Ovarian
Other
Hematologic
DNA/RNA
Leukemia
Lymphoma
Peptide
Multiple myeloma
Neurologic
Recombinant
microbial
Glioblastoma
Other
Respiratory
Tumor cell
Lung
Other
Skin
Melanoma
0
5
10
15
20
Number of ongoing Phase II trials
25
30
Self peptide-based melanoma
vaccines: Conclusions
• A phase III studys showed statistically
significant benefit for vaccinated patients.
• The use of a) multipeptides, b) patients
with limited tumor burden, c) a
concomitant modulation of immune
suppressive cells, d) combination of
chemotherapy/anti-angiogenic agents and
vaccines are providing new and promising
clinical results.
New targets in cancer
vaccination
• Stroma: a site of complex and often
immune inhibitory interactions among
tumor cells, infiltrating lymphocytes,
macrophage and fibroblast and different
soluble factors (e.g. chemokines).
• Cancer stem cells
The role of stroma
• Stromal cells take up tumor-derived exosomes
becoming susceptible (H. Schreiber) or resistant ( G.
Parmiani, L. Rivoltini) to CTL.
• Endothelial cell antigens (e.g.VEGFR-2; D.Schadendorf).
• Fibroblast antigens
• Inflammatory/Immune cells (Tcells, DCs, Monocytes,
MDSC, mast cells?)
• Chemokines (CCL2-5, -19,-21; CxCL9-13; T. F.
Gajewski)
• Aim: Altering the tumor stroma to the benefit of the
host
Cancer stem cells: A new target
of immunotherapy?
• Objectives: Identification of molecules
with immunological relevance expressed
by cancer stem cells and validation of their
role as target molecules of
immunotherapy.
• See Ruggero De Maria presentation?
Rationale for a new generation of
cancer vaccines
•
•
•
•
Early disease
Multiple antigens
New TLR targeting adjuvants (CpG, HSPs)
Down-regulation of Tregs and/or Myeloid Derived
Suppressor Cells
• Immune-monitoring in blood, LNs and tumor tissue.
• Assessment of patient polymorphisms and tumor
gene signatures
• Combination with other therapeutic
agents
• Combination with other
therapeutic agents (e.g. antivascular agents)
Targeted delivery of TNF to tumor vasculature
by coupling with the NGR-peptide
(a ligand of CD13 expressed by angiogenic vessel)
NGR-TNF
TNF
CNGRCG peptide
The antitumor activity of NGR-TNF is >10 times greater than that of TNF
in several mouse models
Curnis et al., Nat. Biotechnol., 2000
Sacchi et al., Clin. Cancer Res. 2006
Combination of vaccine and NGR-TNF in animal models
NGRTNF
% Surviving Animals
DC/OVA
DC/OVA
NGR-TNF
100
80
60
PBS
40
NGR-TNF
DC/OVA
20
DC/OVA + NGR-TNF
0
7
14
21
28
Day since Tumor Challenge
35
PBS (n=6) vs NGR-TNF (n=5): NS
NGR-TNF vs DC/OVA: NS
PBS vs DC/OVA (n=5): NS
NGR-TNF vs DC/OVA+NGR-TNF: p = 0.0061
PBS vs DC/OVA+NGR-TNF (n=6): p = 0.0035
DCOVA vs DC/OVA-NGR-TNF: NS
Calcinotto et al. J Imunology 2012
• A pilot phase I and II study of NGRhTNF in combination with a peptidebased vaccine (MAGE.A3, NA17.A2) for
metastatic melanoma (NGR/Vax/01)
• PI: Giorgio Parmiani, MD
Inclusion criteria (cont.)
• HLA-A*0201- and/or HLA-A1-positive
typing on PBMCs.
• Melanoma cells expressing the NA17.A2
or MAGE-3.A1 melanoma antigens in
HLA-A2 or HLA-A1 patient, respectively,as
defined by PCR analysis on frozen or
paraffinized tissue.
NGR/Vax/01. Early results in the
clinic
• Phase I-II of combination of NGR-hTNF
and melanoma peptide vaccine
• 9 patients enrolled;
• No SAE ; grade I-II local erythema, fever,
chills .
