1. Biomarkers of prognosis, cancer cellkillers of cytotoxic T lymphocytes (CTL)and autologous tumor antigen (vaccines)arising from MAGE genesCheng Luo, Ph. D University of Tartu

2. Why MAGE genes?What is MAGE genes? 3. Metagenes associated to survivalsGenes up-regulated in NSCLCGene ID adj. p-value Fold changeSPP1 2,76E-17 14,52MMP15,20E-1110,74KRT6A 3,85E-0510,39KRT17 1,86E-069,44CTHRC1 2,76E-17 7,62S100A22,21E-057,44MMP11 1,29E-11 7,14TMPRSS4 1,04E-08 6,85CDC201,59E-136,82KRT6B7,41E-046,15Genes down-regulated in NSCLCGene ID adj. p-value Fold changeSFTPC 7,13E-1024,43FCN3 6,77E-1716,71FABP4 3,28E-14 13,54C19ORF595,43E-20 12,51TMEM1001,65E-2112,11CYP4B1 1,35E-12 11,41CLIC5 9,37E-2110,99SFTPD 4,18E-0810,75SFTA2_HUMAN 4,02E-07 10,59CLEC3B 9,66E-22 10,23Urgard E. et al. 2011 4. Melanoma Antigen GEne (MAGE), or Cancer/Testis(CT gene) frequently expressed in Melanoma andother cancer cells, MAGE gene was first reported inmelanoma. CT genes include MAGE(s) and others.CT1:MAGEA, CT3:MAGEB,. 5. MAGE/COX2/Nrf2 genesFrom: Ari Ristimki To: Cheng Luo Sent: Monday, October 24, 2011 9:52 AMSubject: Re: Cluster these genes?Very interesting, you should publish this, Ari from Stockholm (UEGW) 6. Mougiakako D. et al. 2010 7. Andrade VCC et al 2008 (Brazil cohort) percentage of MAGE genes expression in NSCLC (Estonia cohort)706050403020100 8. Gene expression: MAGEB1 (melanomaantigen family B, 1)http://biogps.org 9. Gene expression: MAGEA3(melanoma antigen family A, 3)http://biogps.org 10. Gene expression: MAGEA10(melanoma antigen family A, 10) http://biogps.org 11. Gene expression: MAGEA9 B http://biogps.org 12. Evolutionary History of the CancerImmunity Antigen MAGE Gene Family 13. Evolution (Phylogeny) of Human MAGE genesKatsura et al 2011 (MHD) Can be processed to be epitopes in cancer cells Phase IV (human only)Phase II Phase I 14. 6-7 million years agoPalindrome inMAGE genesReverable palindrome structure play similar roles asmethylation and demethylation 15. MAGE gene is of high CNVCT genes on chromosome X are evolvingMost MAGEfaster than those on other chromosomes genes are coded by X- chromosmeY 16. Cell function of MAGE genesXiao TZ et al. 2011 Autoregulation and accuracy of KRAB-MAGEsXiao T. et al. 2011 Plos One 17. MAGEA10: One of the first Cancerantigens 18. From unknown cancer antigen 1999, J. of Immunolo 19. Antibody producing CTL killer 20. The assembly of MHC Class I molecules (endogenous) 21. Group Leader Pierre van der BruggenMAGE gene was designated in 1991 Contact Pierre van der Bruggen de Duve Institute, Universit catholique de Louvain, and Ludwig Institute for Cancer Research LICR - B1.74.03, Avenue Hippocrate 74, B-1200 Brussels phone (direct) : 32 (0)2 764 74 31 fax : 32 (0)2 792 94 05 e-mail: Pierre van der Bruggen Tumor immunology, or cancer immunotherapy start: van der Bruggen P, et al 1991, A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.. Science. 1991 Dec 13;254(5038):1643-7Barker PA, Salehi A.The MAGE proteins: emerging roles in cell cycle progression, apoptosis, andneurogenetic disease. J Neurosci Res. 2002 Mar 15;67(6):705-12.Since the identification of the first MAGE gene in 1991, the MAGE family has expandeddramatically, and over 25 MAGE genes have now been identified in humans. The focus of studies onthe MAGE proteins has been their potential for cancer immunotherapy, as a result of the finding thatpeptides derived from MAGE gene products are bound by major histocompatibility complexes andpresented on the cell surface of cancer cells. However, the normal physiological role of MAGE proteinshas remained a mystery. Recent studies are now beginning to provide insights into MAGEgenefunction. Necdin acts as a cell cycle regulatory protein and plays a key role in the pathogenesis ofPrader-Willi syndrome, a neurogenetic disorder. MAGE-D1, identified as a binding partner for the p75neurotrophin receptor, the apoptosis inhibitory protein XIAP, and Dlx/MSX homeodomainproteins, blocks cell cycle progression and enhances apoptosis. This review provides an overview of thehuman MAGE genes and proteins, summarizes recent findings on their cellular roles, and provides abaseline for future studies on this intriguing gene family. 