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Forlì, March 1-2, 2007 Anti-cancer vaccines Peptide-based vaccines: The INT experience Giorgio Parmiani, MD Department of ...
Strategies of anti-cancer therapeutic vaccines 1.  I rradiated and genetically modified tumor cells  2. TAAs   3. Antigeni...
Peptide-based vaccines <ul><li>Advantages : </li></ul><ul><li>Known sequence and biochemistry </li></ul><ul><li>Easy to sy...
Peptide-based vaccines ( cont .) <ul><li>Disadvantages : </li></ul><ul><li>Easy degradable in absence of adjuvants </li></...
Issues in peptide-based cancer vaccines <ul><li>Nature of tumor-associated antigens </li></ul><ul><li>Vaccine formulation/...
Tumor-associated  antigens (TAA): Their nature is crucial for anti-tumor immune response
Antigens recognized by T cells  (Novellino et al., Cancer Immunol Immunother 2005): 1)Shared, self  differentiation protei...
1 and 2.   In vitro   and   ex - vivo   T cell natural immune response to  shared “self” TAAs
0.0 0.1 0.2 0.3 0.3 0.8 1.3 1.8 2.3 2.8 3.3 n.s. *** 0.0 0.1 0.2 0.3 0.3 0.8 1.3 1.8 2.3 2.8 3.3 n.s. *** ** gp100 209-217...
<ul><li>CONCLUSIONS </li></ul><ul><li>A hierarchy exists in the natural recognition of   “self”   TAAs by T cells. </li></...
<ul><li>Antigens recognized by T cells: </li></ul><ul><li>Shared, self differentiation proteins expressed also on normal c...
SURVIVIN (SVV) <ul><li>Expression pattern: </li></ul><ul><ul><li>abundant during fetal development </li></ul></ul><ul><ul>...
IN VITRO INDUCTION OF HLA CLASS I-RESTRICTED  ANTI-SVV T CELLS IN PBMCs OF RECTAL CANCER PATIENTS   Autologous   PBMCs SVV...
N. IFN gamma spots/2x10 5  PBMCs In vitro  recognition of prostate antigens-deriving peptides by T lymphocytes of prostate...
<ul><li>Antigens recognized by T cells: </li></ul><ul><li>Shared but expressed on tumor cells only </li></ul><ul><li>Share...
Unique human TAAs recognized by HLA-restricted T cells  (Parmiani et al.,  Journal of   Immunology 2007 ) <ul><li>Tumor No...
Strategies of anti-cancer therapeutic vaccines 1.  I rradiated and genetically modified tumor cells  2. TAAs   3. Antigeni...
Use of shared “self” or “cancer/testis” TAA peptides as vaccines in patients with metastatic melanoma (1995-2004).   Clini...
Phase I/II studies of DC-peptide based  therapeutic vaccines in melanoma DC+ antigen  No.Pts   Responses Ref.   Immunologi...
Rationale for a new generation of peptide-based vaccines at INT of Milan <ul><li>Multiple peptide including universal TAAs...
<ul><li>On the basis of pre-clinical results, the  SVV-1.HLA-A2 peptide is being used , along with other peptides, in clin...
Vaccination HLA-A2-restricted peptides from  PMSA1, PSMA2, survivin  +  Montanide.  CPA  before vaccination   Vaccination ...
Vaccination with peptide deriving from prostate TAAs  in association with Montanide and CPA  Group 1 – Adjuvant Vaccinatio...
Results: Immunologic and clinical responses
Vaccination of prostate cancer patients: Clinical response After the first cycle of vaccination  (PSA 1.04 ng/ml  ->  0.27...
Vaccination of high risk stage IIB/IIIC melanoma patients with multipeptides emulsified in Montanide , and given cyclophos...
<ul><li>Primary end points: Immune response (frequency and intensity)   </li></ul><ul><li>ELISPOT :   Frequency of anti-tu...
Clinical objectives  Primary:   Local and systemic toxicity Secondary:   Disease-free survival after 2/3 years Overall sur...
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 - <ul><li>SVV is expressed by  melanoma;   colon, rectal  and  prostate  cancer cell...
<ul><li>Antigens recognized by T cells: </li></ul><ul><li>Shared but expressed on tumor cells only </li></ul><ul><li>Share...
Vaccination with unique antigens: how is it possible? 1.Autologous gene-modified tumor cells 2. Autologous proteins/peptides
Autologous tumor-derived heat shock-protein/ peptides  complexes
MECHANISMS OF IMMUNE EVASION Why a more strong  immune response  and/or a more frequent  clinical response  was not observ...
