Corso CINBO 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 Tumor destruction by the immune system (are T cells the main player?)(are T cells the main player?)
cTCD8
TCD4
IL-2, IFN, TNF...
IL-2
B ly
Tumor-specific antibodies
Monocytes, DC
NK/NKT
• 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 vivoIn vivo spontaneous tolerance tolerance break (immunogenicity) ofbreak (immunogenicity) of
shared selfshared self MAAs.MAAs.2 examples2 examples
0.0
0.1
0.2
0.3
0.30.81.31.82.32.83.3
n.s.***
0.0
0.1
0.2
0.3
0.30.81.31.82.32.83.3
n.s. *****
gp100209-217 Tyrosinase368-377 Melan-A/Mart-126-35
Patients
Donors StageI-II
StageIII-IV
FREQUENCY OF ANTI-MAA CD8 T LYMPHOCYTES FREQUENCY OF ANTI-MAA CD8 T LYMPHOCYTES AND PROGRESSION OF THE DISEASEAND PROGRESSION OF THE DISEASE
0.0
0.1
0.2
0.3
0.30.81.31.82.32.83.3
Patients
Donors StageI-II
StageIII-IV
Patients
Donors StageI-II
StageIII-IV
*** ********
CONCLUSIONSCONCLUSIONS
• A hierarchy exists in the spontaneous A hierarchy exists in the spontaneous recognition of “self” TAAs.recognition of “self” TAAs.
• Recognition of “self” TAAsRecognition of “self” TAAs increases with increases with the increased tumor burden (e.g. Melan-the increased tumor burden (e.g. Melan-A/MART1) A/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).
Type of peptide MAA
N. of patients
Clinical response (CR+PR) (mean %)
Immune response (%)
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.
Multicentric European StudyLudwig Institute,Brussels; INT and HSR, Milano
Adapted from M.Marchand et al., Int.J. Cancer 1999
Vaccination with MAGE3.A1 Vaccination with MAGE3.A1 peptidepeptide
14
Use of dendritic cells:Use of dendritic cells:which progress which progress ??
seesee Ridolfi’s presentation 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 cell-mediated anti-tumor response
Tumor (Immunosubversion)• Lack of antigen or down-
regulation of HLA • Dysfunction of antigen
presentation• Release of immune-
suppressive 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 down- regulate them in the clinic thus improving efficacy.
• Possible examples
Myeloid suppressor cellsMyeloid suppressor cells are increased are increased
in peripheral blood of stage II-III melanoma patientsin peripheral blood of stage II-III melanoma patients
0
5
10
15
20
% C
D1
4+C
D1
1b
+H
LA-D
R-/
lo
STAGE IIB-IIIC
STAGE IVHD
MELANOMA
p<0-01
p<0.01
N=40
N=40
N=30
Increased frequency of CD4+CD25Foxp3+ Tregis a late event in melanoma patients
HD
Stage
I IB-I
IC
Stage
I IIA
-II IC
Stage
IV0
5
10
15
%C
D4
+C
D25
hig
hFo
xp3
+n.s.
