Forlì, March 1-2, 2007

Anti-cancer vaccines

Peptide-based vaccines: The INT experience

Giorgio Parmiani, MD

Department of Oncology,

San Raffaele Scientific Institute

Strategies of anti-cancer therapeuticStrategies of anti-cancer therapeutic vaccinesvaccines

1. Irradiated and genetically modified tumor cells rradiated and genetically modified tumor cells

2. TAAs

3. Antigenic peptides with adjuvants or dendritic cells

4. Tumor-derived heat shock proteinseat shock proteins

- as whole proteins plus adjuvants

- as genes transduced in normal cells- as plasmid DNA- as DNA contained in viral or non-viral vectors- aS mRNA

Peptide-based vaccines

• Advantages:

• Known sequence and biochemistry

• Easy to synthesize (large availability)

• Allow a specific immune-monitoring of the patient response

• Allow to assess expression of targeted antigens in patient tumor cells

Peptide-based vaccines (cont.)

• Disadvantages:

• Easy degradable in absence of adjuvants

• Require appropriate HLA allele to be recognized (patient selection)

• Induce T cells that may not recognize tumor cells

• Costs

Issues in peptide-based cancer vaccines

• Nature of tumor-associated antigens• Vaccine formulation/adjuvants• Quantity and quality of immune response • First generation of vaccines (1994-

2004): Immmune and clinical response• Immune suppression/tumor escape• Regulatory• Costs

Tumor-associated antigens Tumor-associated antigens (TAA):(TAA):Their nature is crucial Their nature is crucial for anti-tumor immune for anti-tumor immune responseresponse

Antigens recognized by T cells Antigens recognized by T cells (Novellino (Novellino

et al., Cancer Immunol Immunother 2005):et al., Cancer Immunol Immunother 2005):

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 but predominantly expressed on tumor cells (e.g. RAS/m, survivin, hTERT)

4) Unique, expressed only by a single tumor(e.g.CDK4/m,α-actin-m)

1 and 2.1 and 2. In vitroIn vitro andand exex--vivovivo T cellT cell

natural immune response to natural immune response to shared “self” TAAsshared “self” TAAs

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 SPONTANEOUS CIRCULATING ANTI-MAA CD8+FREQUENCY OF SPONTANEOUS CIRCULATING ANTI-MAA CD8+ T LYMPHOCYTES T LYMPHOCYTES ((Mortarini et al., Cancer Res 2004Mortarini et al., Cancer Res 2004))

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 natural A hierarchy exists in the natural recognition ofrecognition of “self”“self” TAAs by T TAAs by T cells.cells.

• MelanA/MART-1 e CEA induce MelanA/MART-1 e CEA induce the most frequent T cell the most frequent T cell response in melanoma and CRC.response in melanoma and CRC.

• Recognition ofRecognition of “self” TAAs“self” TAAs

increases with the increased increases with the increased tumor burden.tumor burden.

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 but predominantly expressed on tumor cells (e.g. RAS/m, survivin, hTERT)

4) Unique, expressed only by a single tumor(e.g. CDK4/m, α-actin-m)

SURVIVIN (SVV)SURVIVIN (SVV)

• Expression pattern:• abundant during fetal development• silenced in normal adult tissues• over-expressed in most common

human cancers

• Member of AIPs (Apoptosis Inhibitory Proteins)

Survivin is a universal TAA that can elicit both HLA-I and HLA-II-restricted T cells

IN VITRO INDUCTION OF HLA CLASS I-RESTRICTED

ANTI-SVV T CELLS IN PBMCs OF RECTAL CANCER PATIENTS

0

25

50

75

100

- + - +

0

25

50

75

100

- + - +

Autologous PBMCs

SVV-1Medium

IFN

γ s

pots

/50,0

00 c

ells Patient 1

Patient 2

+ anti-HLA-I Ab

(Casati et al., Cancer Research 2003)

0

25

50

75

100

med

PSMA1

PSMA2

svv

Her2neu

EpCam

Flu

N.