FACTORS THAT CAN INTERFERE WITH THE RECOGNITION AND LYSIS OF TUMOR CELLS BY T LYMPHOCYTES   <ul><li>Tumor </li></ul><ul><l...
Acknowledgments Chiara Castelli Licia Rivoltini Chiara Camisaschi   Gianluca Cutolo Veronica Huber  Manuela Iero   Andrea ...
 
Take home message. Perspectives 1 <ul><li>Antigens : use both cross-reacting self and unique TAA; use both classes I and I...
Perspectives 2 <ul><li>Escape mechanisms : use compounds which may help in restoring normal immune functions (e.g.  retino...
REGULATORY T LYMPHOCYTES
Treg assessment during peptide-based vaccination and modulation by Cyclophosphamide <ul><li>Recurrent rectal carcinoma pat...
% of CD25+ Foxp3+ (in CD4) Pt#4 Pt#3 Pt#2 Pt#1 Treg (CD4+CD25+Foxp3+) in PBMC of rectal carcinoma  patients and their modu...
CD4+CD25 high  CD4+CD25 high  CD4+CD25 high  % of positive cells CD4+CD25+ CD25+Foxp3+(in CD4) CD4+CD25 brigh Pt#1 Pt#2 Tr...
Studi clinici di vaccinoterapia in assenza di cellule dendritiche in pazienti con carcinoma prostatico Vaccino N. HLA Risp...
Features of peptide vaccines <ul><li>Peptide length :  </li></ul><ul><li>8-10 aa for HLA class I-restricted peptides </li>...
1, 2:  Shared “self” TAAs <ul><li>Normal  subjects and cancer patients  show some forme of tolerance   to “self”  TAAs ( i...
Natural CD8+T cell response of HLA-A2 colorectal cancer (CRC) patients to differentiation antigens (Nagorsen et al., Cance...
N. IFN γ  spots/3x10 4  cells med T2+PSMA T2+PSMA + anti-HLA-I CaP + anti-HLA-I CaP Anti-PSMA T cells CaP Anti-svv T cells...
1 2 3 5 4 6 1 2 3 5 4 6 7 8 9 10 7 8 9 10 T2 + Survivin  T2 + Survivin + anti-HLA-I T2 + PSMA P1 T2 + PSMA P1 + anti-HLA-I...
Ex-vivo  recognition of prostate antigens by patient T cells <ul><li>No. of patients tested:  112 </li></ul><ul><li>No. of...
Studio di fase III di vaccinazione con DC di pazienti con carcinoma della prostata ND: non disponibile *Provenge= Proteina...
Patient 8- 3R MHC-CLASS II RESTRICTED RESPONSE OF COLORECTAL CANCER PATIENTS TO rSVV PROTEIN OR CANCER LYSATE IFN-   ELIS...
The response of autologous T cells to a human melanoma is dominated by mutated neoantigens ( Lennerz V et al.,  PNAS ,  20...
Dendritic cells (DC)- based peptide vaccines Characteristics : * Direct stimulation of T cells in lymph nodes. *  Ex-vivo ...
Are unique  TAAs  spontaneously recognized  in v ivo ? T lymphocytes recognizing unique TAAs have been described in long-t...
Phase I-II studies of DC based   therapeutic   vaccines in prostate cancer DC loaded with    No.  % Immune Clinical % Refe...
Autologous monocytes  pulsed with  melanoma-derived   HSP70   are recognized  by specific T cell clones (from Castelli et ...
  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...
Phase II study of vaccination of hormone-refractory metastatic  prostate cancer  patients with GVAX ® Cell Genesys , ASCO ...
A phase IIb randomized study of vaccination with L-BLP25 in stage IIIb and IV  NSCLC  after first line chemo <ul><li>Vacci...
Phase II randomized trial of autologous formalin-fixed tumor vaccine for postsurgical recurrence of  hepatocellular carcin...
Istituto Nazionale Tumori Milano Vaccinazione con  4 Peptidi  emulsionati in Montanide  (MART-1, gp100, NY-ESO, survivin) ...
Cancer Vaccine Pipeline (www.bioseeker.com) <ul><li>There are  more than 260  non-antibody based vaccine drugs, therapeuti...
Clinical studies of vaccination  with HSPPC-96 1. Liver metastases of colon cancer 2. Metastatic melanoma (HSPPC-96 only) ...
Vaccination of patients with liver metastases of CRC  with autologous HSPPC-96 -  TREATMENT SCHEME  -
In vivo  expansion of anti-CEA T cells in HLA-A2 CRC patients vaccinated with HSPPC-96 CD8 2.14 HLA/ CEA  tetramers 0.22 C...
Resection  Purification of HSP-GP96 Vaccination Vaccination of metastatic patients with HSPPC-96 derived from the autologo...
