pivac-15 programme
TRANSCRIPT
Programme Book
06 - 08 October 2015Tübingen, Germany
15th International Conference on
Progress in Vaccination Against Cancer
Scientific Organising Committee
Graham Pawelec (Chair) • Suzanne Ostrand-Rosenberg
Robert C. Rees • Per thor Straten
PIVAC-15
CTL Europe GmbHHans-Boeckler-Str. 19-2953225 Bonn, Germany
Tel +49 (0) 228 85 44 78 - 0Fax +49 (0) 228 85 44 78 - 22
Immune Monitoringby ELISPOT
Cryopreservation ofPBMC samples
Assay development
Assay qualification & validation
GLP-compliant testing
Assay consultation
PBMC Reference Sample QC Set
Serum-free test platform
ELISPOT analyzers & kits
15th International Conference on
Progress in Vaccination Against CancerCasino am Neckar, Tübingen, Germany
06 – 08 October 2015
Tuesday 06 October 2015
14.00 – 18.00 Registration - Posters may be put up between 14.00 & 16.00
14.00 – 16.00 Welcome Refreshments (Room “Neckar”)
16.00 – 16.10 “Welcome to 15 Years Progress of PIVAC”Graham Pawelec (Germany)
16.10 – 16.30“35 years hunting the peptides”Hans-Georg Rammensee (Germany)
Session I Chair: Graham Pawelec 16.30 – 16.50 “Immunotherapy and the Immunosuppressive Tumor Network”Gosse J. Adema (Netherlands) 16.50 – 17.00 Questions
17.00 – 17.20 “The evolution of HER-2/neu peptide vaccines: from in vitro models to preclinical studies and clinical trials” Constantin N. Baxevanis (Greece) 17.20 – 17.30 Questions
17.30 – 17.50 “Designing effective subunit cancer vaccines by mimicking systemic infections”Esteban Celis (USA) 17.50 – 18.00 Questions 18.00 – 20.00 Welcome Reception, Posters & Opening of Trade Exhibition (Room “Neckar”) Light Buffet & Drinks19.00 – 20.00 Poster Defence Session 1
Wednesday 07 October
Session II Chair: Rolf Kiessling
09.15 – 09.35“Antigenicity and immunogenicity of human tumors”Pierre G. Coulie (Belgium) 09.35 – 09.45 Questions
09.45 – 10.05“Vaccination based strategies for immune therapy of acute myeloid leukaemia.” Farzin Farzaneh (UK) 10.05 – 10.15 Questions
10.15 – 10.35“Dendritic cell vaccines; lessons learned, new ideas, & future prospects”Carl Figdor (Netherlands) 10.35 – 10.45 Questions
10.45 – 11.15 Coffee Break (Room “Neckar”)
Session III Chair: Constantin N. Baxevanis 11.15 – 11.35 “15 years of PIVAC and 40 years of looking into tumour MHC expression”Federico Garrido (Spain) 11.35 – 11.45 Questions
11.45 – 12.05 “From single agent peptide vaccines to combination trials with checkpoint inhibitors and TcR therapy: Progress after 22 years of clinical trials of cancer vaccines”Gustav Gaudernack (Norway) 12.05 – 12.15 Questions
12.15 – 12.35“Identification of CD4CD8aa DP8a T cells as a new human regulatory T cell subset induced by Faecalibacterium prausnitzii and deficient in patients with inflammatory Bowel Disease”Francine Jotereau (France) 12.35 – 12.45 Questions
12.45 – 14.00 Lunch, Posters & Trade Exhibition (Room “Neckar”)
Session IV Chair: Suzanne Ostrand-Rosenberg
14.00 – 14.20 “Immunotherapy post-haematopoetic stem cell transplantation using cord blood cell products”J. Alejandro Madrigal (UK) 14.20 – 14.30 Questions
14.30 – 14.50 “Fifteen years of efforts at Karolinska Institutet to improve cancer vaccines”Rolf Kiessling (Sweden) 14.50 – 15.00 Questions
15.00 – 15.15Proffered Paper 1“With a little help from CD4 T cells in adoptive T-cell transfer”Else Marit Inderberg-Suso (Norway)15.15 – 15.20 Questions
15.20 – 15.35 Proffered Paper 2“ATM kinase governs CREB activity in human Dendritic Cells to regulate IL-23 and Th17 responses”Andrew Jackson (UK) 15.35 – 15.40 Questions
15.40 – 16.05 “The emerging role of regulatory T cells (Treg) in human cancer progression and immune therapies”Theresa L. Whiteside (USA) 16.05 – 16.15 Questions 16.15 – 16.35 Satellite Session Sponsored by CTL “Direct detection of T and B memory lymphocytes by ELISPOT reveals HCMV exposure that serum antibodies fail to identify”Paul V. Lehmann (USA) 16.35 – 16.45 Questions
16.45 – 18.00 Coffee Break, Posters & Trade Exhibition (Room “Neckar”)17.00 – 18.00 Poster Defence Session 2
18.00 Free Evening to explore Tübingen
18.00 – 19.00 Cancer Immunology, Immunotherapy Board Meeting (Editorial Board Members Only)
Thursday 08 October
Session V Chair: Theresa L. Whiteside