IFN

gam

ma

spo

ts/2

x10

5 P

BM

Cs

In vitro recognition of prostate antigens-deriving peptides byT lymphocytes of prostate cancer patients

In vitro recognition of prostate antigens-deriving peptides byT lymphocytes of prostate cancer patients

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)

Unique human TAAs recognized by HLA-restricted T cells (Parmiani et al.,

Journal of Immunology 2007)

Tumor No. Class I No. Class II

Melanoma 11 6NSCLC 3 1H/N 2 0RCC 2 1Bladder cancer 1Total 19 8

Most of these unique TAA have a pathogenetic role in neoplastic transformation/ progression.

Strategies of anti-cancer therapeuticStrategies of anti-cancer therapeutic vaccinesvaccines

1. Irradiated and genetically modified tumor cells rradiated and genetically modified tumor cells

2. TAAs

3. Antigenic peptides with adjuvants or dendritic cells

4. Tumor-derived heat shock proteinseat shock proteins

- As whole proteins plus adjuvants

- As genes transduced in normal cells- As plasmid DNA- As DNA contained in viral or non-viral vectors- AS mRNA

Use of shared “self” or “cancer/testis” TAA peptides as

vaccines in patients with metastatic melanoma

(1995-2004). Clinical RR 18%.

CD8+T cell response in20-50% of patients but

limited expansion of T cells (<1%).

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.

Rationale for a new generation of peptide-based vaccines at INT of

Milan

• Multiple peptide including universal TAAs

• New TLR targeting adjuvants (CpG, HSP)

• Down-regulation of Tregs and/or Myeloid Suppressor Cells (see Licia Rivoltini)

• Immune-monitoring in blood and LNs

• Assessment of patient polymorphisms

On the basis of pre-clinical results, the SVV-1.HLA-A2 peptide is being used, along with other peptides, in clinical studies of vaccination of patients with prostate and rectal carcinoma and melanoma.

Vaccination

HLA-A2-restricted peptides from PMSA1, PSMA2, survivin + Montanide. CPA before vaccination

Vaccination

HLA-A2-restricted peptides from PMSA1, PSMA2, survivin + Montanide. CPA before vaccination

Vaccination with peptides deriving from prostate TAAs in association with Montanide and cyclophosphamide (CPA)

Peptides

Activation ofspecific T cells

Unit of Immunotherapy of Human Tumors

CPA(300 mg/m2)

Blocking of suppressive effectof CD4+ CD25+T regon lymphocyte proliferation

Montanide

Recruitment of pro-infliammatory cells(APC)

Vaccination with peptide deriving from prostate TAAs in association with Montanide and CPA

Group 1 – Adjuvant Vaccination

•Malattia localizzata a livello prostatico, resecabile•Vaccinazione in regime neoadiuvante ed adiuvante dopo chirurgia

Unita’ di Immunoterapia dei Tumori Umani

1° cycle vaccine 2° cycle vaccine

C C-3 1 w

C C-3 1 month

C C

Diagnostic biopsy

ProstatectomyProstatectomy

Analysis of induction of T lymphocytes recognizing vaccine peptides in PBLs

Analysis of tumor infiltrate

Results: Immunologic and clinical responses

Vaccination of prostate Vaccination of prostate cancer patients: Clinical cancer patients: Clinical

responseresponsePatientPatientss

Clinical outcomeClinical outcome

001 Biochemical SD at 10 months from interruption of hormone therapy

002 Biochemical SD at 8 months from interruption of hormone therapy

003 Biochemical SD at 6 months from interruption of hormone therapy

004 Biochemical SD at 6 months and CR of a prostatic lesion

005 PD

006 PD

007 After the first cycle of vaccination (PSA 1.33 ng/ml → 1.01 ng/ml)

008 Aftrer the first cycle of vaccination (PSA 0.51 ng/ml → 0.44 ng/ml)

009 After the first cycle of vaccination (PSA 1.04 ng/ml → 0.27 ng/ml)

Vaccination of high risk stage IIB/IIIC melanoma patients with multipeptides

emulsified in Montanide , and given cyclophosphamide and IL-2 vs.