VACCINATION WITH HSPPC-96 IN METASTATIC COLORECTAL CARCINOMA (CRC) Clinical and immunologic responses (Mazzaferro et al., ...
DISEASE-FREE SURVIVAL   ACCORDING TO T CELL IMMUNE RESPONSE IN PATIENTS WITH LIVER METASTASES FROM CRC 17 14 11 7 6 16 12 ...
OVERALL SURVIVAL   ACCORDING TO T CELL IMMUNE RESPONSE IN PATIENTS WITH LIVER METASTASES FROM CRC 17 17 17 14 11 Patients ...
HSPPC-96 vaccination increases CD8+T cell response to class I HLA-matched CRC lines in 16 out of 29 patients 501 537 698 4...
CONCLUSIONS Vaccination of patients with liver metastases of CRC with autologous HSPPC-96 is feasible and safe. Approximat...
TUMORS FOUND TO BE INFILTRATED BY T REGULATORY  LYMPHOCYTES <ul><li>Tumor Reference </li></ul><ul><li>NSCLC  Woo/June, 200...
Giorgio Parmiani Istituto Nazionale Tumori, Milan - Italy Mario Santinami Lorenzo Pilla Roberto Patuzzo Elisabetta Pennacc...
Phase III studies  of DC-based vaccines in patients with melanoma or prostate carcinoma  **Provenge (Dendreon) = Fusion pr...
DC-BASED VACCINATION IN PATIENTS WITH METASTASES -  C O N C L U S I O N S - <ul><li>DC-peptide based vaccines induced a fr...
Istituto Nazionale Tumori Milano Vaccination with 4 peptides  emulsified in Montanide   (MART-1, gp100, NY-ESO, survivin) ...
PATIENTS ENROLLED
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Transcript of "Giorgio Parmiani"

  1. 1. Forlì, March 1-2, 2007 Anti-cancer vaccines Peptide-based vaccines: The INT experience Giorgio Parmiani, MD Department of Oncology, San Raffaele Scientific Institute
  2. 2. Strategies of anti-cancer therapeutic vaccines 1. I rradiated and genetically modified tumor cells 2. TAAs 3. Antigenic peptides with adjuvants or dendritic cells 4. Tumor-derived h eat shock proteins - as whole proteins plus adjuvants <ul><li>as genes transduced in normal cells </li></ul><ul><li>as plasmid DNA </li></ul><ul><li>as DNA contained in viral or non-viral vectors </li></ul><ul><li>aS mRNA </li></ul>
  3. 3. Peptide-based vaccines <ul><li>Advantages : </li></ul><ul><li>Known sequence and biochemistry </li></ul><ul><li>Easy to synthesize (large availability) </li></ul><ul><li>Allow a specific immune-monitoring of the patient response </li></ul><ul><li>Allow to assess expression of targeted antigens in patient tumor cells </li></ul>
  4. 4. Peptide-based vaccines ( cont .) <ul><li>Disadvantages : </li></ul><ul><li>Easy degradable in absence of adjuvants </li></ul><ul><li>Require appropriate HLA allele to be recognized (patient selection) </li></ul><ul><li>Induce T cells that may not recognize tumor cells </li></ul><ul><li>Costs </li></ul>
  5. 5. Issues in peptide-based cancer vaccines <ul><li>Nature of tumor-associated antigens </li></ul><ul><li>Vaccine formulation/adjuvants </li></ul><ul><li>Quantity and quality of immune response </li></ul><ul><li>First generation of vaccines (1994-2004): Immmune and clinical response </li></ul><ul><li>Immune suppression/tumor escape </li></ul><ul><li>Regulatory </li></ul><ul><li>Costs </li></ul>
  6. 6. Tumor-associated antigens (TAA): Their nature is crucial for anti-tumor immune response
  7. 7. Antigens recognized by T cells (Novellino 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 )
  8. 8. 1 and 2. In vitro and ex - vivo T cell natural immune response to shared “self” TAAs