• Ex-vivo immune response to vaccine 6/8;
• Clinical response 5 Long Term SD (4, 5, 8,
11, 15+, 16+, 17+, 22+)
Antigens recognized by T cells:
1) Shared but expressed on tumor cells only
2) Shared Cancer/Testis expressed by different
tumors and by normal testis or placenta
3) Shared, differentiation proteins expressed also
on normal cells
4) Unique, mutationderived,expressed only by a single
tumor (e.g. CDK4/m, α-actin-m)
Proteins derived from somatically mutated cancer
genes give raise to neo-peptide antigens
presented by MHC molecules
neopeptide
neopeptide
A reverse immunology approach to go from
mutated cancer genes to neo-peptide antigens
and specific T cell immunity
Massive sequencing of CAN genes ->
Somatically mutated TAAs
Bioinformatic epitope prediction
(peptides)
Investigation of autologous
T cell responses in vitro/ex vivo
Clinical trial vaccination
PREDICTED EPITOPES DERIVED FROM APC MUTATION MISSENSE A3079T (Y->L)
BINDING SOME RAPRESENTATIVE HLA-A ALLELES
MUTATED SEQ
HLA-A*01
DTPINLSLKY
ELDTPINLSL
HLA-A*01 11
LDTPINLSLKY
ELDTPINLSLK
HLA-A*0201
ELDTPINLSL
NLSLKYSDEQ
HLA-A*03
NLSLKYSDE
TPINLSLKY
LDTPINLSLK
ELDTPINLSL
HLA-A*1101
DTPINLSLK
HLA-A*26
DTPINLSLKY
ELDTPINLSL
DGELDTPINL
HLA-A*6801
DTPINLSLK
score
W.T. SEQ score
25
14
DTPINYSLKY
ELDTPINYSL
25
11
20
14
LDTPINYSLKY
ELDTPINYSLK
20
13
20
12
ELDTPINYSL
NYSLKYSDEQ
20
2
15
13
16
15
NYSLKYSDE
TPINYSLKY
LDTPINYSLK
ELDTPINYSL
5
13
16
12
22
DTPINYSLK
22
28
20
17
DTPINYSLKY
ELDTPINYSL
DGELDTPINY
28
19
19
24
DTPINYSLK
20
The maximal score for peptides binding HLA-A*0201 is 36.
Influenza A matrix GILGFVFTL scores 30
Vaccination with unique TAAs.
A new genomic strategy
• Cancer cells contain somatic mutations
detectable by new sequencing technology
(Sjoblom et al., Science 2006; Parsons et al., Science
2008). These mutations can generate candidate
new T cell epitopes.
• Breast and colorectal cancers may accumulate
7-10 new HLA-A*0201 epitopes that may define
an individual tailored polyvalent vaccine (Segal et
al., Cancer Res 2008)
• Driver mutations/each melanoma in 120
genes
P. Robbins Trial?
• At the moment I am not aware of any
ongoing cancer vaccination program
based on individually mutated cancer
gene-products but I know that several
research groups are pursuing this idea.
Acknowledgments
• Financial support:
•
•
•
•
AIRC, Milano
Alleanza contro il Cancro (ACC), Rome
Italian Network for Biotherapy of Tumors (NIBIT), Siena
European Commission (ATTACK)
• Collaborators:
• Cristina Maccalli, Vincenzo Russo, Paolo Dellabona,
Gloria Sovena, (San Raffaele Scientific Institute, Milano)
• Licia Rivoltini, Chiara Castelli, Mario Santinami, (National
Tumor Institute, Milano)
• END OF THE PRESENTATION.
• Thank you for your attention!
• A pilot phase I and II study of NGRhTNF in combination with a peptidebased vaccine for metastatic
melanoma.