09.15 – 09.35 “Promoting cancer cell death to benefit immunotherapy”Thomas J. Sayers (USA) 09.35 – 09.45 Questions
09.45 – 10.05 “Conditions for successful therapeutic peptide vaccines” Cornelis J. M. Melief (Netherlands) 10.05 – 10.15 Questions
10.15 – 10.35“Immunogenetics of cancer - from polymorphisms to clinic and beyond”Elissaveta J. Naumova (Bulgaria) 10.35 – 10.45 Questions
10.45 – 11.15 Coffee Break (Room “Neckar”)
Session VI Chair: Cornelis J. M. Melief
11.15 – 11.30 Proffered Paper 3“Citrullinated vimentin, which is presented on MHC-II on tumor cells, is a novel rejection target for CD4 T cells”Victoria Brentville (UK)11.30 – 11.35 Questions
11.35 – 11.50Proffered Paper 4 “T-cell receptor (TCR) repertoire profiling by deep sequencing as a biomarker for immunotherapy and a starting point for TCR gene therapy”Isabel Poschke (Germany) 11.50 – 11.55 Questions
11.55 – 12.15“Fifteen years’ worth of lessons on the immunogenicity of tumors and tumor antigens; from melanoma to pancreatic cancer” Rienk Offringa (Germany) 12.15 – 12.25 Questions
12.25 – 12.45 “The continuing saga of understanding and improving anti-tumor immunity: From cancer vaccines to myeloid-derived suppressor cells”Suzanne Ostrand-Rosenberg (USA) 12.45 – 12.55 Questions
13.00 – 14.15 Lunch, Posters & Trade Exhibition (Room “Neckar”)
Session VII Chair: Farzin Farzaneh
14.15 – 14.30 Proffered Paper 5 “xCT is a new cancer stem cell immunotherapeutic target for breast cancer” Roberto Ruiu (Italy) 14.30 – 14.35 Questions
14.35 – 14.50Proffered Paper 6 “Mass Cytometry: High dimensional identification of cellular immune signatures in healthy individuals and stage IV melanoma patients turned out to correlate with patients survival”Kilian Wistuba-Hamprecht (Germany) 14.50 – 14.55 Questions
14.55 – 15.10 Proffered Paper 7 “Developing an awareness of the mechanism of Epithelial to Mesenchymal Transition (EMT) in prostate cancer; implications for immunotherapy” Jayakumar Vadakekolathu (UK) 15.10 – 15.15 Questions
15.15 – 16.00 Coffee break (Room “Neckar”) Posters must be removed by 16.45
Session VIII Chair: Graham Pawelec
16.00 – 16.20 “NK receptors and their ligands in cancer. Towards personalised NK cell based immunotherapy” Rafael Solana (Spain) 16.20 – 16.30 Questions
16.30 – 16.50“15 years of tinkering with T cells”Per thor Straten (Denmark) 16.50 – 17.00 Questions
17.00– 17.20“The next 15 years of PIVAC”Graham Pawelec (Germany)17.20 – 17.30 Questions
19.30 – 22.00 Festive Conference Dinner “Banquet Hall” & Awards Ceremony
Congratulations to the winners of the Meeting Bursaries provided by the European Federation of Immunological Societies (EFIS) and the European Journal of Immunology (EJI). Each winner received a full registration free of charge and funds of up to 700 Euros to assist with the cost of travel and accomodation.
EFIS / EJI Meeting Bursary Award winners
Mr. Timothy Spear (USA)Dra. Pinelopi Samara (Greece)Dra. Maurenis Hernández Perez (Cuba)
Mrs. Sabaria Shah (UK)
The bursary offer is designed to support participants with less than four years post-doctoral experience - including late entrants to cancer research - who have difficulty in securing the necessary funding to participate in the Conference. The bursaries provide either a standard (€400) or a student registration (€300), plus funds towards travel and accommodation. The total maximum amount of the award is €1,000.
The organisers wish to express their appreciation for the significant support provided by sponsors at the 15th International Conference on Progress in Vaccination Against Cancer. Their interest and enthusiasm for the meeting has enabled the organisers to provide an impressive scientific programme.
Premium Sponsor with Satellite Symposium
Gold sponsors
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Meeting Sponsor
Proffered Paper Awards Sponsor
Meeting Bursary Support
Immatics Biotechnologies is a leading cancer immunotherapy company and the global leader in the discovery of novel targets for various types of cancer immunotherapies.
Immatics’ XPRESIDENT® platform and pipeline:
The powerful discovery engine XPRESIDENT® is to date the only known high-throughput research technology to directly identify, quantify, and prioritize naturally presented tumor-associated and T-cell targets.