  9. 9. 0.0 0.1 0.2 0.3 0.3 0.8 1.3 1.8 2.3 2.8 3.3 n.s. *** 0.0 0.1 0.2 0.3 0.3 0.8 1.3 1.8 2.3 2.8 3.3 n.s. *** ** gp100 209-217 Tyrosinase 368-377 Melan-A/MART-1 26-35 FREQUENCY OF SPONTANEOUS CIRCULATING ANTI-MAA CD8+ T LYMPHOCYTES ( Mortarini et al., Cancer Res 2004 ) 0.0 0.1 0.2 0.3 0.3 0.8 1.3 1.8 2.3 2.8 3.3 *** *** ** *** Patients Donors Stage I-II Stage III-IV Patients Donors Stage I-II Stage III-IV Patients Donors Stage I-II Stage III-IV
  10. 10. <ul><li>CONCLUSIONS </li></ul><ul><li>A hierarchy exists in the natural recognition of “self” TAAs by T cells. </li></ul><ul><li>MelanA/MART-1 e CEA induce the most frequent T cell response in melanoma and CRC. </li></ul><ul><li>Recognition of “self” TAAs increases with the increased tumor burden. </li></ul>
  11. 11. <ul><li>Antigens recognized by T cells: </li></ul><ul><li>Shared, self differentiation proteins expressed also on normal cells (e.g. MART-1, CEA, PSA) </li></ul><ul><li>Shared self Cancer/Testis expressed by different tumors and by normal testis or placenta (e.g. MAGE, NY-ESO-1) </li></ul><ul><li>Shared but predominantly expressed on tumor cells (e.g. RAS/m, survivin , hTERT) </li></ul><ul><li>Unique, expressed only by a single tumor (e.g. CDK4/m, α -actin-m) </li></ul>
  12. 12. SURVIVIN (SVV) <ul><li>Expression pattern: </li></ul><ul><ul><li>abundant during fetal development </li></ul></ul><ul><ul><li>silenced in normal adult tissues </li></ul></ul><ul><ul><li>over-expressed in most common human cancers </li></ul></ul>• Member of AIPs (Apoptosis Inhibitory Proteins ) Survivin is a universal TAA that can elicit both HLA-I and HLA-II-restricted T cells
  13. 13. IN VITRO INDUCTION OF HLA CLASS I-RESTRICTED ANTI-SVV T CELLS IN PBMCs OF RECTAL CANCER PATIENTS Autologous PBMCs SVV-1 Medium IFN γ spots/50,000 cells Patient 1 Patient 2 + anti-HLA-I Ab ( Casati et al., Cancer Research 2003 )
  14. 14. N. IFN gamma spots/2x10 5 PBMCs In vitro recognition of prostate antigens-deriving peptides by T lymphocytes of prostate cancer patients
  15. 15. <ul><li>Antigens recognized by T cells: </li></ul><ul><li>Shared but expressed on tumor cells only </li></ul><ul><li>Shared Cancer/Testis expressed by different tumors and by normal testis or placenta </li></ul><ul><li>Shared, differentiation proteins expressed also on normal cells </li></ul><ul><li>Unique , mutation-derived,expressed only by a single tumor ( e.g. CDK4/m, α -actin-m ) </li></ul>
  16. 16. Unique human TAAs recognized by HLA-restricted T cells (Parmiani et al., Journal of Immunology 2007 ) <ul><li>Tumor No. Class I No. Class II </li></ul><ul><li>Melanoma 11 6 </li></ul><ul><li>NSCLC 3 1 </li></ul><ul><li>H/N 2 0 </li></ul><ul><li>RCC 2 1 </li></ul><ul><li>Bladder cancer 1 </li></ul><ul><li>Total 19 8 </li></ul>Most of these unique TAA have a pathogenetic role in neoplastic transformation/ progression.
  17. 17. Strategies of anti-cancer therapeutic vaccines 1. I rradiated and genetically modified tumor cells 2. TAAs 3. Antigenic peptides with adjuvants or dendritic cells 4. Tumor-derived h eat shock proteins - As whole proteins plus adjuvants <ul><li>As genes transduced in normal cells </li></ul><ul><li>As plasmid DNA </li></ul><ul><li>As DNA contained in viral or non-viral vectors </li></ul><ul><li>AS mRNA </li></ul>
  18. 18. 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 in 20-50% of patients but limited expansion of T cells (<1%).
  19. 19. Phase I/II studies of DC-peptide based therapeutic vaccines in melanoma DC+ antigen No.Pts Responses Ref. Immunologic Clinical Peptides 1 or lysate 32 11 2 CR, 6 PR Nestle 2001 HLA-A2 peptides 1 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.
