The immune biology of MSI cancers and its clinical implications

Transcription

1 The immune biology of MSI cancers and its clinical implications Magnus von Knebel Doeberitz, MD Department of Applied Tumor Biology Institute of Pathology Universityy of Heidelberg CCU G105 German Cancer Research Center Molecular Medicine Partner Unit EMBL Danish-HNPCC Registry 26th Annual Meeting Copenhagen, October 12th 2017

2 Major Pathways of Carcinogenesis CIN MSI e.g. HPV E6-E7 activation DNA mismatch repair deficiency chromosomal instability microsatellite instability

3 Molecule of the Year: The DNA Repair Enzyme Sience 1994 Altonen et al., Science 1993 Yonov et al., Nature 1993

4 DNA mismatch repair deficiency causes high-level microsatellite instability (MSI-H)

5 Lynch syndrome the most common inherited colon cancer syndrome ATB Henry T. Lynch Germ line mutations of DNA mismatch repair genes Allele frequency between 1:350 and 1:1500

6 The history of Lynch syndrome Aldred S. Warthin Warthin et al Lynch and Krush 1971 Douglas et al Family G familial cancer predisposition to colon, endometrial and other cancers hereditary non-polyposis colon cancer (HNPCC-syndrome, Lynch syndrome)

7 A typical HNPCC-Pedigree (penetrance only about 50 %) bladder (53) ureter (45) M M colon (45) M cervix (53) breast (52) M M endometrium (29) endometrium (54)

8 A C T G A T T C A A G A C G A G C T A G T A I I I I I I I I I I I I I I I I I I I I I I T G A C T A A G T T C T G C T C G A T C A T

9 A C T G A T T C A A G A C G A G C T A G T A I I I I I I I I I I I I I I I I I I I I I I T G A C T A A G T T C T G C T C G A T C A T A C T G A A A A A A A A A A A G C T A G T A I I I I I I I I I I I I I I I I I I I I I I T G A C T T T T T T T T T T T C G A T C A T

10 A C T G A T T C A A G A C G A G C T A G T A I I I I I I I I I I I I I I I I I I I I I I T G A C T A A G T T C T G C T C G A T C A T A C T G A A A A A A A A A A A G C T A G T A I I I I I I I I I I I I I I I I I I I I I T G A C T T T T T T T T T T C G A T C A T T T

11 Pathogenesis of DNA Mismatch Repair (MMR) Deficient Cancer normal MMR deficient MMR microsatellite Instability (MSI) Kloor et al., Expert Rev Mol Diagn Jul;5(4):

12 Kloor and von Knebel Doeberitz, Trends in Cancer, March 2016, Vol. 2, No. 3http://dx.doi.org/ /j.trecan ATB

13 Loss of DNA mismath repair results in the generation of frameshift peptide (FSP) antigens coding microsatellite TGT. AAA. AAA. AAA. ACG. TGC. TGG. CTA. GCT. GA... C K K K T C W L A... amino acid sequence FSP neo-sequence

14 Microsatellite instability is associated with local lymphocyte infiltration and low frequency of distant metastases Buckowitz et al., British Journal of Cancer (2005)

15 The concept of FSP neo-antigens FSP neo-antigens are highly immunogenic due to long mutational antigens that encompass multiple potential epitopes point mutation-induced antigen FSP antigen T cell T cells antibodies e.g. mutant p53 or kras genes

16 MSI-H cancers have an exceptionally high mutational load ATB Alexandrov et al., Nature 2013

17 _2 Ersteze durch andere folie ATB FSP antigens are a unique source of cancer antigens The neo-antigen load appears to predict the efficacy of immune interventions (Schumacher and Schreiber, 2015) MSI-H cancers show an abundance of antigenic mutations(vogelstein et al., 2013) Anti-PD1 checkpoint blockade highly efficient in MSI-H cancers (Le et al., 2015)

18 Clinical response rates of MSS and MSI CRCs to PD-L1 blockade by Pembrolizumab (Keytruda ) Le et al., NEJM 2016

19 Evolution of MSI-H cancers ATB Selection of cell clones with distinct combinations of FSPs? shared comon FSP neo-antigens on MSI cancer cells