observation only. A randomized phase II study

• G. Parmiani, M. Santinami, L. Pilla, G. Cutolo, A. Maurichi, R. Patuzzo, A. Carbone, G. Tragni, C. Castelli, L. Rivoltini (INT);

• M. Maio (Siena),V. Chiarion (Padua)

• Ethics Committee approval: May 2004.

• Delayed due to the long term negotiation with Coley to obtain CpG-ODN (not provided).

• First patient enrolled in July 2005

Primary end points: Immune response (frequency and intensity)

ELISPOT: Frequency of anti-tumor specific T lymphocytes

Flow-cytometry: Phenotype of anti-tumor T cells

-HLA tetramers-Cytokine intracellular staining

-Immunohistochemistry: characterization of tumor cells and of TILs

Clinical objectives

Primary:

Local and systemic toxicity

Secondary:

Disease-free survival after 2/3 years

Overall survival at 5 years

HLA- A2

negative

patients

60%

HLA- A2

positive

patients

40%

No. of assessed patients: 210

ENROLLED PATIENTS: 28

Stage II: 6 Stage III: 22

Control arm: 13 Vaccination arm: 15

SVV as universal TAA : - Conclusions -

• SVV is expressed by melanoma; colon, rectal and prostate cancer cells

• Class I and II HLA-restricted SVV epitopes can be recognized both ex-vivo and in vitro by patient T though to a variable extent

• SVV can then be considered a shared, universal tumor-restricted antigen that is being used in clinical studies of vaccination

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)

Vaccination withVaccination withunique antigens:unique antigens:

how is it possible?how is it possible?1.Autologous gene-modified tumor 1.Autologous gene-modified tumor

cellscells

2. Autologous 2. Autologous proteins/peptidesproteins/peptides

Autologous tumor-derived

heat shock-protein/peptides complexes

MECHANISMS OFMECHANISMS OFIMMUNE EVASIONIMMUNE EVASION

Why a more strong Why a more strong immune immune responseresponse and/or a more frequent and/or a more frequent

clinical responseclinical response was not observed was not observed in vaccination trialsin vaccination trials

??

FACTORS THAT CAN INTERFERE WITH THE RECOGNITIONFACTORS THAT CAN INTERFERE WITH THE RECOGNITIONAND LYSIS OF TUMOR CELLS BY T LYMPHOCYTESAND LYSIS OF TUMOR CELLS BY T LYMPHOCYTES

TumorTumor Lack of antigenic peptide or

down-regulation of HLA

Dysfunction of antigen presentation

Release of immune-suppressive factors (IL-10, TGF, VEGF)

Tumor counterattack (Fas/FasL)

Cannibalization of T lymphocytes

Immune systemImmune system Immune anergy or

ignorance

Lack of tissue homing molecules; defective adhesion

TCR dysfunction

Inactivation of T cells within the tumor (granzyme B)

Induction of anti-peptide but not anti-tumor T cells

T regulatory cells

Myeloid suppressive cells

AcknowledgmentsAcknowledgmentsChiara CastelliLicia RivoltiniChiara Camisaschi Gianluca CutoloVeronica Huber Manuela Iero

Andrea MarrariLuisa NovellinoLorenzo PillaRoberta Valenti

Agata CovaFrancesca Rini Gloria SovenaPaola Squarcina Istituto Nazionale Tumori

Milan - Italy

Vincenzo MazzaferroJorgelina CoppaMario SantinamiRoberto PatuzzoErmanno LeoFiliberto BelliFlavio ArientiAlfonso Marchianò

Grazia BarpAnnabella Di Florio

Pramod K. SrivastavaAntigenics, New York

Michele MaioSiena

Take home message. Perspectives 1

• Antigens: use both cross-reacting self and unique TAA; use both classes I and II HLA-restricted peptides.