Immatics has a broad pipeline of cancer immunotherapies in clinical and preclinical development for the treatment of various tumor types.
Approaches include vaccines, adoptive cell therapy and development of soluble TCRs and monoclonal antibodies.
Immatics Biotechnologies GmbH Immatics US Inc. Paul-Ehrlich-Str. 15 7000 Fannin Suite 2115 72076 Tuebingen Houston, Texas 77030 Germany USA
Phone +49 7071 5397 0 +1 (832) 871-5270 Fax +49 7071 5397 900 +1 (832) 871-5271 www.immatics.com
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A21659
Cancer Immunology, ImmunotherapySince its inception in 1976, Cancer Immunology, Immunotherapy (CII) has reportedsigni� cant advances in the � eld of tumor immunology. The journal serves as a forum fornew concepts and advances in basic, translational, and clinical cancer immunology andimmunotherapy. CII is keen to publish broad-ranging ideas and reviews, results whichextend or challenge established paradigms, as well as negative studies which fail toreproduce experiments that support current paradigms, and papers that do succeed inreproducing others’ results in di� erent contexts.
In addition to publishing original research articles and reviews, CII also o� ers the followingunique publication formats:
• Focussed Research Reviews,” published together with a conference meetingreport, which provide individual speakers with the opportunity to expand on the ideasand concepts they presented at the conference
• “Symposia-in-Writing,” which are virtual workshops consisting of a series ofcomplimentary papers centered on a speci� c issue and addressing controversial butstrategic topics in the � eld, usually consisting of 4-8 papers and an accompanyingcommentary by the convener
CII is a� liated with the Association for Cancer Immunotherapy (CIMT), Canadian CancerImmunotherapy Consortium (CCIC), The Japanese Association for Cancer Immunology (JACI), Network Italiano per la Bioterapia dei Tumori (NIBIT), and Sociedad Española de Inmunologia-Grupo Española de InmunoTerapia (SEI-GEIT).
On the homepage of Cancer Immunology, Immunotherapy at springer.com you can
• Read selected articles for free • Sign up for our Table of Contents Alerts • Get to know the complete Editorial Board • Find submission information
Editors-in-Chief: G. Pawelec S. Ostrand-Rosenberg
94% of authors who answered a survey reported that they wouldde� nitely publish or probably publish in the journal again
Impact Factor: 3.941 (2014), Journal Citation Reports®, Thomson Reuters
12 issues/year
Electronic access:springerlink.com
Subscription information:springer.com/librarians
springer.com
€ (D) sind gebundene Ladenpreise in Deutschland und enthalten 7 % MwSt. € (A) sind gebundene Ladenpreise in Österreich und enthalten 10 % MwSt. Die mit * gekennzeichneten Preise sind unverbindliche Preisempfehlungen und enthalten die landesübliche MwSt. Preisänderungen und Irrtümer vorbehalten.
A21659
Cancer Immunology, ImmunotherapySince its inception in 1976, Cancer Immunology, Immunotherapy (CII) has reportedsigni� cant advances in the � eld of tumor immunology. The journal serves as a forum fornew concepts and advances in basic, translational, and clinical cancer immunology andimmunotherapy. CII is keen to publish broad-ranging ideas and reviews, results whichextend or challenge established paradigms, as well as negative studies which fail toreproduce experiments that support current paradigms, and papers that do succeed inreproducing others’ results in di� erent contexts.
In addition to publishing original research articles and reviews, CII also o� ers the followingunique publication formats:
• Focussed Research Reviews,” published together with a conference meetingreport, which provide individual speakers with the opportunity to expand on the ideasand concepts they presented at the conference
• “Symposia-in-Writing,” which are virtual workshops consisting of a series ofcomplimentary papers centered on a speci� c issue and addressing controversial butstrategic topics in the � eld, usually consisting of 4-8 papers and an accompanyingcommentary by the convener
CII is a� liated with the Association for Cancer Immunotherapy (CIMT), Canadian CancerImmunotherapy Consortium (CCIC), The Japanese Association for Cancer Immunology (JACI), Network Italiano per la Bioterapia dei Tumori (NIBIT), and Sociedad Española de Inmunologia-Grupo Española de InmunoTerapia (SEI-GEIT).