22. TIB and MAGE-B2 antibody in serum 23. http://www.immunocore.com/technology/specificity/ This video was filmed over a period of 11 hours and demonstrates the specificity of the MAGE-A3 ImmTAC. The target cancer cells with HLA-A1 and the MAGE A3 antigen are dyed red; control cells which are also HLA-A1 but which do not have the antigen MAGE A3 are dyed green. CD8+ T cells are unstained and appear grey. When the MAGE specific ImmTAC is added to the experiment at 50pM concentration all of the target cells (red) are killed by the T cells whilst the innocent bystanders (green) are unaffected. 24. Cancer cells loadedanti-tumor with MHC I andT cells in MAGE3Amelanoma A CTL clone (pulsedpatients by MAGE3A) that recognize MAGE3 ACTLisolatedfromtumors andexpanded Cancer cell without MHCfor severalor MAGE3Aweeks invitro, lysedautologousmelanomacells Start of the video 25. End of the video 26. Non-specific T cells (blue) recognise and kill target melanoma cells (red) but ignore non-target cells (green) in the presence ofthe drug, IMCgp100. http://www.immunocore.com/pipeline/imcgp100/ 27. For antibody (or humoral) mediated immunology 28. MAGE antigen (epiopes) presentedby CD8+ T-cellsTCR 29. >100 million TCR5.3. MAGE-A3 vaccineMAGE-A3 is a tumor-associated antigen that is notexpressed in most normal cells [31,61]. Approximately 3550% of lung cancers express MAGE-A3, and expression hasbeen associated with poor prognosis [31]. This providedthe rationale for the development of a TCV comprisingMAGE-A3 recombinant protein combined with the AS02Bimmunoadjuvant. Mellstedt H. et al 2011 30. Source of tumor antigens:MAGE Epitopeshttp://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm 31. MAGE antigens: From nuclearprotein to epitopes 32. By experimentalhttp://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm 33. Immunotherapy database (successful, or the peptide with potential because ofhigh stringency):http://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm By experimental 34. Table 3. Differentiation antigens By experimental 35. Table 4. Antigens overexpressed in tumors By experimental 36. Potential epitopes of HLA-A (10AA) of MAGEA10for lung cancer patiensts by BIMAS algorithms BioInformatics & Molecular Analysis Section (BIMAS) 37. MAGE antigens for HLA-II were also determined (The A, B, C, E, F, and G genes belong to MHC class I, six D genes belong to class II)HLA II alsoin CTA, orMAGEantigensMAGE genescan beimprinted 38. An alignment of primate MAGE-A amino acid sequences for an epitope coding region. In humans, based onreferences (116), MAGE-A epitopes for HLA alleles are denoted by squares (magenta; HLA class , light blue; HLAclass II). HLA alleles that recognize each epitope are indicated in parallel below. Among 13 amino acid substitutionsbetween MAGE-A3 and -A6, 11 substitutions marked by stars occur in the alignment whereas two substitutions(P303L, A308V) occured outside of the region. Among the 11 substitutions, ten that contribute to the production ofepitopes for different HLA alleles(E115K, D156L, L175V, T199A, L201F, V205I, K211R, D249H/D249Y, L279V/L279I, H298R) are indicated by green stars.The other substitution within this region (indicated by a blue star; F239L) does not contribute to the production ofepitopes of MAGE-A3 and -A6 [5368]. 39. Correlation of MAGE gene expression and survivals 40. 