• PI: Giorgio Parmiani
Expression of MHC and APM molecules and NKG2DLs in GBM-derived CSCs
and FBS tumor cells
• The expression of:
MHC class I and II;
Antigen processing machinery (APM), using 21
different mAbs directed against HLA molecules, their
heavy chains, b2-microglobulin immunoproteasome,
constitutive proteasome subunits, chaperon molecules,
TAPs etc.;
NKG2DLs;
has been tested in 11 different GBM CSCs and, for 5 of
them, in their paired tumor cells grown in the presence
of FBS (FBS tumor cells).
Antigens recognized by T cells:
1) Shared, self differentiation proteins expressed also on normal
cells (e.g. MART-1, CEA, PSA)
2) Shared self Cancer/Testis expressed by different tumors and
by normal testis or placenta (e.g. MAGE, NY-ESO-1)
3) Shared, universal TAAs predominantly
expressed by tumor cells (e.g. hTERT,
survivin)
4) Unique, expressed only by a single tumor
(e.g. CDK4/m, α-actin-m)
Exploiting known frequently somatically mutated
CAN genes in CRC
- From massive sequencing of each exons of about
20.000 protein-coding genes
- In 22 + 48 (only some genes) tumor samples
- Identified 142 genes that bear non-synonymous
mutations with driver characteristics
- On average of about 15 CAN-genes are mutated
in each CRC
Wood LD et al Science 2007
Massive parallel pyrosequencing
Roche 454 GS-FLX
Obtain up to 500Mb per run with between one and sixteen samples
300~500bp average read length, in 10 hours
20 most frequently mutated CAN-gene in CRC
APC
KRAS
TP53
PIK3CA
FBXW7
CSMD3
TNN
NAV3
SMAD4
EPHA3
MAP2K7
EPHRB6
PTEN
ADAMTSL3
GUCY1A2
SMAD2
OR51E1
LAMA1
C10orf137
TCF7L2
Mutated in 100% to 20% of CRCs
Mutated in fewer than 5% of CRCs
Most frequently mutated CAN genes are
associated with colorectal tumorigenesis
Jones S. et.al. PNAS 2008
Somatically mutated CD4 and CD8 epitopes may
not only display improved affinity for MHC
but also for TCR binding
neopeptide
neopeptide
Risultati e conclusione del primo
anno di attività.Studi pre-clinici
• Sono state identificate 9 sequenze di SVV e
sintetizzati i peptidi sintetici corrispondenti.
• I peptidi sono stati utilizzati in vitro per stimolare,
in colture a breve termine, linfociti T CD4+ isolati
dal sangue di 7 soggetti normali e 7 pazienti
con melanoma metastatico.
• Linfociti CD4 di 2/7 pazienti e 4/7 donatori hanno
risposto. Nel caso dei pazienti con melanoma la
reattività anti-SVV dei linfociti T CD4+ appare
quindi ridotta rispetto a quella dei soggetti sani.
Risultati del primo anno di attività.
Studi pre-clinici
• Unità di Biologia dei Tumori e Targeting
Vascolare HSR (A. Corti);
• Obbiettivo: Verificare se la combinazione
di NGR-TNF con vaccinazione può
indurre, in modelli animali, una più forte e
duratura risposta immune e una migliore
risposta clinica in confronto alla sola
vaccinazione .
Vaccination of metastatic patients with mutated peptide
HSPPC-96 derived from the autologous tumor
Resection
Tumor
sample
Purification of
HSP-GP96
Tumor cell
suspension
Immunologic monitoring
in vivo: DTH
in vitro: ELISPOT, tetramers staining
Vaccination
Phase I/II clinical studies of
vaccination with HSPPC-96
1. Liver metastases of colon
cancer (29 patients)
2. Metastatic melanoma
(39 patients)
3.
Metastatic melanoma
(HSPPC-96 + GM-CSF + IFN,
30 patients)
Regressioni complete di metastasi da melanoma in seguito a
vaccinazione con HSP96
Prima del vaccino
12 mesi dopo
34 mesi dopo
da Belli et al. J Clin Oncol., 2002
66
In vivo expansion of anti-MART-1 T cells in a
CR patient (02-003) vaccinated with HSPPC-96
HLA/MART-1 tetramer
Day 1--7--14--21---------------49--------------------------63
V1
V6
V7
0.31
2.81
CD8
CD8
4.42
CD8
*
400
T2 alone
300
T2+Mart-1
*
N. spots/5000 cells
T2+gp100
200
501mel
colo705
*
*
100
0
V1
V6
V7
Rivoltini et al.