20 Identification of cms and prediction of FSP neo-antigens In silico identification of all cms in the human genome mrna expression analysis of cms in colon epithelium prediction of FSP neo-antigens Clinical trial for FSP based lynch vaccine monitoring of immune responses against FSP neo-antigens in MSI cancer patients

21 Prediction of frameshift peptides in silico > candidates SELECTION OF TARGETS Determination of mutation frequency >500 candidates Evaluation of expression and immunogenicity 40 candidates Analysis of immune response in patients 13 candidates in biopsies in vitro Analysis of immune escape mechanisms in vivo

22 Mutations of defined coding microsatellites drive MMRdeficient cancer development shared mutations shared FSP neoantigens

23 Do tumor infiltrating T-cells recognize the predicted FSPs? Hypothesis and Project Design Prediction of Targets Characterization Immune analysis In vitro IFN-γ ELISpot analysis Preparation of Clinical trial Schwitalle et al. Gastroenterology 2008 (4):988-97

24 A typical HNPCC-Pedigree (penetrance only about 50 %) bladder (53) ureter (45) M M colon (45) M cervix (53) breast (52) M M endometrium (29) endometrium (54)

25 Distribution of FSP-specific responses in MSI-H CRC patients Only half of the Lynch syndrome gene carriers develop MSI-H cancers during their lives. Are the others protected by immune surveillance? Schwitalle et al., Gastroenterology 2008

26 Are FSP-specific T-cells cytotoxic for FSPpositive target cells? * * * * * * Schwitalle et al. Gastroenterology 2008 Linnebacher et al. Int J Cancer 2001 Schwitalle et al. Gastroenterology 2008

27 Figure 5 ATB Immune responses against predicted FSP neo-antigens Trends in Cancer , DOI: ( /j.trecan ) Kloor and von Knebel Doeberitz, Trends in Cancer 2016

28 Identification of novel cancer precursors in Lynch syndrome MMR-deficient crypt foci Kloor et al. J Clin Oncol 2011, Kloor et al. Lancet Oncol 2012

29 Immune escape during evolution of MSI-H lesions Selection of cell clones with distinct combinations of FSPs? shared comon FSP neo-antigens on MSI cancer cells outgrowth of cells that become resistant against FSP specific immune attack activated T-cell targeting FSP neoantigen presenting tumor cell

30 Mechanisms to evade FSP specific T-cell attack Kloor and von Knebel Doeberitz, Trends in Cancer 2016

31 Stage distribution of ATB ß2-microglobulin (ß2m) mutations 50,0 37,5 25,0 non-truncating mutations truncating mutations p=0.01 Only silent mutations or single AA exchanges, w/o functional loss of ß2m 12,5 0,0 UICC III 9/22 (40.9%) UICC III 9/22 (40.9%) UICC IV 2/26 (7.7%) UICC IV 2/26 (7.7%) UICC IV, M1: No truncating mutations observed in CRC with distant metastasis ß2m mutation frequency is significantly lower in M1 than M0 MSI CRC Intact ß2m may be needed for distant metastasis formation in MSI-H CRC Kloor et al. Int. J. Cancer 2007

32 ß2M mutations predict lack of distant metastases B2M wild type B2M mutant Kloor et al. Int J Cancer 2007, Kloor et al. Int J Cancer 2010

33 A FSP-based vaccine: Can amplification of FSP-reactive T-cells provide protection? Hypothesis: MSI induces the generation of immunogenic frame shift induced -neopeptides (FSPs). If a sufficient number of reactive T-cells is generated before a MSI-lesion occurs, these T-cells may be capable to defeat potentially emerging MSIcell clones even before they get a chance to undergo immune evasion?