2 12 4 8 0 6 10 lung, squamous cell carcinoma Lung-Ca 617 lung, adenocarcinoma Lung-Ca 674 lung, squamous cell carcinoma Lung-Ca 619lung, adenocarcinoma Lung-Ca 1104lung, squamous cell carcinoma Lung-Ca 746 lung, adenocarcinoma Lung-Ca 572 Lung Cancerlung, squamous cell carcinoma Lung-Ca 576lung, squamous cell carcinoma Lung-Ca 268lung, large cell carcinoma Lung-Ca 735 lung, adenocarcinoma Lung-Ca 743lung, squamous cell carcinoma Lung-Ca 1086 lung, adenocarcinoma Lung-Ca 814MAGEA3 expression lung, adenocarcinoma Lung-Ca 481 lung, adenocarcinoma Lung-Ca 721 lung, adenocarcinoma Lung-Ca 562lung, squamous cell carcinoma Lung-Ca 554 lung, adenocarcinoma Lung-Ca 865 lung, adenocarcinoma Lung-Ca 670 lung, squamous cell carcinoma Lung-Ca 777lung, adenocarcinoma Lung-Ca 1102 Determinant of Worse Survival in Stage IA Non-Small Cell lung, squamous cell carcinoma Lung-Ca 591 Port JL et al. 2009, MAGE-A3 Expression is an Independentlung, normal Lung Normal 1050 lung, normal Lung Normal 567lung, squamous cell carcinoma Lung-Ca 265lung, squamous cell carcinoma Lung-Ca 582lung, normal Lung Normal 1054 lung, adenocarcinoma Lung-Ca 729 lung, adenocarcinoma Lung-Ca 593lung, squamous cell carcinoma Lung-Ca 494lung, squamous cell carcinoma Lung-Ca 1087lung, normal Lung Normal 551 lung, adenocarcinoma Lung-Ca 715 lung, normal Lung Normal 549 lung, carcinoma Lung-Ca 863lung, squamous cell carcinoma Lung-Ca 625 lung, normal Lung Normal 548 lung, adenocarcinoma Lung-Ca 820lung, squamous cell carcinoma Lung-Ca 560lung, squamous cell carcinoma Lung-Ca 676 lung, large cell carcinoma Lung-Ca 578 41. Example of MAGEexpression isassociated to goodprognosisGrau et al., MAGE-A1expression is associatedwith good prognosis inneuroblastoma tumors, J. of Cancer Researchand ClinicOncology, 2009 42. Example of MAGEexpression isassociated to goodprognosis 43. Condomines et al. 2007, J. of Immunology 44. MAGE Expressions Mediated by Demethylation of MAGE Promoters Induce Progression of Non-small Cell Lung CancerYANAGAWA N. et al. 2011Correlation between MAGE expression and overall survival of 67 NSCLC patients using the Kaplan-Meier method.The patients with any expression had poorer prognosis than those with no expression. 45. Boehmer et al. 2011, MAGE-C2/CT10 Protein Expression Is anIndependent Predictor of Recurrence in Prostate Cancer, PLos One 46. Gure A. et al. 2005, Clinic CancerResearch,Survival of patients with NSCLCstratified according to CT-Xexpression and pathologic stage.Distributions were estimated usingthe Kaplan-Meier method. Tick marksrepresent the time of last follow-upfor patients who remained alive.Representative series. A, high-level MAGE-A3 expression inadenocarcinoma patients of stage I(P = 0.04). B, high level NY-ESO-1 expression in adenocarcinomapatients of stage II (P =0.02). C, SSX2 expression in patientswith adenocarcinoma of stage III (P =0.05). 47. Estonia cohort By Krista Fischer & Reedik Mgi 48. Estonia cohort By Krista Fischer & Reedik Mgi 49. Estonia cohort By Krista Fischer & Reedik Mgi 50. Estonia cohort By Krista Fischer & Reedik Mgi 51. Estonia cohort By Krista Fischer & Reedik Mgi 52. Estonia cohort By Krista Fischer & Reedik Mgi 53. Estonia cohort By Krista Fischer & Reedik Mgi 54. The barrier of immunotherapy 55. Grgoire Wiers, Nathalie Demotte, Danile Godelaine and Pierre vander Bruggen * 2011 Immune Suppression in Tumors as aSurmountable Obstacle to Clinical Efficacy of Cancer Vaccines 56. Immuneactivationin favorCytokines influence T-helpersImmune suppressivein favorPanel A: Immune-mediated tumour killing requires a Th1 microenvironment. In the presence of Th1 cytokines, tumours upregulate MHC I and co-stimulatory molecules necessary for cytolytic T-cells (CTL) to recognise and kill the tumours. In addition, the inflammatory Th1 environment cannon-specifically activate T-cells to kill tumours through FasL and TRAIL effector molecules. Panel B: Tumours condition the microenvironment tohave a dominant Th2-bias as a strategy to escape immune mediated attack. In the presence of Th2 cytokines, tumours down-regulate MHC and co-stimulatory molecules, attract suppressor cells such as Treg and myeloid suppressors which suppress killer cell function. The Th2 environmentdown-regulates co-stimulatory molecules on APC which serves to anergise any Th1 cells that may infiltrate the tumour, whether as a naturalimmune response or through tumour vaccination strategies. Therefore, strategies to boost Th1 immunity alone are not sufficient to mediate anti-tumour immunity. Sustained Th1 cytokine production is required in the tumour microenvironment. 