J Immunol 2004
Tumor-derived HSPPC-96 mediate cross-presentation
by HLA-A*0201+ monocytes of peptides from
know tumor antigens to specific T cell clones
Anti-MART-1 HLA-A2-restricted CD8+ T cell clone
HSPPC-96-colon ca
HSPPC-96-501mel
CEA peptide
14%
0.9%
5.3%
IFN
1.3%
MART-1 peptide
CD8
CD8
CD8
CD8
Anti-CEA HLA-A2 restricted CD8+ T cell clone
HSPPC-96-colon ca
HSPPC-96-501mel
2%
MART-1 peptide
1.5%
12%
IFN
73%
CEA peptide
CD8
CD8
CD8
CD8
PHASE III RANDOMIZED TRIAL OF AUTOLOGOUS
TUMOR-DERIVED HSPPC-96 vs. PHYSICIAN CHOICE
IN METASTATIC MELANOMA (Testori A, Parmiani G
et al. JCO 2008)
• Trial features: Randomization 2:1 favoring vaccination (215 vs. 107
patients). Physician choice included IL-2 and/or
dacarbazine/temozolomide-based therapy and/or surgery
• Results. Overall, patient in ITT vaccination arm fared similarly to
those in the physician choice arm in terms of survival.
. Subset of patients who received at least 10 doses of vaccine showed
an extension in median survival of 29% compared with those
receiving physician choice treatment.
Oncophage® was associated with clinical benefit (P = 0.017) in
stage M1a and M1b patients who received at least 10 doses.
• Conclusion: Signs of potential survival benefit in M1a/b patients.
Potential therapeutic targets for
metastatic melanoma
• Target
• BRAF
• RAS
•
•
•
•
•
•
VEGF
CTLA4
PI3K/AKT
PTEN loss
BCL2
PTK
•
•
•
•
•
•
•
•
•
•
Agent
Sorafenib
Farnesyl transferase
inhibitors
Bevacizumab
Anti-CTLA4 (Ipilimumab)
CCI-779
17-AAG
Antisense (Oblimersen)
Gleevec
Trial
+
+
+
+
+
+
+
+
With the possible exception of Anti-CTLA$ none of the trials showed
a statistically significant clinical benefit.
Phase I/II studies of DC-peptide based
therapeutic vaccines in melanoma
DC+ antigen
No.Pts
Responses
Immunologic
Clinical
Ref.
Peptides1 or lysate
32
11
2 CR, 6 PR
Nestle 2001
HLA-A2 peptides1
18
16
3 CR
Banchereau 2001
Tumor lysate
11
5
1 PR
Mulé 2002
Tumor lysate
17
5
2 CR, 3PR
Ridolfi, 2006
MAGE-DR
16
15
1 CR
Schuler 2002
Gp100, tyrosinase
12
1/9
1 PR
Slingluff 2003
Melan-A + IL-12
20
5
2 CR
Gajewski 2003
Gp100-9V
12
12
2 PR
Haluska 2005
Allo-Tumor cells
13
3
1CR, 1PR
Total
151
73 (51%)
1
Peptides included MART-1, MAGE, tyrosinase, gp100.
25 (13%)
Banchereau 2006
DC-peptide based cancer
vaccines
• Ex-vivo DCs+ tumor cell lysate/apoptotic
cells
• Ex-vivo DCs + purified MAA (proteins,
peptides)
• DC+mRNA
• DCs Gene-modifed to express MAA
DCs delivered ex-vivo (Ab-mediated) or in vitro
Phase III prospective trial with
Sepuleucel-T in prostate cancer
• Vaccine: Autologous DC pulsed with
PAP/GM-CSF hybrid protein.
• Statistically significant benefit in survival at
3 years of observation.
