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Corso CINBO Recent advances in the medical treatment of melanoma Roma 21/06/2013

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 (are T cells the main player?). NK/NKT. TCD8.

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Corso CINBO Recent advances in the medical treatment of melanoma Roma 21/06/2013

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  1. Corso CINBORecent advances in the medical treatment of melanomaRoma 21/06/2013 Pre-clinical and clinical aspects of peptide-based melanoma vaccines Giorgio Parmiani San Raffaele Foundation, Milano

  2. Tumor destruction by the immune system (are T cells the main player?) NK/NKT TCD8 Monocytes, DC c IL-2, IFN, TNF... B ly TCD4 Tumor-specific antibodies IL-2

  3. The Melanoma-Associated Antigens

  4. Melanoma-associated antigens (MAAs) recognized by T cells. Which is the best MAA or combination of? • Shared/self/differentiation MAAs (e.g. Mart1, tyrosinase) • Shared/self/cancer testis or germinal MAAs (e.g.MAGE, NY-ESO1) • Universal MAAs (survivin, hTERT) • Mutated, unique MAAs

  5. 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

  6. 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

  7. In vivospontaneoustolerance break (immunogenicity) ofshared selfMAAs. 2 examples

  8. StageI-II StageI-II StageI-II StageIII-IV StageIII-IV StageIII-IV Donors Donors Donors Patients Patients Patients FREQUENCY OF ANTI-MAA CD8 T LYMPHOCYTES AND PROGRESSION OF THE DISEASE gp100209-217 Tyrosinase368-377 Melan-A/Mart-126-35 *** *** *** *** ** ** n.s. *** n.s. 3.3 3.3 3.3 2.8 2.8 2.8 2.3 2.3 2.3 1.8 1.8 1.8 1.3 1.3 1.3 0.8 0.8 0.8 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.1 0.1 0.1 0.0 0.0 0.0

  9. CONCLUSIONS • A hierarchy exists in the spontaneous recognition of “self” TAAs. • Recognition of “self” TAAsincreases with the increased tumor burden (e.g. Melan-A/MART1)

  10. Break of tolerance to shared self TAAs by vaccination

  11. 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

  12. 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)

  13. Results of first generation (1998-2006) of self peptide-based vaccination of metastatic melanoma patients (Phase I/II studies). In a recent study Slingluff et al.(2008) reported 100% immune response and survival benefit in melanoma patients vaccinated with 12 peptides.

  14. Vaccination with MAGE3.A1 peptide Multicentric European Study Ludwig Institute,Brussels; INT and HSR, Milano Adapted from M.Marchand et al., Int.J. Cancer 1999 14

  15. Use of dendritic cells:which progress ? see Ridolfi’s presentation

  16. 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?

  17. Reasons for the limited clinical response

  18. 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

  19. …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.

  20. Possible examples

  21. Myeloid suppressor cells are increasedin peripheral blood of stage II-III melanoma patients N=30 p<0.01 p<0-01 N=40 % CD14+CD11b+HLA-DR-/lo N=40 STAGE IV STAGE IIB-IIIC HD MELANOMA

  22. Increased frequency of CD4+CD25Foxp3+Treg is a late event in melanoma patients p= 0.0002 n.s. p= 0.002 p=0.04

  23. Monitoring T regs : unpredictable effect of low-dose cyclophosphamide on the frequency of CD4+CD25highFoxp3+ 10 IIB-IIIC HIGH RISK MELANOMA PATIENTS 8 300mg/m2 3-5 days before vaccination 6 % CD4+CD25highFoxP3+ cells (gated on lymphocytes) cyclophosphamide 4 2 0 P1 P4 cyclophosphamide P0 HD VACCINATION ARM

  24. Cyclophosphamide is effective in a different trial with RCC patients (Walter et al., Nature Med 2013)

  25. de010203040020406080100Time (months)Percentage survival3+Cy 1+Cy 0–Cy 0–Cy 1210010203040020406080Percentage survival100Time (months)bc0246810020406080100Time (months)Percentageprogression-free survival+ Multipeptide vaccination In RCC patients

  26. Conclusions Myeloid-derived suppressor cells are increasedin the peripheral blood of stage II-III melanoma patients Increased frequency of CD4+CD25FoxP3+Tregs is a late event in melanoma patients Not clear the effect of low-dose cyclophosphamide on the frequency of CD4+CD25+Foxp3+

  27. 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)

  28. Phase III study of gp100 peptide vaccine in melanoma • A phase III multi-institutionalrandomized 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

  29. 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)

  30. EVIDENCE FOR CLINICAL ACTIVITY OF CANCER VACCINES

  31. Ongoing Phase II trials of active cancer immunotherapy: number of trials by immunotherapy and cancer type

  32. Self peptide-based melanoma vaccines: Conclusions • A phase IIIstudys 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.

  33. 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

  34. 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

  35. 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?

  36. 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

  37. Combination with other therapeutic agents (e.g. anti-vascular agents)

  38. Targeted delivery of TNF to tumor vasculature by coupling with the NGR-peptide (a ligand of CD13 expressed byangiogenic vessel) TNF NGR-TNF CNGRCG peptide The antitumor activity of NGR-TNF is >10 times greater than that of TNF in several mouse models Curnis et al., Nat. Biotechnol., 2000 Sacchi et al., Clin. Cancer Res. 2006

  39. Combination of vaccine and NGR-TNF in animal models NGR-TNF DC/OVA DC/OVA NGR-TNF % Surviving Animals Day since Tumor Challenge 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 Calcinotto et al. J Imunology 2012

  40. 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

  41. 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.

  42. 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+)

  43. Antigens recognized by T cells: • Shared but expressed on tumor cells only • Shared Cancer/Testis expressed by different tumors and by normal testis or placenta • Shared, differentiation proteins expressed also on normal cells • Unique, mutation-derived,expressed only by a single tumor(e.g. CDK4/m, α-actin-m)

  44. Proteins derived from somatically mutated cancer genes give raise to neo-peptide antigens presented by MHC molecules neo- peptide neo- peptide

  45. 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

  46. PREDICTED EPITOPES DERIVED FROM APC MUTATION MISSENSE A3079T (Y->L) BINDING SOME RAPRESENTATIVE HLA-A ALLELES MUTATED SEQ score W.T. SEQ score HLA-A*01 DTPINLSLKY25DTPINYSLKY 25 ELDTPINLSL 14ELDTPINYSL 11 HLA-A*01 11 LDTPINLSLKY 20 LDTPINYSLKY 20 ELDTPINLSLK 14 ELDTPINYSLK 13 HLA-A*0201 ELDTPINLSL 20 ELDTPINYSL 20 NLSLKYSDEQ 12 NYSLKYSDEQ 2 HLA-A*03 NLSLKYSDE 15 NYSLKYSDE 5 TPINLSLKY 13 TPINYSLKY 13 LDTPINLSLK 16 LDTPINYSLK 16 ELDTPINLSL 15 ELDTPINYSL 12 HLA-A*1101 DTPINLSLK 22 DTPINYSLK 22 HLA-A*26 DTPINLSLKY 28 DTPINYSLKY 28 ELDTPINLSL 20 ELDTPINYSL 19 DGELDTPINL 17 DGELDTPINY 19 HLA-A*6801 DTPINLSLK 24DTPINYSLK 20 The maximal score for peptides binding HLA-A*0201 is 36. Influenza A matrix GILGFVFTL scores 30

  47. 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

  48. 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.

  49. 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)

  50. END OF THE PRESENTATION. • Thank you for your attention!

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