p= 0.0002
p=0.04
p= 0.002
% C
D4
+C
D25
hig
hFo
xP3
+ c
ells
(gate
d
on lym
phocy
tes)
Monitoring T regs : unpredictable effect of low-dose Monitoring T regs : unpredictable effect of low-dose cyclophosphamide on the frequency of CD4cyclophosphamide on the frequency of CD4++CD25CD25highhighFoxp3Foxp3++
0
2
4
6
8
10
HD P0 P1 P4
10
8
6
4
2
0
VACCINATION ARMVACCINATION ARM
cyclophosphamide
300mg/m2 3-5 days before vaccination
IIB-IIIC HIGH RISK MELANOMA PATIENTS
cyclophosphamide
• Cyclophosphamide is effective in a different trial with RCC patients (Walter et al., Nature Med 2013)
de010203040020406080100Time (months)Percentage survival3+Cy 1+Cy 0–Cy 0–
Cy 1210010203040020406080Percentage survival100Time
(months)bc0246810020406080100Time (months)Percentageprogression-free survival+
Multipeptide vaccination In RCC patients
Conclusions
1.1. Myeloid-derived suppressor cells are Myeloid-derived suppressor cells are increasedincreased
in the peripheral blood of stage II-III in the peripheral blood of stage II-III melanoma patientsmelanoma patients
2. Increased frequency of CD4+CD25FoxP3+ Tregs is a late event in melanoma patients
3.3. Not clear the effect of low-dose Not clear the effect of low-dose cyclophosphamidecyclophosphamide on the frequency of on the frequency of
CD4+CD25+Foxp3+CD4+CD25+Foxp3+
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/neu
Breast cancer
IIR 101/75 IV P<0.04
DC/NHL NHL II 18 3CR,3PR,8SD
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
Number of ongoing Phase II trials
Breast
Gastrointestinal
Genitourinary
Gynecologic
Hematologic
Neurologic
Respiratory
Skin
300 5 10 15 20 25
Autologous T-cell/lymphocyte infusion
Dendritic cell
DNA/RNA
Peptide
Recombinant microbial
Tumor cell
Type of active immunotherapy
Breast
Colorectal
Other
Prostate
Renal
Ovarian
Other
Leukemia
Lymphoma
Multiple myeloma
Glioblastoma
Other
Lung
Other
Melanoma
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. anti-
vascular agents)
TNFNGR-TNF
CNGRCG peptide
Targeted delivery of TNF to tumor vasculatureby coupling with the NGR-peptide
(a ligand of CD13 expressed by angiogenic vessel)
Curnis et al., Nat. Biotechnol., 2000
The antitumor activity of NGR-TNF is >10 times greater than that of TNFin several mouse models
Sacchi et al., Clin. Cancer Res. 2006
0
20
40
60
80
100
7 14 21 28 35
PBS
NGR-TNF
DC/OVA
DC/OVA + NGR-TNF
Day since Tumor Challenge
% S
urv
ivin
g A
nim
als
DC/OVA DC/OVANGR-TNF
NGR-TNF
PBS (n=6) vs NGR-TNF (n=5): NS
PBS vs DC/OVA (n=5): NS
PBS vs DC/OVA+NGR-TNF (n=6): p = 0.0035
NGR-TNF vs DC/OVA: NS
NGR-TNF vs DC/OVA+NGR-TNF: p = 0.0061
DCOVA vs DC/OVA-NGR-TNF: NS
Combination of vaccine and NGR-TNF in animal models
Calcinotto et al. J Imunology 2012
• A pilot phase I and II study of NGR-hTNF in combination with a peptide-based 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: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, mutation-derived,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
neo-peptide
neo-peptide
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 score W.T. SEQscoreHLA-A*01DTPINLSLKY 25 DTPINYSLKY 25ELDTPINLSL 14 ELDTPINYSL 11HLA-A*01 11 LDTPINLSLKY 20 LDTPINYSLKY 20ELDTPINLSLK 14 ELDTPINYSLK 13HLA-A*0201ELDTPINLSL 20 ELDTPINYSL 20NLSLKYSDEQ 12 NYSLKYSDEQ 2HLA-A*03NLSLKYSDE 15 NYSLKYSDE 5TPINLSLKY 13 TPINYSLKY 13LDTPINLSLK 16 LDTPINYSLK 16ELDTPINLSL 15 ELDTPINYSL 12HLA-A*1101DTPINLSLK 22 DTPINYSLK 22HLA-A*26DTPINLSLKY 28 DTPINYSLKY 28ELDTPINLSL 20 ELDTPINYSL 19DGELDTPINL 17 DGELDTPINY 19HLA-A*6801DTPINLSLK 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 NGR-hTNF in combination with a peptide-based 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, 2-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: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)
- 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
Exploiting known frequently somatically mutated CAN genes in CRC
Roche 454 GS-FLXObtain up to 500Mb per run with between one and sixteen samples
300~500bp average read length, in 10 hours
Massive parallel pyrosequencing
APCKRASTP53PIK3CAFBXW7CSMD3TNNNAV3SMAD4EPHA3MAP2K7EPHRB6PTENADAMTSL3GUCY1A2SMAD2OR51E1LAMA1C10orf137TCF7L2
20 most frequently mutated CAN-gene in CRC
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
neo-peptide
neo-peptide
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 .