Publications
•
Sacchi et al. Phase I Study of NGR-hTNF, a
Selective Vascular Targeting Agent, in
Combination with Cisplatin in Refractory Solid
Tumors. Clin Cancer Res 2011; 17:1964;
Calcinotto A, Grioni M, Jachetti E, Curnis F, Mondino A,
Parmiani G, Corti A, Bellone M. Targeting tumor
necrosis factor–α to neoangiogenesis vessels
enhances lymphocyte infiltration in tumors and
increases the therapeutic potential of immunotherapy.
J Immunol, 2012; 188: www.jimmunol.org.
Reactivity against CSC or FBS tumor cell lines in autologous setting by T lymphocytes
isolated from 4 GBM patients
+: ÅÜ50 < 100 spots/ 4x104 T cells; ++: ÅÜ100 < 200 spots/ 4x10 4 T cells; +++: ÅÜ200 < 800 spots/ 4x104 T cells;
N.A.: FBS tumor cells are not available;
Immunobiological differences between CSC and FBS tumor cell lines.
CSCs
FBS
CSCs
FBS
a: modulation of the expression of MHC, NKG2D and APM molecules following in vitro treatment of the cells with either IFN- or-;
b: modulation of the expression of MHC, NKG2D and APM molecules following in vitro treatment of the cells with the demethylating
agent 5-Aza.-(CdR).
CSCs
Conclusions
A Low immunogenic profile was found in both
CSCs and FBS tumor cells isolated from GBM
patients, with higher defective APC pattern in
CSCs;
the immune profile can be rescued, though more
efficiently in FBS tumor cells, by treatment of GBM
cell lines with IFNs or 5-Aza-CdR ;
T cell-mediated immune responses can be
obtained from GBM patients, though mostly in the
TH2-mediated subset;
Differential gene signature, including immune
related genes, was detected in CSCs vs FBS tumor
cells.
Reclutamento pazienti
1. Disponibilità dei pazienti in rapporto a
trattamenti competitivi (es. GSK1021202 e
Vemurafenib nel melanoma);
2. Complessità del trattamento
(interdisciplinarietà degli operatori, ecc.) e
costi.
Inclusion criteria
• Histologically confirmed AJCC (modified) stage
IV melanoma;
• ECOG performance status 0-1;
• Life expectancy of at least 6 months;
• Hematopoietic, liver and renal normal functions
• No brain mets
• Signed written informed consent.
•
Obiettivi e Razionale
• Il progetto è basato sull’attivazione di studi
clinici di combinazione tra farmaci biologici
e vaccinazione con peptidi antigenici, oltre
a studi associati o paralleli di laboratorio
destinati: 1) a migliorare le conoscenze sui
meccanismi alla base della combinazione
proposta; 2) al monitoraggio
immuno/biologico dei pazienti; c) a
valutare polimorfismi di geni che possono
influire sulla risposta al trattamento.
Immune response as assessed
by 4 different assays
• Tabella (Silvia)
Criticità per il trasferimento
clinico (cont.)
•
Scarsa partecipazione industriale dovuta
a difficoltà di standardizzazione della
combinazione di farmaci e brevettabilità
(fornitura gratuita del farmaco NGRhTNF da parte di MolMed, Milano).
Somatically mutated tumor antigens:
Best candidates with limitations
1. Somatically mutated TAAs are in principle the best antigens
to use for cancer immunotherapy;
2. Surprisingly, still a limited number of such antigens have
been identified.
Cumbersome, lengthy and poorly processing
approaches for the identification of mutated TAAs (shared
or unique) have prevented as yet their massive characterization
3. As a consequence, the T cell responses specific for mutated
TAAs remains largely undefined
Somatic mutations in CAN genes fulfil two critical
requirements for cancer immunotherapy
1. They involve driver mutations:
– causally implicated in cancer development.
– confer growth advantage.
– positively selected in the microenvironment of the tissue in
which the cancer arise
This is relevant to avoid immune escape
2. They (at least some) seem to be expressed by cancer
stem/initiating cells
Probability to generate novel unique class I
epitopes from somatically mutated
CAN genes in CRC
- Individual CRC accumulate, on
average, 10 novel and unique HLAA*0201 epitopes, including genes
implicated in the neoplastic process.