• Adjuvants: CpG-ODN plus IFA are likely to be the most potent; cytokines: how to use them?(IFN-, GM-CSF?, IL-12, IL-15, IL-21, IL-23).

• Tumor burden: avoid excess tumor burden to prevent release of suppressive factors.

1

Perspectives 2

• Escape mechanisms: use compounds which may help in restoring normal immune functions(e.g. retinoic acid, cytokines, anti-Treg Ab).

• Combination therapy: vaccination may be combined with anti-angiogenic drugs, radiotherapy and immune-modulating chemotherapy(non-myeloablative lymphodepletion?)

• Modulation of the immune response by anti-Treg and/or anti-CTLA4 antibodies.

2

REGULATORY T LYMPHOCYTES

Treg assessment during peptide-based vaccination and

modulation by Cyclophosphamide

• Recurrent rectal carcinoma patients receiving a peptide-based vaccine(no IL-2)

• Stage III melanoma patients receiving a multipeptide-based vaccine (with IL-2)

4

6

8

10

12

14

CTX1 V1 CTX2 V4 CTX3 V5 post

8

9

10

11

12

13

CTX1 V1 CTX2 V4 CTX3 V5 post

7

8

9

10

CTX1 V1 CTX2 V4 CTX3 V5 post

8

9

10

11

12

13

14

CTX1 V1 CTX2 V4 CTX3 V5 post

% o

f C

D25

+ F

oxp

3+ (

in C

D4)

Pt#4 Pt#3

Pt#2Pt#1

Treg (CD4+CD25+Foxp3+) in PBMC of rectal carcinoma patients and their modulation by CTX treatment

10

15

20

25

30

35

CTX1V1

Surge

ryCTX2

V3 V4

post

IL-2

5

10

15

20

CTX1V1

Surge

ryCTX2

V3 V4

post

IL-2

5

10

15

20

CTX1V1

Surge

ryCTX2

V3 V4

post

IL-2

% o

f p

os

itiv

e ce

lls

CD4+CD25+ CD25+Foxp3+(in CD4) CD4+CD25brigh

Pt#1 Pt#2

Treg in PBMC of Melanoma patients: modulation by CTX and IL-2 treatment

Studi clinici di vaccinoterapia in assenza di cellule dendritiche

in pazienti con carcinoma prostaticoVaccino N. HLA Risposta Risposta Ref.

pazienti immune clinica

SART, p56LCK, 10 A2402 4/10 1 PR* Naguchi, ’03ART, CyB 5 SD

SART, p56LCK, 16 A2402 10/14 13/13 ** Naguchi, ’05ART, CyB, PSA, PAP, (71%) (6/13 PR)PSMA estramustine

Allo-prostata + BCG 26 A*0201 11/26 11/26§ Pandha, ’05

pVAX/PSA + GM-CSF 9 A*0201 2/9 Non Pisa, ‘04+ IL-2 valutabile

Auto-Prostate/GM-CSF 8 NV 7/8 Non Simons, ’99 DTH valutabile

FowlPox+VacPSA 64 NA 46% 18 PFS Kaufman ‘04 (78%)

* 89% PSA - ** Tutti PSA, con 6/13 > 50%.§ Riduzione prolungata e statisticamente significativa di PSA.

Features of peptide vaccines

• Peptide length:

• 8-10 aa for HLA class I-restricted peptides

• 12-23 aa for HLA class II-restricted peptides

• Modified peptides (heteroclitic and/or long peptides)

• Highly and rapidly degradable in vivo (adjuvants are required)

1, 2: Shared “self” TAAs• Normal subjects and cancer patients show

some forme of tolerance to “self” TAAs (immune ignorance, peripheral tolerance, central tolerance, low frequency of T cells, etc.).