  20. 20. Rationale for a new generation of peptide-based vaccines at INT of Milan <ul><li>Multiple peptide including universal TAAs </li></ul><ul><li>New TLR targeting adjuvants (CpG, HSP) </li></ul><ul><li>Down-regulation of Tregs and/or Myeloid Suppressor Cells ( see Licia Rivoltini) </li></ul><ul><li>Immune-monitoring in blood and LNs </li></ul><ul><li>Assessment of patient polymorphisms </li></ul>
  21. 21. <ul><li>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 . </li></ul>
  22. 22. 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 of specific T cells Unit of Immunotherapy of Human Tumors CPA (300 mg/m 2 ) Blocking of suppressive effect of CD4+ CD25+T reg on lymphocyte proliferation Montanide Recruitment of pro-infliammatory cells (APC)
  23. 23. Vaccination with peptide deriving from prostate TAAs in association with Montanide and CPA Group 1 – Adjuvant Vaccination <ul><li>Malattia localizzata a livello prostatico, resecabile </li></ul><ul><li>Vaccinazione in regime neoadiuvante ed adiuvante dopo chirurgia </li></ul>1° cycle vaccine 2° cycle vaccine C C -3 1 w C C -3 1 month C C Diagnostic biopsy Prostatectomy Unita’ di Immunoterapia dei Tumori Umani Analysis of induction of T lymphocytes recognizing vaccine peptides in PBLs Analysis of tumor infiltrate
  24. 24. Results: Immunologic and clinical responses
  25. 25. Vaccination of prostate cancer patients: Clinical response After the first cycle of vaccination (PSA 1.04 ng/ml -> 0.27 ng/ml) 009 Aftrer the first cycle of vaccination (PSA 0.51 ng/ml -> 0.44 ng/ml) 008 After the first cycle of vaccination (PSA 1.33 ng/ml -> 1.01 ng/ml) 007 PD 006 PD 005 Biochemical SD at 6 months and CR of a prostatic lesion 004 Biochemical SD at 6 months from interruption of hormone therapy 003 Biochemical SD at 8 months from interruption of hormone therapy 002 Biochemical SD at 10 months from interruption of hormone therapy 001 Clinical outcome Patients
  26. 26. 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 <ul><li>G. Parmiani, M. Santinami, L. Pilla, G. Cutolo, A. Maurichi, R. Patuzzo, A. Carbone, G. Tragni, C. Castelli, L. Rivoltini (INT); </li></ul><ul><li>M. Maio (Siena),V. Chiarion (Padua) </li></ul><ul><li>Ethics Committee approval: May 2004. </li></ul><ul><li>Delayed due to the long term negotiation with Coley to obtain CpG-ODN (not provided). </li></ul><ul><li>First patient enrolled in July 2005 </li></ul>
  27. 27. <ul><li>Primary end points: Immune response (frequency and intensity) </li></ul><ul><li>ELISPOT : Frequency of anti-tumor specific T lymphocytes </li></ul><ul><li>Flow-cytometry: Phenotype of anti-tumor T cells </li></ul><ul><li>- HLA tetramers </li></ul><ul><li>Cytokine intracellular staining </li></ul><ul><li>Immunohistochemistry : characterization of tumor cells and of TILs </li></ul><ul><li> </li></ul>
  28. 28. Clinical objectives Primary: Local and systemic toxicity Secondary: Disease-free survival after 2/3 years Overall survival at 5 years
  29. 29. No. of assessed patients: 210
  30. 30. ENROLLED PATIENTS: 28 Stage II: 6 Stage III: 22 Control arm: 13 Vaccination arm: 15
  31. 31. SVV as universal TAA : - Conclusions - <ul><li>SVV is expressed by melanoma; colon, rectal and prostate cancer cells </li></ul><ul><li>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 </li></ul><ul><li>SVV can then be considered a shared, universal tumor-restricted antigen that is being used in clinical studies of vaccination </li></ul>
  32. 32. <ul><li>Antigens recognized by T cells: </li></ul><ul><li>Shared but expressed on tumor cells only </li></ul><ul><li>Shared Cancer/Testis expressed by different tumors and by normal testis or placenta </li></ul><ul><li>Shared, differentiation proteins expressed also on normal cells </li></ul><ul><li>Unique , mutation-derived,expressed only by a single tumor ( e.g. CDK4/m, α -actin-m ) </li></ul>
  33. 33. Vaccination with unique antigens: how is it possible? 1.Autologous gene-modified tumor cells 2. Autologous proteins/peptides
  34. 34. Autologous tumor-derived heat shock-protein/ peptides complexes
  35. 35. MECHANISMS OF IMMUNE EVASION Why a more strong immune response and/or a more frequent clinical response was not observed in vaccination trials ?