On the homepage of Cancer Immunology, Immunotherapy at springer.com you can
• Read selected articles for free • Sign up for our Table of Contents Alerts • Get to know the complete Editorial Board • Find submission information
Editors-in-Chief: G. Pawelec S. Ostrand-Rosenberg
94% of authors who answered a survey reported that they wouldde� nitely publish or probably publish in the journal again
Impact Factor: 3.941 (2014), Journal Citation Reports®, Thomson Reuters
12 issues/year
Electronic access:springerlink.com
Subscription information:springer.com/librarians
springer.comSpeaker abstracts
Speaker abstracts
Speaker abstracts
35 years hunting the peptides
Hans-Georg Rammensee1
1 Univ. of Tübingen, Tübingen, GERMANYIn 1979 we started to work on minor histocompatibility antigens, not knowing that we are dealing with peptides. In 1990, we showed that they are, and also idnetified the first viral peptides naturally presented by MHC to CD8 T cells. In 1991 we showed that MHC presented peptides are usually 9 amino acids long, and that they display allele specific motifs, defined by anchor residues at defined positins. In 1995 we started to systematically analyse the HLA ligandome of human tumors and normal tissues.In the recent years, we have analyzed tumor samples of intermediate mutation load, in particular hepatocellular carcinomas, renal cell carcinomas, ovarian carcinomas and several leukemia types, by exome sequencing for somatic mutations and by mass spectrometry analysis for the identification of HLA ligands. Careful attention was given to HLA ligands possibly containing mutations; sequences containing nonsynonymous mutations on DNA level were subjected to prediction of peptides fitting to the relevant HLA class I molecules. In no instance we were able to find mutated peptides presented by tumor HLA molecules, although we had shown that the approach works within an experimental tumor model. These findings are in accordance with the assumption that many human tumors expressing an intermediate or low number of somatic mutations do not express immunogenic mutated antigens visible for T cells.In contrast, in analysing the entire detectable landscape of HLA ligands on these tumor samples, consisting of 1000 through 5000 peptides per sample, we do find dozens to hundreds of peptides in germline sequence with apparently tumor specific expression, based on the absence of these peptides on adjacent autologous benign tissue and absence on a large number of normal tissue samples from all organs and tissue types available for analysis, all, of course, within the sensitivity limits of our technology.
Immunotherapy and the Immunosuppressive Tumor Network
Gosse Adema1
1 Radboud University Medical Center, Nijmegen, NETHERLANDS
Abstract not available at the time of printing.
Speaker abstracts
The evolution of HER-2/neu peptide vaccines: from in vitro models to preclinical studies and clinical trials
Constantin N. Baxevanis1
1 Cancer Immunology and Immunotherapy Center, St Savas Cancer Hospital, Athens, GREECEIn the late 90s’ and early 2000, work in our laboratory has focussed on the role of CD4+ T (Th) cells for opmizing cytotoxic CD8+ T lymphocyte (CTL) responses against autologous tumor targets from patients with various types of cancer. To this end, we have demonstrated that human CTL can be effectively activated to mediate cytotoxicity against the autologous tumor via a Th-dependent activation of the autologous tumor antigen (Ag)-bearing dendritic cells (DCs). In particular, interactions between CD40 ligand and CD40 on Th cells and DCs, respectively, appeared to be essential for the subsequent activation of the DCs to present Ag to and costimulate the priming of CD8+ CTL to lyse autologous tumor targets. These data showed for the first time in humans that a critical pathway for delivery of help to CTLs is dependent on CD4+ T cells and uses DCs as an intermediary. Moreover, they paved the way for the inclusion of Th-stimulating epitopes in vaccine formulations. Later on, our studies have focused on translating the knowledge from basic science to the preclinical setting and on the development of novel cancer vaccines for clinical testing in cancer patients. For achieving these goals we have used the HER-2/neu receptor as a model molecule for analyzing in depth cellular immune responses against tumor-associated antigens. A great part of our research work we devoted to the discovery of immunogenic peptides representing HER-2/neu epitopes recognized by T cells to be used both as prophylactic and therapeutic tumor vaccines in HER-2/neu tolerant triple (HER-2/neu, HLA-A2.1, HLA-DR1) transgenic animals which were produced in our laboratory. These studies explored the possibility that HER-2/neu peptide-activated Th cells assist in breaking tolerance against HER-2/neu CTL epitopes representing tumor rejection molecules. When immunized with CTL plus Th HER-2/neu peptide combination vaccines, these animals retarded tumor growth or became long-term survivors by converting tolerogenic into immunogenic conditions. These experimentations were the first defining the in vivo effect of combined vaccination with HER-2/neu peptides representing CTL and Th epitopes on the dynamic relationship between vaccine-specific tumor infiltrating lymphocytes. As a continuation of the studies mentioned above, we conducted the first phase I trial of a HER-2/neu hybrid peptide vaccine (AE37) with recombinant
granulocyte macrophage colony-stimulating factor (GM-CSF) as adjuvant in patients with HER-2/neu+ prostate cancer. The primary end points of the study were to evaluate toxicity and monitor patients’ immune responses to the vaccine. In addition, we are participating in a multicenter prospective, randomized, single blinded Phase II trial evaluating the AE37 vaccine in breast cancer (NCT00524277). The primary endpoint is disease recurrence and the trial enrolled node-positive or high-risk node-negative breast cancer patients. The results from these studies have highlighted the role of pre-existent immunity to AE37 as well as of certain MHC class I and II alleles as factors predicting immunological and clinical responses. A summary of the results from these studies will be presented.