• Tolerance needs to be broken in order to induce a T cell immune response against “self” TAAs.

• Thus, these TAAs are considered to be “weak antigens”

Natural CD8+T cell response of HLA-A2 colorectalNatural CD8+T cell response of HLA-A2 colorectalcancer (CRC) patients to differentiation antigenscancer (CRC) patients to differentiation antigens

(Nagorsen et al., Cancer Res 2000)(Nagorsen et al., Cancer Res 2000)

Antigen Clinical N. of respondingstage N. of tested*

Ep-CAM Donors 0/8A-B 0/9C-D 4/13

Her2/neu Donors 0/8A-B 0/9C-D 5/13

CEA Donors 0/8A-B 0/9C-D 6/13

* ELISPOT assay.

0

25

50

75

100

N.

IFN

γ s

pots

/3x1

04

cells

med

T2+

PS

MA

T2+

PS

MA

+ a

nti

-HL

A-I

CaP

+ a

nti

-HL

A-I

CaP

Anti-PSMA T cells

CaP

Anti-svv T cells

0

25

50

75

100

med

T2+

svv

T2+

svv

+ a

nti

-HL

A-I

CaP

+ a

nti

-HL

A-IN.

IFN

γ s

pots

/3x1

04

cells

Fresh PBMC

0

25

50

75

100

med

PS

MA

sv

v

Flu

N.

IFN

γ s

pots

/10

6 c

ells

A

B

A. Analisi della frequenza di precursori linfocitari T anti-tumore specifici nel sangue periferico in pazienti con carcinoma della prostata. I PBMC isolati attraverso centrifugazione in gradiente di densità dal sangue periferico di pazienti affetti da carcinoma della prostata HLA-A*0201 sono stati incubati con peptidi di PSMA, survivina e dalla proteina del virus influenzale (M1), come controllo positivo. Il riconoscimento dei vari antigeni da parte dei linfociti e’ stato valutato come rilascio di IFNγ, mediante Elispot. I dati rappresentano 1 dei 2 casi di pazienti con carcinoma prostatico che hanno mostrato un’elevata immunogenicita’ per gli antigeni analizzati.

B. Analisi quantitativa dell’attivazione di linfociti T anti-PSMA-specifici derivati da pazienti affetti da carcinoma prostatico. I PBMC di pazienti HLA-A*0201 sono stati stimolati per due settimane con i peptidi tumorali derivanti da PSMA e survivina per due settimane. Al termine di tale periodo la specificità dei linfociti T nei confronti di cellule T2 e di cellule di carcinoma prostatico LNCaP (?), è stata valutata quantificando il rilascio di IFNattraverso analisi immunoenzimatica Elispot.

1 2 3 54 6 1 2 3 54 67 8 9 10 7 8 9 10

T2 + Survivin T2 + Survivin + anti-HLA-I

T2 + PSMA P1T2 + PSMA P1 + anti-HLA-I

N.

of

sp

ots

per

30

4 c

ells

N.

of

sp

ots

per

30

4 c

ells

Frequency of TAA-specific T cells precursorsafter in vitro stimulation with prostatic peptides

No. of microcultures tested/patient

Ex-vivo recognition of prostate antigens by patient T cells

• No. of patients tested: 112

• No. of patients whose peripheral blood T lymphocytes recognized prostate antigens: 65 (58%)

Studio di fase III di vaccinazione con DC di Studio di fase III di vaccinazione con DC di pazienti con carcinoma della prostatapazienti con carcinoma della prostata

N. Pazienti Antigene Risposta Risposta P.I.immune clinica

110 vs 108 Provenge* ND Mediana E.Small, Metastatici, vs placebo sopravvivenza 2005ormone 25.9 vs 21.9resistenti OS 34% vs 11%

ND: non disponibile*Provenge= Proteina di fusione PAP+GM-CSF su cellule dendritiche