57. CD8 T (for MHC I) cells are neededMDSC: myeloid-derived suppressor cells 58. Some successfulexamples even though the obstacles ofimmunotherapy incancer medication because of immunosuppressiveenvironmentWiers, der Bruggen et al2011, Immune Suppression in Tumorsas a Surmountable Obstacle toClinical Efficacy of Cancer Vaccines 59. patients who received at30patients least four vaccinations with ALVAC (attenuated 1/30canarypox virus ) cured miniMAGE-1/3. Metastasis at 8/30study entry: Dark gray, measurable metastases at study entry; light gray, metastasis removed before study. n-visc., non- visceral distant metastasis; visc., visceral metastasis; C, cutaneous; S, subcutaneous; L, lymph node; Lu, lung; O, other visceral localization; Prog., progression; Reg., regression. Baren et al 2005 60. Frequencies and target antigens of antitumor CTLsfrom patient EB81.The frequencies of antitumor and anti-vaccineCTLps are indicated in the top. Frequencies ofantitumor CTLps were measured by MLTCwith a tumor cell line derived from the invadedPierre G. Coulie, de Duve Institute,lymph node, whereas those of antiMAGE-3.A1 Universit catholique de Louvain,Avenue Hippocrate 74, UCL 7459, B-120and antiMAGE-C2336344 CTLps were Brussels, Belgium.measured by clonotypic PCR. The bottomTel: 32(2)7647581, Fax: 32(2)7647590,Email: pierre.coulie@uclouvain.bepanels represent antitumor CTL clones withdifferent numbers for each TCR sequence andthe occurrence of repeated clones. Most ofthese CTL clones were derived fromlymphocytes collected in September 1999 andMarch 2000 and were stimulated in autologousMLTC. Additional CTL clones were derivedfrom lymphocytes collected in October 2000by stimulation with MAGE-C2 peptides andidentification of the positive microcultureswith the appropriate tetramer. For peptideMAGE-C2336344, 15 additional CTL clones were TCRobtained. 14 turned out to be the CTL 16clonotype, whereas one, CTL 40, had anotherTCR. A similar experiment performed withMAGE-C2191200 revealed a new highly repeatedclonotype, CTL 41.Germeau C et al. J Exp Med2005;201:241-248 61. Current Objectives:Dr. Krista Frisher: Kaplan Meier survival curve based on MAGE genesexpression in NSCLC of Estonia cohort. An independent biomarker ? After this,epitope screening?Stringency testing? 2D and 3D MAGE gene ? Serum title of living subjects? ? 62. gspagnoli-at-uhbs.chDepartment of BiomedicineUniversity Hospital BaselHebelstrasse 20CH - 4031 BaselOffice phone+41 61 265 23 78Office fax+41 61 265 30 90 63. Expression of MAGE-A Cancer/Testis Antigens in Esophageal Squamous Cell CarcinomasJ. HAIER1, M. OWZCARECK1, U. GULLER3, G. C. SPAGNOLI3,H. BRGER2, N. SENNINGER1 and TH. KOCHER4 1Molecular Biology Laboratory, Department of General Surgery and2Department of Pathology, University Hospital, Mnster, Germany;3Institute for Surgical Research and Hospital Management, University Hospital Basel;4Department of General, Visceral and Vascular Surgery, Kantonsspital, Baden, Switzerland 64. Bujas T et al. European Journal of Histochemistry 2011; volume 55:e7Immunohistochemical expression of MAGEA 3/4 and NY-ESO-1 in esophageal squamous cell carcinoma and lymph node metastasis. Expression of A) MAGE-A 3/4 andB) NY-ESO-1 in a primary tumor was cytoplasmic and limited to tumor cells. Similar immunohistochemicalreaction for C) MAGE-A 3/4 and D) NY-ESO-1 was also observed in corresponding lymph node metastases. 65. Normally MAGE genes are highly hypermethylated 66. Thank you for your attention!