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.