Speaker abstracts
Designing effective subunit cancer vaccines by mimicking systemic infections
Zili Wang1, Takumi Kumai1, Hussein Sultan1, Valentyna Fesenkova1, Juan Wu1, Esteban Celis1
1 Georgia Regents University Cancer Center, Augusta, GA, USANumerous CD8 cytotoxic T lymphocyte (CTL) epitopes have been identified allowing the development of epitope-based cancer immunotherapies such as the use of synthetic peptide-based vaccines. However, peptide vaccines have been notoriously weakly immunogenic, providing suboptimal therapeutic effects. In contrast, as a response to viral and bacterial infections, the immune system can produce massive numbers of antigen-specific CTLs eliminating disease and providing memory responses to prevent future infections. Our strategy to optimize peptide vaccines is to design immunization strategies that mimic infections by providing the necessary immune activation signals together with the appropriate immunogenic peptides. Furthermore, it has been proposed that minimal peptide epitopes are poorly immunogenic because they are presented to T cells by non-professional APCs. Therefore, it is suggested that using long peptides vaccines will improve immunogenicity by forcing antigen presentation by professional APCs. Using several mouse tumor models, we observe that peptide composition (hydrophobicity, amphipathicity), adjuvant and route of administration are more critical than peptide size for generating strong CTL responses that limit tumor growth. Two separate events are required for peptides to generate huge CTL responses, similar to those observed during acute infections: 1) Peptide priming mediated professional APCs, where CD40 activation and TLR signals are critical; 2) T cell expansion, which can be mediated by either professional and non-professional APCs and where type-I interferon induced by retinoic acid-inducible (RIG-I)-like receptor stimulation by poly-IC plays a critical role. Effective anti-tumor CTL responses were accomplished by 2 systemic injections (i.v. or i.m. 5-7 days apart) of peptide/poly-IC. Lastly, in some instances even in the presence of huge numbers of tumor-reactive CTLs anti-tumor effects remained suboptimal, but could be significantly enhanced by implementing PD1 blockade. Resulting in complete tumor eradication. After 15 years we are learning how to motivate the immune system to reject tumors.
Antigenicity and immunogenicity of human tumors.
Pierre G. Coulie1, David Schröder1, Orian Bricard1, Gérald Hames1, Nathalie Rémy1, Tiphanie Gomard1, Javier Carrasco2
1 de Duve Institute, Brussels, BELGIUM, 2 Grand Hôpital de Charleroi, Charleroi, BELGIUMMost human tumors bear antigens that can be recognized by T cells. Some of these antigens, notably those encoded by genes that are mutated in the tumor, are truly tumor-specific and T cells naturally obtained against them do not damage normal tissues. Such ‘mutated’ antigens recognized by T cells were first described on the mouse mastocytoma cells P815 and, exactly 20 years ago, mutated antigens were found to be present also on human tumors, namely melanomas. Mutated antigens were repeatedly identified on human tumors of different types. Most of them are unique and result from single nucleotide substitutions. Their absolute tumor-specificity qualified them to (re)stimulate antitumor T cells in patients but their identification was laborious. Today they are back on center stage as they appear to be a key determinant to the clinical success of immunostimulatory antibodies. Patients with a tumor containing many candidate ‘antigenic’ mutations, i.e. non-synonymous in an HLA-binding peptide, are more susceptible to display a clinical response to anti-CTLA-4 or anti-PD-1 antibodies.Most if not all tumors are antigenic. In addition many of them are immunogenic: they naturally prime tumor-specific T cells in vivo. The best example, and certainly the best studied, is melanoma. We are currently exploring primary breast carcinomas. The importance of spontaneous antitumor T cell responses prior to immunotherapy with immunostimulatory antibodies is yet to be explored and might vary with the targeted inhibitory or costimulatory pathway.It is a tautology that in patients tumors progress despite their antigenicity and immunogenicity. They are automatically selected for variants that escape or blunt immune attack. Immunotherapy should therefore be used as early as possible and include compounds that counteract intratumoral immunosuppression.
Speaker abstracts
Vaccination based strategies for immune therapy of acute myeloid leukaemia.
Kyriaki Ioannou1, Lucas Chan1, Yuqian Ma1, Sabine Domning1, Ruby Quartey-Papafio1, Linda Barber1, Ghulam Mufti1, Farzin Farzaneh1
1 King’s College London, London, UKIn pre-clinical studies we have demonstrated that tumour cells expressing immune co-stimulatory molecules and the appropriate Th1 cytokines can induce immune mediated rejection of previously established tumours. We are now assessing this autologous AML cell vaccination strategy in two Phase-I clinical studies. The first, in poor prognosis AML patients who have relapsed following allogeneic haematological stem cell transplantation (HSCT). The second, in poor prognosis patients who are not eligible for HSCT. In both studies patients in temporary chemotherapy induced remission, are vaccinated with autologous AML cells that are genetically modified to express CD80 and IL-2. Early results indicate, feasibility, safety and stimulation of immunological responses against the unmodified AML blasts. We have also developed a new vaccination strategy based on employing combinations of adjuvants for synergistic activation of cellular immunity (CASAC). Pre-clinical studies show that subcutaneous vaccination with CASAC and target peptides is able to break tolerance to chronically experienced antigens that are associated with cancer (e.g. TRP2, WT1, Glypican-3). CASAC based vaccination can induce antigen-specific immunity, including in vivo cytolytic activity and lysis of antigen positive cells, even in immune senescent aged mice.