  36. 36. FACTORS THAT CAN INTERFERE WITH THE RECOGNITION AND LYSIS OF TUMOR CELLS BY T LYMPHOCYTES <ul><li>Tumor </li></ul><ul><li>Lack of antigenic peptide or down-regulation of HLA </li></ul><ul><li>Dysfunction of antigen presentation </li></ul><ul><li>Release of immune-suppressive factors (IL-10, TGF  , VEGF) </li></ul><ul><li>Tumor counterattack (Fas/FasL) </li></ul><ul><li>Cannibalization of T lymphocytes </li></ul><ul><li>Immune system </li></ul><ul><li>Immune anergy or ignorance </li></ul><ul><li>Lack of tissue homing molecules; defective adhesion </li></ul><ul><li>TCR dysfunction </li></ul><ul><li>Inactivation of T cells within the tumor (granzyme B) </li></ul><ul><li>Induction of anti-peptide but not anti-tumor T cells </li></ul><ul><li>T regulatory cells </li></ul><ul><li>Myeloid suppressive cells </li></ul>
  37. 37. Acknowledgments Chiara Castelli Licia Rivoltini Chiara Camisaschi Gianluca Cutolo Veronica Huber Manuela Iero Andrea Marrari Luisa Novellino Lorenzo Pilla Roberta Valenti Agata Cova Francesca Rini Gloria Sovena Paola Squarcina Istituto Nazionale Tumori Milan - Italy Vincenzo Mazzaferro Jorgelina Coppa Mario Santinami Roberto Patuzzo Ermanno Leo Filiberto Belli Flavio Arienti Alfonso Marchianò Grazia Barp Annabella Di Florio Pramod K. Srivastava Antigenics, New York Michele Maio Siena
  38. 39. Take home message. Perspectives 1 <ul><li>Antigens : use both cross-reacting self and unique TAA; use both classes I and II HLA-restricted peptides. </li></ul><ul><li>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 ). </li></ul><ul><li>Tumor burden : avoid excess tumor burden to prevent release of suppressive factors. </li></ul>1
  39. 40. Perspectives 2 <ul><li>Escape mechanisms : use compounds which may help in restoring normal immune functions (e.g. retinoic acid, cytokines, anti-Treg Ab ). </li></ul><ul><li>Combination therapy : vaccination may be combined with anti-angiogenic drugs, radiotherapy and immune-modulating chemotherapy ( non-myeloablative lymphodepletion? ) </li></ul><ul><li>Modulation of the immune response by anti-Treg and/or anti-CTLA4 antibodies. </li></ul>2
  40. 41. REGULATORY T LYMPHOCYTES
  41. 42. Treg assessment during peptide-based vaccination and modulation by Cyclophosphamide <ul><li>Recurrent rectal carcinoma patients receiving a peptide-based vaccine ( no IL-2) </li></ul><ul><li>Stage III melanoma patients receiving a multipeptide-based vaccine ( with IL-2 ) </li></ul>
  42. 43. % of CD25+ Foxp3+ (in CD4) Pt#4 Pt#3 Pt#2 Pt#1 Treg (CD4+CD25+Foxp3+) in PBMC of rectal carcinoma patients and their modulation by CTX treatment
  43. 44. CD4+CD25 high CD4+CD25 high CD4+CD25 high % of positive cells CD4+CD25+ CD25+Foxp3+(in CD4) CD4+CD25 brigh Pt#1 Pt#2 Treg in PBMC of Melanoma patients: modulation by CTX and IL-2 treatment
  44. 45. Studi clinici di vaccinoterapia in assenza di cellule dendritiche in pazienti con carcinoma prostatico Vaccino N. HLA Risposta R i s p o s t a Ref. pazienti immune clinica SART, p56LCK , 10 A2402 4/10 1 PR* Naguchi, ’03 ART, CyB 5 SD SART, p56LCK, 16 A2402 10/14 13/13  ** Naguchi, ’05 ART, 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.
  45. 46. Features of peptide vaccines <ul><li>Peptide length : </li></ul><ul><li>8-10 aa for HLA class I-restricted peptides </li></ul><ul><li>12-23 aa for HLA class II-restricted peptides </li></ul><ul><li>Modified peptides (heteroclitic and/or long peptides) </li></ul><ul><li>Highly and rapidly degradable in vivo (adjuvants are required) </li></ul>
  46. 47. 1, 2: Shared “self” TAAs <ul><li>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 .). </li></ul><ul><li>Tolerance needs to be broken in order to induce a T cell immune response against “self” TAAs. </li></ul><ul><li>Thus, these TAAs are considered to be “ weak antigens ” </li></ul>
  47. 48. Natural CD8+T cell response of HLA-A2 colorectal cancer (CRC) patients to differentiation antigens (Nagorsen et al., Cancer Res 2000) * ELISPOT assay. Antigen Clinical N. of responding stage N. of tested* Ep-CAM Donors 0/8 A-B 0/9 C-D 4/13 Her2/neu Donors 0/8 A-B 0/9 C-D 5/13 CEA Donors 0/8 A-B 0/9 C-D 6/13
  48. 49. N. IFN γ spots/3x10 4 cells med T2+PSMA T2+PSMA + anti-HLA-I CaP + anti-HLA-I CaP Anti-PSMA T cells CaP Anti-svv T cells med T2+svv T2+svv + anti-HLA-I CaP + anti-HLA-I N. IFN γ spots/3x10 4 cells Fresh PBMC med PSMA svv Flu N. IFN γ spots/10 6 cells 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 IFN γ , 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 IFN  attraverso analisi immunoenzimatica Elispot.