34 Three MSI-H triggered FSPS may represent a unique vaccine covering > 95 % of all MSI-H cancers Micoryx vaccine combines three FSP antigens covers 98.5% of MSI-H colorectal / endometrial cancers targets shared antigens likely related to tumor progression AIM2 HT % 9.0 % 8.2 % 32.0 % 8.5 % 29.5 % Subcutaneous injection of FSPs and Montanide ISA % TAF1B Study week Pre Monitoring of toxicity, immune response (including DTH) and tumor response

35 Results of the Micoryx phase I/Iia trial ATB All patients vaccinated per protocol CD4 CD8 humoral response develop significant T cell responses As expected, T cell responses are mainly CD4, with occasional CD8 responses in 8 out of 22 patients Strong induction of humoral immune responses against all antigens Antibody titers increase over the time of the study not done negative positive Kloor et al., submitted

36 Conclusions ATB Vaccination with FSPs is well tolerated Safety and immunological efficacy have been demonstrated in phase I/IIa trial No FSP-related systemic side effects have been observed FSP-specific humoral and T cell responses were induced in all patients vaccinated per protocol

37 A Mouse Model for MSI Cancers MMR-proficient intestinal stem cells MMR-deficient intestinal stem cells normal phenotype (no cancer) normal proteins MSI cancer FSP proteins in tumors Kucherlapati et al., 2010 VCMsh2 LoxP/LoxP In collaboration with Steve Lipkin Cornell University, NYC about 90 % of mice develop intestinal neoplasms within 12 month Identification of cms mt frequency in cms FSP in cms genes vaccination against FSP monitoring of cancer incidence

38 A Mouse Model for Lynch Syndrome identification of cms in the mouse genome mrna expression analysis of cms in intestinal epithelium vaccination of mice and monitoring of tumor development prediction of FSP neo-antigens comparison of tumor incidence in vaccinated and non-vaccinated mice monitoring of immune responses against FSP neo-antigens

39 T-cell response to 4 FSP neo-antigens of cms ATB Xirp(-1) Maz(-1) Nacad(-1) Senp6(-1) wt neopeptide sequence aa Legend low wt sequence likelihood of immunogenic epitopes high FSP neoantigen sequence

40 MSI cancer projects - Summary Microsatellite-unstable (MSI) cancers harbor an excessively high number of frameshift mutations, which result from DNA mismatch repair deficiency. Frameshift peptide neo-antigens (FSPs) resulting from a key set of functionally relevant driver mutations are shared by the majority of MSI-H cancers. These FSPs elicit strong immune reactions in mice and men and can be used as vaccine to trigger T-cell responses. Immune checkpoint modulation and vaccination with MSI-H-specific frameshift peptide antigens are promising strategies for the treatment and potentially prevention of MSI-H cancers. Hereditary MSI-H cancers developing in Lynch syndrome are an ideal model to evaluate the feasibility of cancer-preventive vaccines in general.

41 Heidelberg Applied Tumor Biology Anna Schulz Cathrin Huth Laura Staffa Fabian Echterdiek Aysel Ahadova Jonas Janikovits Mine Özcan Meike Müller Miriam Reuschenbach Johannes Gebert Matthias Kloor Department of Surgery Mirjam Tariverdian Markus W. Büchler EMBL Heidelberg Peer Bork DKFZ Heidelberg Axel Benner Bioquant TIGA Center Niels Grabe Acknowledgements University Hospital Charité, Berlin Hendrik Bläker University of Newcastle John Burn Richard Gallon Ben Hartog University of Leeds Tim Bishop Harvard Medical School, Boston Soldano Ferrone Krankenhaus Nordwest, Frankfurt Elke Jäger German HNPCC Consortium Gabriela Möslein Markus Löffler Christoph Engel Cornell University NYC Steven Lipkin National Cancer Institute (NCI) Division Cancer Prevention Shizuko Sei Robert Shoemaker ATB hnpcc-heidelberg.de

42 Acknowledgements ATB Universitätsklinikum Heidelberg Magnus von Knebel Doeberitz EMBL Heidelberg Peer Bork NCT Heidelberg Georg Martin Haag Krankenhaus Nordwest Frankfurt Elke Jäger Markus Löffler Elke Holinski-Feder Gabriela Möslein CaPP Consortium John Burn Weill Cornell M. C. Steve Lipkin NCI Cancer Prevention Asad Umar InSiGHT group Funding and Awards KFO227 Deutsche Krebshilfe (German Cancer Aid)

43 ATB Thank you very much for your attention