Dendritic cell vaccines; lessons learned, new ideas, & future prospects.
Carl G. Figdor1, Gerty Schreibelt1, Kalijn Bol1, Harm Westdorp1, Steve Boudewijns1, Angela Vasaturo1, Altuna Halilovic1, Mark Gorris1, Winald Gerritsen1, Jolanda de Vries1
1 Radboud University Medical Center, Nijmegen, NETHERLANDSTo date, Dendritic Cell (DC)-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, So far, predominantly ex vivo-cultured monocyte- or Cd34+ derived DCs have been used. Although during the past 15 years the concept of DC vaccination has been clearly proven and found safe, the number of patients that have long-term benefit is limited.Instead of monocyte derived DC, we recently performed studies with two major types of naturally occurring DCs: myeloid DCs (mDCs) and plasmacytoid (pDCs). These different natural DC subsets, expressing distinct TLRs, do not require extensive in vitro culture, and provide us with a novel toolbox to more precisely explore the therapeutic potential or natural DC in cancer immunotherapy. The first results indicate that these cells are extremely potent in initiating immune responses in cancer patients.Despite these encouraging results, the recent introduction of immune checkpoint inhibitors in the clinic raises questions such as; How DC based vaccinations perform compared to immune checkpoint inhibition? And based on this, what is the future perspective of DC based cancer immunotherapy at all? These, and related questions will be discussed, also in view of the developments of biomarkers that may predict which patients might benefit from immunotherapy.
Speaker abstracts
15 years of PIVAC and 40 years of looking into tumour MHC expression
Federico Garrido1
1 Dept Analisis Clinicos & Inmunologia,Hospital Universitario Virgen Nieves; Dept. Bioquímica, Biología Molecular e Inmunologia, Facultad de Medicina, Universidad de Granada, Granada, SPAINAfter 40 years of studying MHC antigen expression in tumors we have learned that primary tumor cells are heterogeneous and frequently loose these molecules by a variety of molecular mechanisms. MHC-I loss variants are immunoselected in vivo by antitumor CTLs and escape T cell recognition and destruction during the natural history of tumor development. The discovery of NK cells with capability to eliminate MHC class I deficient tumor or virus infected cells adds a biological significance to the role MHC class I in tumor rejection. Both reversible (soft) and irreversible (hard) defects of MHC class I have been described in solid tumors and in cancer cell lines. Tumor dormancy it is also associated with MHC class I loss variants with MHC-I soft lesions. After 15 years of PIVAC we have come to a conclusion that the resistance to immunotherapy and generation of progressing metastases seems to be correlated with class I altered expression and dissemination of MHC-negative tumor cells. Immunotherapy is effective in eliminating MHC positive tumor cells and MHC deficient tumor cells with reversible/soft molecular lesions, while cells with loss or downregulation of MHC antigens with irreversible/hard lesions escape therapy-induced T cell mediated immune attack and produce new distant tumor lesions. It is important to know the precise molecular mechanism causing MHC-I loss for designing proper therapeutic strategies aimed at increasing both tumor immunogenicity and anti-tumor immunity. Reduced MHC class I expression linked to “soft” lesions can be upregulated using immunotherapy, radiotherapy, low dose chemotherapy or a combination of these types of treatments. However, structural defects in β2m or MHC genes will require genetic transfer strategies to recover normal expression of MHC class I antigens. We believe that the reexpression and enhancement of tumor MHC is responsible for tumor regression observed in patients under different immunotherapy protocols. Finally, we recommend closely monitoring the HLA class I tumor profile before, during and after cancer immunotherapy in contact with surgeons, pathologists and oncologists. One has to keep in mind that the tumor HLA analysis is a complex diagnostic approach that requires tissue cryopreservation, microdissection, application of a wide panel of specific antibodies and various molecular techniques.