  49. 50. 1 2 3 5 4 6 1 2 3 5 4 6 7 8 9 10 7 8 9 10 T2 + Survivin T2 + Survivin + anti-HLA-I T2 + PSMA P1 T2 + PSMA P1 + anti-HLA-I N. of spots per 30 4 cells N. of spots per 30 4 cells Frequency of TAA-specific T cells precursors after in vitro stimulation with prostatic peptides No. of microcultures tested/patient
  50. 51. Ex-vivo recognition of prostate antigens by patient T cells <ul><li>No. of patients tested: 112 </li></ul><ul><li>No. of patients whose peripheral blood T lymphocytes recognized prostate antigens: 65 (58%) </li></ul>
  51. 52. Studio di fase III di vaccinazione con DC di pazienti con carcinoma della prostata ND: non disponibile *Provenge= Proteina di fusione PAP+GM-CSF su cellule dendritiche N. Pazienti Antigene Risposta Risposta P.I. immune clinica 110 vs 108 Provenge* ND Mediana E.Small, Metastatici, vs placebo sopravvivenza 2005 ormone 25.9 vs 21.9 resistenti OS 34% vs 11%
  52. 53. Patient 8- 3R MHC-CLASS II RESTRICTED RESPONSE OF COLORECTAL CANCER PATIENTS TO rSVV PROTEIN OR CANCER LYSATE IFN-  ELISA IFN-  pg/ml Patient 1- 3R IFN-  pg/ml (Casati C et al., Cancer Research, 2003) 0 500 1000 1500 2000 2500 = 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
  53. 54. The response of autologous T cells to a human melanoma is dominated by mutated neoantigens ( Lennerz V et al., PNAS , 2005 ) <ul><li>Five neoantigens deriving from somatic mutations were found at different time points in different metastases of a melanoma patient. </li></ul><ul><li>CD8+ cytotoxic T cells against mutated TAA were predominant over shared differentiation self TAA over 4-5 years of testing. </li></ul>
  54. 55. 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.
  55. 56. Are unique TAAs spontaneously recognized in v ivo ? T lymphocytes recognizing unique TAAs have been described in long-term surviving patients previously affected by melanoma; NSCLC; head/neck, bladder and renal carcinoma (T. Wolfel, P. Coulie, G. Parmiani, F. Triebel)
  56. 57. Phase I-II studies of DC based therapeutic vaccines in prostate cancer DC 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
  57. 58. Autologous monocytes pulsed with melanoma-derived HSP70 are recognized by specific T cell clones (from Castelli et al., Cancer Res. 2001) IFN-  (pg/ml) Anti-gp100 APC Medium HSP70 HSP70 + W6.32 Auto-Me Anti-MART-1 APC Medium HSP70 Auto-Me Statistically significant inhibition. HSP70 + W6.32 W6.32 = anti-HLA-I mAb Auto-Me = autologous melanoma
  58. 59. Some recent good news in anti-cancer vaccination
  59. 60. 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 - * Well balanced for pathological state, primary lesion thickness, etc.