Resection Purification of
HSP-GP96
Vaccination
Vaccination of metastatic patients with mutated peptide Vaccination of metastatic patients with mutated peptide HSPPC-96 derived from the autologous tumorHSPPC-96 derived from the autologous tumor
Tumor sample
Tumor cell suspension
Immunologic monitoringin vivo: DTHin vitro: ELISPOT, tetramers staining
Phase I/II clinical studies of Phase I/II clinical studies of vaccination with HSPPC-96vaccination with HSPPC-96
1.1. Liver metastases of colon Liver metastases of colon cancer (29 patients)cancer (29 patients)
2.2. Metastatic melanomaMetastatic melanoma(39 patients)(39 patients)
3.3. Metastatic melanomaMetastatic melanoma(HSPPC-96 + GM-CSF + IFN,(HSPPC-96 + GM-CSF + IFN,30 patients)30 patients)
Regressioni complete di metastasi da melanoma in seguito a Regressioni complete di metastasi da melanoma in seguito a vaccinazione con HSP96vaccinazione con HSP96
da Belli et al. J Clin Oncol., 2002
Prima del vaccino 12 mesi dopo 34 mesi dopo
66
0.31 2.81 4.42
Day 1--7--14--21---------------49--------------------------63 V1 V6 V7
CD8 CD8 CD8
HL
A/M
AR
T-1
tet
ram
er
0
100
200
300
400
V1 V6 V7
T2 alone
T2+Mart-1
T2+gp100
501mel
colo705
N. s
pots
/500
0 ce
lls
*
*
*
*
In vivo expansion of anti-MART-1 T cells in aIn vivo expansion of anti-MART-1 T cells in aCR patient (02-003) vaccinated with HSPPC-96CR patient (02-003) vaccinated with HSPPC-96
Rivoltini et al.J Immunol 2004
IFN
CD8 CD8 CD8 CD8
1.3% 14% 0.9% 5.3%
Tumor-derived HSPPC-96 mediate cross-presentationTumor-derived HSPPC-96 mediate cross-presentationby HLA-A*0201+ monocytes of peptides fromby HLA-A*0201+ monocytes of peptides fromknow tumor antigens to specific T cell clonesknow tumor antigens to specific T cell clones
Anti-MART-1 HLA-A2-restricted CD8+ T cell clone
CD8 CD8 CD8 CD8
HSPPC-96-colon ca HSPPC-96-501mel MART-1 peptideCEA peptide
IFN
73% 2% 12% 1.5%
Anti-CEA HLA-A2 restricted CD8+ T cell cloneHSPPC-96-colon ca HSPPC-96-501mel MART-1 peptideCEA peptide
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 Trial• Sorafenib + • Farnesyl transferase • inhibitors +• Bevacizumab +• Anti-CTLA4 (Ipilimumab) +• CCI-779 +• 17-AAG + • Antisense (Oblimersen) +• Gleevec +
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 Phase I/II studies of DC-peptide based therapeutic vaccines in melanomatherapeutic vaccines in melanoma
DC+ antigen No.Pts Responses Ref. Immunologic Clinical
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 Banchereau 2006
Total 151 73 (51%) 25 (13%)
1 Peptides included MART-1, MAGE, tyrosinase, gp100.
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 lymphocytesisolated from 4 GBM patients
+: ÅÜ 50 < 100 spots/ 4x104 T cells; ++: ÅÜ 100 < 200 spots/ 4x104 T cells; +++: ÅÜ 200 < 800 spots/ 4x104 T cells;
N.A.: FBS tumor cells are not available;
Immunobiological differences between CSC and FBS tumor cell lines.
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 demethylatingagent 5-Aza.-(CdR).
CSCs
CSCs
CSCs
FBS
FBS
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 NGR-hTNF da parte di MolMed, Milano).
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 processingapproaches 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
Somatically mutated tumor antigens:Best candidates with limitations
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 HLA-A*0201 epitopes, including genes implicated in the neoplastic process.