From single agent peptide vaccines to combination trials with checkpoint inhibitors and TcR therapy: Progress after 22 years of clinical trials of cancer vaccines
Gustav Gaudernack1
1Institute for Cancer Research Oslo University Hospital, The Norwegian Radium Hospital, Oslo, NORWAYTesting blood samples from cancer patients with tumours frequently harbouring mutations in the RAS oncogens, we were able to show 25 years ago that some of these patients had circulating memory T cells specific for single mutations. This lead to our first clinical trial, started in 1993 and published in the Lancet in 1995. Due to the complexity of the approach, which involved multiple different mutations an potentially also multiple different HLA molecules, we decided tright from the start to use long synthetic peptides containing multiple epitopes capable of eliciting CTL and Th responses. This allowed each patient to select the motif best fitting his/her HLA makeup, and permitted us to avoid selelection of patients with a narrow set of predefined HLA types. In the following years, a large number of small Phase I/II clinical trials in different patient groups were performed aimed at optimizing the clinical setting and the vaccination regime. Several general findings emerged from these clinical trials: Survival allways correlated with immune responses against the vaccine. Depending on the patient population, 50-80% of the patients were able to mount an immune response after vaccination.The immune response was diverse with multifunctional CD8 and CD4 T cell responses, involving a broad spectrum of HLA molecules. The T cells were generally specific for individual RAS point mutations, and with two exeptions did not recognize wt RAS. Very similar findings were subsequently seen in clinical trials, using the long peptide approach, targeting frameshift mutations in TGF-betaRII and Bax, and targeting hTERT. Lately, with the aid of the clinical databank and the patient biobank, we have observed memory responses >10 years after vaccination in long term survivors and extensive intramolecular epitope spreading following hTERT vaccination. This lead to a second generation of hTERT vaccine now being tested in patients with prostate and lung carcinomas and in melanoma patients in combination with Ipilimumab. In a different approach, we are now generating a TcR repository for TcR transfer, starting the first clinical trial soon.
Speaker abstracts
Identification of CD4CD8aa DP8a T cells as a new human regulatory T cell subset induced by Faecalibacterium prausnitzii and deficient in patients with inflammatory Bowel Disease.
Francine Jotereau1, Guillaume Sarrabayrouse1, Frédéric Altare1
1 UMR 892 INSERM, Nantes, FRANCERegulatory T cells and IL-10 are important contributors to colonic homeostasis. In mice this results in part from the local induction of IL-10 secreting Foxp3 Treg by Clostridium members of the fecal microbiota. In humans, changes in the abundance of the fecal Clostridium leptum group and in particular of Faecalibacterium prausnitzii (F prau) are associated with inflammatory bowel diseases (IBD). It is however unclear if these alterations contribute to inflammation or results from it and whether inflammation may arise from disturbed Treg inductionWe report that Foxp3 negative CD4CD8aa (DP8a) TCRab lymphocytes present in the human colonic lamina propria exhibit most regulatory markers and functions of Foxp3 Treg and secrete IL-10. Surprisingly, the majority of these cells, despite a high TCR Vb diversity specifically recognized F prau in a MHC II restricted manner. Moreover, IBD patients who are known to exhibit decreased F prau abundance in their fecal microbiota also showed decreased frequencies of DP8a T cells in their inflamed colonic mucosa compared to healhy colonic mucosa. We also report the presence of F prau -specific DP8a Treg in blood and a decrease of these cells in IBD patients compared with healthy controls.These findings argue that DP8a colonic Treg are induced locally by cognate recognition of F prau and play a role in the prevention or the control of inflammation, opening the road to new diagnostic and therapeutic strategies for IBD. DP8a Treg represent new tools to address the impact of the gut microbiota in colon diseases such as cancer and IBD.
Immunotherapy post-haematopoietic stem cell transplantation using cord blood cell products
Alejandro Madrigal11 Anthony Nolan Research Institute, London, UKTherapeutic manipulation of the immune system may overcome morbidity and mortality caused by a deficient capacity for adaptive immunity and by malignancies not eradicable by other therapeutic interventions, surgery, radiotherapy or chemotherapy. In addition, interventions in order to modulate immune reactivity may be valuable to provide adaptive immunity to reduce the heavy burden of morbidity and mortality associated to haematopoietic stem cell transplantation (HSCT).At the Anthony Nolan Research Institute, new strategies are being developed to optimise the use of allogeneic HSCT, based on the prediction and modulation of the immune response. This is in order to reduce the risk of graft versus host disease, generate the selective anti-tumour immune response and to provide protective immunity against opportunistic infections.Our research has focussed on cord blood (CB) products to define cell subpopulations useful for therapy with minimally manipulated procedures. Hence, it may be possible to generate ‘off-the-shelf’ products to be tested clinically for application in regenerative medicine (using CB after CD133+ selection) or tolerance (using natural naïve T regulatory cells) after CD25+ selection, or natural killer cells.Obviously, this approach requires understanding of the potential immunoreactions produced by infusing allogeneic cells from one or multiple donors to a particular patient in the case of single or double CB transplantation. Altogether, knowledge acquired by basic research studies on stem cells and immunomodulatory cells contained in CB, would help with the translational challenges before application of CB cells in adoptive cell therapy. Data will be presented on the identification and isolation of CB stem cells, regulatory T cells (Tregs) and NK cells with the objective of targeting GvL, GVI and immunomodulation in HSCT.