Patient 8- 3R

MHC-CLASS II RESTRICTED RESPONSE OF COLORECTAL MHC-CLASS II RESTRICTED RESPONSE OF COLORECTAL

CANCER PATIENTS TO rSVV PROTEIN OR CANCER LYSATECANCER PATIENTS TO rSVV PROTEIN OR CANCER LYSATE

IFN- ELISA

0

500

1000

1500

2000

2500

IFN

- p

g/m

l

Patient 1- 3R

IFN

- p

g/m

l

= medium

= auto LCL

= HCT 116 lysate

= melanoma lysate

= LCL + rSVV

= LCL + rSVV + L243

= LCL + HCT 116 lysate

= LCL + HCT 116 lysate + L243

= LCL + C1R/SVV lysate

= LCL + C1R/SVV lysate + L243

0

500

1000

1500

2000

2500

(Casati C et al., Cancer Research, 2003)

The response of autologous T cells to a human melanoma is dominated by

mutated neoantigens

(Lennerz V et al., PNAS, 2005)

• Five neoantigens deriving from somatic mutations were found at different time points in different metastases of a melanoma patient.

• CD8+ cytotoxic T cells against mutated TAA were predominant over shared differentiation self TAA over 4-5 years of testing.

Dendritic cells (DC)-based peptide vaccines

Characteristics:

* Direct stimulation of T cells in lymph nodes.

* Ex-vivo preparation of high numbers of DCs

and their phenotipic and functional

characterization.

* High cost and time consuming.

Are unique TAAs spontaneously recognized in vivo ?

T lymphocytes recognizing unique TAAs have been described in long-term surviving patients previously

affected bymelanoma; NSCLC;

head/neck, bladder and renal carcinoma

(T. Wolfel, P. Coulie, G. Parmiani, F. Triebel)

Phase I-II studies ofDC based therapeutic vaccines in prostate

cancerDC loaded with No. % Immune Clinical % Reference

pts response response

PAP/GM-CSF 31 38 3 (↓PSA) 10 Small ’00

PSMA/A2 37 NE 1 CR, 10 PR 31 Murphy ’99

PSA/mRNA 13 100 3 CR 23 Vieweg ’02

Mouse PAP 21 49 6 SD 0 Fong ’01

PSA 24 45 11 (↓PSA) 46 Barrou ’04

hTERT/mRNA 20 95 9 (↓PSA) 45 Vieweg ‘05

T O T A L 142 46 23

Autologous monocytes pulsed with Autologous monocytes pulsed with melanoma-derivedmelanoma-derived HSP70HSP70 are recognizedare recognized

by specific T cell clones by specific T cell clonesIF

N-

(p

g/m

l)

0

1000

2000

3000

Anti-gp100

APC

Med

ium

HS

P7

0

HS

P7

0+

W6

.32

Au

to-M

e

0

250

500

Anti-MART-1

APC

Med

ium

HS

P7

0

Au

to-M

e

Statistically significant inhibition.

HS

P7

0+

W6

.32

W6.32 = anti-HLA-I mAb Auto-Me = autologous melanoma

(from Castelli et al., Cancer Res. 2001)

Some recent good news in

anti-cancer vaccination

Recombinant NY-ESO-1 protein with ISCOMATRIX

adjuvant induces broad integrated antibody and CD4+ and CD8+ T cell responses in melanoma

Davis et al., (Old LJ, Cebon JS), PNAS 101: 10697-10702, 2004

Treatment Disease-free survival at median follow-up of 2

years*

Placebo 2/7(28%)NY-ESO-1 protein only 7/16 (48%)NY-ESO-1 + IMX 17/19 (89%)

- Clinical results -- Clinical results -

* Well balanced for pathological state, primary lesion thickness, etc.

Phase II study of vaccination of hormone-refractory metastatic prostate cancer patients

with GVAX®Cell Genesys, ASCO 2005

• No. of patients 22• Median survival time of vaccinated patients was

over 29 months.• Median survival time of Taxotere-treated patients

is 19 months.• Phase III trials ongoing.