  60. 61. Phase II study of vaccination of hormone-refractory metastatic prostate cancer patients with GVAX ® Cell Genesys , ASCO 2005 <ul><li>No. of patients 22 </li></ul><ul><li>Median survival time of vaccinated patients was over 29 months. </li></ul><ul><li>Median survival time of Taxotere-treated patients is 19 months. </li></ul><ul><li>Phase III trials ongoing . </li></ul>
  61. 62. A phase IIb randomized study of vaccination with L-BLP25 in stage IIIb and IV NSCLC after first line chemo <ul><li>Vaccine : Liposome-MUC1 </li></ul><ul><li>Overall median survival : 17.4 vs . 13 months in 88 vs . 83 patients </li></ul><ul><li>Overall 2 years survival : 43.2 vs . 28.9 %; subset stage IIIb : 60 vs .37 % </li></ul>Biomira/Merck KGaA, ESMO 2004
  62. 63. Phase II randomized trial of autologous formalin-fixed tumor vaccine for postsurgical recurrence of hepatocellular carcinoma Kuang et al. Clin Cancer Res 2004 <ul><li>No. of patients 41 , after curative resection </li></ul><ul><li>Vaccine treatment N= 19 ; no treatment N= 22 </li></ul><ul><li>Median follow-up of 15 months </li></ul><ul><li>Recurrence: Vaccinated 3/18 vs. Control 13/21 </li></ul><ul><li>Recurrence-free survival P=0.003; OS P= 0.01 </li></ul>
  63. 64. 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
  64. 65. Cancer Vaccine Pipeline (www.bioseeker.com) <ul><li>There are more than 260 non-antibody based vaccine drugs, therapeutic and preventive vaccines included. 87 are cancer vaccines. </li></ul><ul><li>Vaccines are being developed for over 30 different cancers; 38 companies are involved. </li></ul><ul><li>The number of vaccine targets has increased to over 50 different molecular targets </li></ul><ul><li>Vaccine market is predicted to be of 6 billions USD by 2010 </li></ul>
  65. 66. Clinical studies of vaccination with HSPPC-96 1. Liver metastases of colon cancer 2. Metastatic melanoma (HSPPC-96 only) 3. Metastatic melanoma (HSPPC-96 + GM-CSF + IFN)
  66. 67. Vaccination of patients with liver metastases of CRC with autologous HSPPC-96 - TREATMENT SCHEME -
  67. 68. In vivo expansion of anti-CEA T cells in HLA-A2 CRC patients vaccinated with HSPPC-96 CD8 2.14 HLA/ CEA tetramers 0.22 CD8 HLA/ CEA tetramers Day 1------7-------14------21 V1 V5 Patient 045 N. spots/10 4 T cells V1 V5 * 0 100 200 T2 alone T2+CEA T2+CEA+W6.32 Colo206 N. spots/5x10 4 T cells CD8 CD8 Day 1---7—-14—21-------51 V1 V6 HLA/ CEA tetramers 0.11 2.31 V1 V6 * HLA/ CEA tetramers Patient 038
  68. 69. Resection Purification of HSP-GP96 Vaccination Vaccination of metastatic patients with HSPPC-96 derived from the autologous tumor Tumor sample Tumor cell suspension Immunologic monitoring in vivo : DTH in vitro : ELISPOT, tetramers staining
  69. 70. VACCINATION WITH HSPPC-96 IN METASTATIC COLORECTAL CARCINOMA (CRC) Clinical and immunologic responses (Mazzaferro et al., Clin Cancer Res 2003)
  70. 71. DISEASE-FREE SURVIVAL ACCORDING TO T CELL IMMUNE RESPONSE IN PATIENTS WITH LIVER METASTASES FROM CRC 17 14 11 7 6 16 12 9 7 12 1 1 9 1 1 p < 0.0001 Immune responders Immune non-responders Patients at risk Immune responders Immune non-responders September 2004
  71. 72. 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 14 Immune responders 12 9 6 3 11 6 5 2 p < 0.0001 2 Immune responders Immune non-responders Immune non-responders September 2004
  72. 73. HSPPC-96 vaccination increases CD8+T cell response to class I HLA-matched CRC lines in 16 out of 29 patients 501 537 698 449 N. spots/1.67 x 10 5 PBMC Patient number
  73. 74. CONCLUSIONS Vaccination of patients with liver metastases of CRC with autologous HSPPC-96 is feasible and safe. Approximately 60% of patients developed 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.
  74. 75. TUMORS FOUND TO BE INFILTRATED BY T REGULATORY LYMPHOCYTES <ul><li>Tumor Reference </li></ul><ul><li>NSCLC Woo/June, 2001 </li></ul><ul><li>Pancreatic/breast cancer Liyanage, 2002 </li></ul><ul><li>Colorectal cancer D. Herlyn, 2002 </li></ul><ul><li>Gastric/esophageal cancer Ichihara, 2003 </li></ul><ul><li>Melanoma Javia/Rosenberg, 2003 </li></ul><ul><li>Ovarian Cancer Curiel, 2004 </li></ul><ul><li>HCC Ormandy, 2005 </li></ul><ul><li>Breast cancer Bates, 2006 </li></ul>
  75. 76. Giorgio Parmiani Istituto Nazionale Tumori, Milan - Italy Mario Santinami Lorenzo Pilla Roberto Patuzzo Elisabetta Pennacchioli Alfonso Marchianò Licia Rivoltini Chiara Castelli
  76. 77. Phase III studies of DC-based vaccines in patients with melanoma or prostate carcinoma **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. 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, 2005 Metastatic, vs placebo survival hormone 25.9 vs 21.9 resistant OS 34% vs 11%
  77. 78. DC-BASED VACCINATION IN PATIENTS WITH METASTASES - C O N C L U S I O N S - <ul><li>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). </li></ul>
  78. 79. 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 : 3x10 6 IU (s.c.): to expand T cells activated by the vaccine
  79. 80. PATIENTS ENROLLED
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