A phase IIb randomized study of vaccination with L-BLP25 in stage IIIb and IV NSCLC

after first line chemo

• Vaccine: Liposome-MUC1

• Overall median survival: 17.4 vs. 13 months in

88 vs. 83 patients

• Overall 2 years survival: 43.2 vs. 28.9 %;

subset stage IIIb: 60 vs.37 %

Biomira/Merck KGaA, ESMO 2004

Phase II randomized trial of autologous

formalin-fixed tumor vaccine for postsurgical recurrence of hepatocellular carcinoma

Kuang et al. Clin Cancer Res 2004

• No. of patients 41, after curative resection• Vaccine treatment N=19; no treatment N=22• Median follow-up of 15 months• Recurrence: Vaccinated 3/18 vs. Control 13/21• Recurrence-free survival P=0.003; OS P= 0.01

Istituto Nazionale Tumori Milano

Vaccinazione con 4 Peptidi emulsionati in

Montanide (MART-1, gp100, NY-ESO, survivin) allo scopo di diminuire l’emergere di varianti antigene negative

Razionale

Cancer Vaccine Pipeline(www.bioseeker.com)

• There are more than 260 non-antibody based vaccine drugs, therapeutic and preventive vaccines included. 87 are cancer vaccines.

• Vaccines are being developed for over 30 different cancers; 38 companies are involved.

• The number of vaccine targets has increased to over 50 different molecular targets

• Vaccine market is predicted to be of 6 billions USD by 2010

Clinical studies of vaccinationClinical studies of vaccination with HSPPC-96 with HSPPC-96

1.1. Liver metastases of colon Liver metastases of colon cancercancer

2.2. Metastatic melanomaMetastatic melanoma(HSPPC-96 only)(HSPPC-96 only)

3.3. Metastatic melanomaMetastatic melanoma(HSPPC-96 + GM-CSF + IFN)(HSPPC-96 + GM-CSF + IFN)

Vaccination of patients with liver metastases of CRC Vaccination of patients with liver metastases of CRC with autologous HSPPC-96with autologous HSPPC-96

- - TREATMENT SCHEMETREATMENT SCHEME - -

In vivoIn vivo expansion of anti-CEA T cells in HLA- expansion of anti-CEA T cells in HLA-A2 CRC patients vaccinated with HSPPC-96A2 CRC patients vaccinated with HSPPC-96

CD8

2.14

HL

A/ C

EA

tet

ram

ers

0.22

CD8

HL

A/ C

EA

tet

ram

ers

Day 1------7-------14------21 V1 V5

Patient 045Patient 045

N. s

pots

/104 T

cel

ls

V1 V5

*0

100

200T2 alone

T2+CEA

T2+CEA+W6.32

Colo206

0

100

200

300

400

500T2 alone

T2+CEA

T2+CEA+W6.32

Colo206

N. s

pots

/5x1

04 T c

ells

CD8CD8

Day 1---7—-14—21-------51 V1 V6

HL

A/ C

EA

tet

ram

ers

0.11 2.31

V1 V6

*

HL

A/ C

EA

tet

ram

ers

Patient 038Patient 038

Resection Purification of

HSP-GP96

Vaccination

Vaccination of metastatic patients with HSPPC-96 Vaccination of metastatic patients with HSPPC-96 derived from the autologous tumorderived from the autologous tumor

Tumor sample

Tumor cell suspension

Immunologic monitoringin vivo: DTHin vitro: ELISPOT, tetramers staining

VACCINATION WITH HSPPC-96 IN VACCINATION WITH HSPPC-96 IN METASTATIC COLORECTAL METASTATIC COLORECTAL

CARCINOMACARCINOMA(CRC)(CRC)

Clinical and immunologic responsesClinical and immunologic responses

(Mazzaferro et al., Clin Cancer Res 2003)(Mazzaferro et al., Clin Cancer Res 2003)

DISEASE-FREE SURVIVAL ACCORDING TO T CELL IMMUNERESPONSE IN PATIENTS WITH LIVER METASTASES FROM CRC

17 14 11 7 616 12 9 7

12 1 19 1 1

p < 0.0001

Immune responders

Immune non-responders

Patients at risk

Immune respondersImmune non-responders September 2004

OVERALL SURVIVAL ACCORDING TO T CELL IMMUNE RESPONSE

IN PATIENTS WITH LIVER METASTASES FROM CRC

17 17 17 14 11

Patients at risk

17 17 17 14Immune responders 12 9 6 311 6 5 2

p < 0.0001

2

Immune responders

Immune non-responders

Immune non-responders September 2004

HSPPC-96 vaccination increases CD8+T cell HSPPC-96 vaccination increases CD8+T cell response to class I HLA-matchedresponse to class I HLA-matchedCRC lines in 16 out of 29 patientsCRC lines in 16 out of 29 patients

0

50

100

150

200

250

300

350

400

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Pre Post (p < 0.05) Post (p > 0.05)501 537 698 449

N.

spots

/1.6

7 x

10

5 P

BM

C

Patient number

CONCLUSIONSCONCLUSIONSVaccination of patients with liver metastases of CRC with autologousHSPPC-96 is feasible and safe.

Approximately 60% of patientsdeveloped a T cell response to CRC

antigens and CRC cells.

Both OS and DFS were longer in T cell responding than in non-responding subjects,independently from other

prognostic factors.

TUMORS FOUND TO BE INFILTRATED BYT REGULATORY LYMPHOCYTES

Tumor Reference

NSCLC Woo/June, 2001

Pancreatic/breast cancer Liyanage, 2002

Colorectal cancer D. Herlyn, 2002

Gastric/esophageal cancer Ichihara, 2003

Melanoma Javia/Rosenberg, 2003

Ovarian Cancer Curiel, 2004

HCC Ormandy, 2005

Breast cancer Bates, 2006

Giorgio Parmiani

Istituto Nazionale Tumori, Milan - Italy

Mario SantinamiLorenzo Pilla

Roberto PatuzzoElisabetta Pennacchioli

Alfonso Marchianò

Licia Rivoltini Chiara Castelli

Phase III studiesPhase III studies of DC-based vaccines of DC-based vaccinesin patients with melanoma or prostate carcinoma in patients with melanoma or prostate carcinoma

No. Patients Antigen Immune Clinical P.I.response response

55 vs 53 MAGE1,3; Not available No advantage Schadendorf, 2006* Stage IV MART1, gp100,

tyrosine+DTIC vs DTIC

110 vs 108 Provenge** Not done Median Small, 2005Metastatic, vs placebo survivalhormone 25.9 vs 21.9resistant OS 34% vs 11%

**Provenge (Dendreon) = Fusion protein of PAP+GM-CSF loaded on autologous DCs.

*But in the vaccinated arm, a significant benefit in OS was found in the subset of HLA-A2+/B44 patients vs.non-HLA-A2/B44 ones.

DC-BASED VACCINATIONIN PATIENTS WITH METASTASES

- C O N C L U S I O N S -

• DC-peptide based vaccines induced a frequency of CD8+T cells response higher (up to 100%) than that obtained by peptide only-based vaccines but without a parallele increase in the clinical outcome (tumor response).

Istituto Nazionale Tumori Milano

Vaccination with 4 peptides emulsified in Montanide (MART-1, gp100, NY-ESO, survivin) with the aim of reducing the emergence of antigenic negative variants.

Rationale

Cyclophosfamide: 300 mg/m2: with the aim of down-regulating T regs

IL-2: 3x106 IU (s.c.): to expand T cells activated by the vaccine

67%9%

24% Others

Stage IIB/C

Stage IIIB

PATIENTS ENROLLED