Speaker abstracts
Fifteen years of efforts at Karolinska Institutet to improve cancer vaccines
Rolf Kiessling1
1 Karolinska Institutet, Stockholm, SWEDENAlthough we recently have witnessed a remarkable development in the field of immune therapy of cancer, the majority of patients do not respond and only in certain types of tumors has this type of therapy been successful. One of the major underlying causes for this limited success of immunotherapy is the phenomenon of tumor-induced immune suppression. This term was coined during the 90th to distinguish it from the immunosuppression induced by chemotherapy, radiotherapy or cachexia often observed in late-stage cancer patients. Tumors are often regarded as an “immune privileged site” with an internal tumor milieu which is hostile to T cells and NK cells. To what extent a progressing cancer also results in a generalized effect on systemic immunity is more controversial. An increasing amount of data indicate that this appears to be the case, and alterations in the functions and phenotypes of circulating T cells and NK cells in several cancer types have been observed. A broad variety of underlying mechanisms appear to be responsible for these alterations. Recently, we are beginning to understand the central role that myelomonocytic cells producing inflammatory factors play in the phenomenon of tumor induced immune suppression, as will be discussed.
Proffered Paper 1
With a little help from CD4 T cells in adoptive T-cell transfer
Else Marit Inderberg1, Sébastien Wälchli1,3, Marit Renée Myhre1, Kari Lislerud1, Gunnar Kvalheim1, Gustav Gaudernack3
1 Dept. of Cellular Therapy, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, NORWAY, 2 Oslo University, Oslo, NORWAY, 3 Section for Immunology, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, NORWAYAdoptive transfer of genetically engineered T cells offers great opportunities in cancer immunotherapy. Most studies have focused on transfer of HLA class I-restricted T-cell receptors (TCRs); however, several studies have shown the importance of CD4 T-cell help for tumour elimination.We have identified and cloned HLA class II-restricted CD4+ T cells isolated from patients vaccinated with long peptides derived from antigens such as hTERT, survivin and frameshift mutated TGFβRII.Several cancer patients demonstrated outstanding clinical responses to peptide- or dendritic cell based cancer vaccines with >10-year survival. In these patients strong T-cell responses against peptides other than those used for vaccination were detected, suggesting epitope spreading. Responses against certain peptides associated with clinical benefit were identified and CD4+ T-cell clones recognising such peptides were isolated.These HLA-DR and -DQ restricted T-cell clones recognised target cells loaded with long peptides at low concentrations. For one of the CD4+ T-cell clones where a melanoma cell line with the corresponding HLA allele was available we could show direct tumour recognition.mRNA encoding TCRs from these CD4+ T-cell clones was electroporated into expanded T cells. Both CD8+ and CD4+ T cells expressing the TCRs produced TNF-α, IFN-γ and degranulated (CD107a+) following co-incubation with peptide-loaded targetsWe have demonstrated that choosing highly functional CD4+ T-cell clones specific for tumour-associated or -specific antigens from patients with clinical responses after immunotherapy treatment is a successful method for identifying highly functional HLA class II restricted TCRs for adoptive T-cell transfer.The use of HLA class II-restricted TCRs may
Speaker abstracts
be of therapeutic value both in haematopoietic malignancies and in melanoma where tumour cells often express HLA class II. In addition, combining the redirection of T cells with both HLA class I- and class II-restricted TCRs may provide a more powerful therapeutic effect in adoptive T cell therapy.
Proffered Paper 2
ATM kinase governs CREB activity in human Dendritic Cells to regulate IL-23 and Th17 responses
Qunwei Wang3, Sabaria Shah3, Anna Malecka3, Jane Goodall2, Xiangshu Xiao1, Poulam Patel3, Andrew Jackson3
1 Knight Cancer Institute, Portland, OR, USA, 2 University of Cambridge, Cambridge, UK, 3 University of Nottingham, Nottingham, UKTh17 responses can either drive anti-tumour immunity or promote tumour progression [1]. We recently described [2] an alternate function of Ataxia telangiectasia mutated (ATM) kinase in dendritic cells (DC) showing regulation of unfolded protein responses (UPR) and IL-23 repression, a key Th17-polarizing cytokine. Here we dissect the interaction of ATM with TLR-signalling pathways and identify molecular alterations in the CREB transcription factor responsible for IL-23 regulation.Inhibition of ATM (ATMi, KU55933) selectively enhanced IL-23 (but not IL-1b, -6, -10, -12, -27) secretion by activated DC. In contrast, inhibition of the functionally related ATR (ATM and RAD3-related) kinase did not affect IL-23. ATMi-dependent IL-23 was restricted to TLR4 and didn’t occur with other TLR agonists. Whilst the IRE1 and PERK UPR pathways were activated upon ATMi they were not responsible for increased IL-23. Furthermore, whilst a role for the CHOP transcription factor in TLR4-dependent IL-23 was confirmed, ATMi-dependent IL-23 was surprisingly found to be CHOP-independent.Exposure of DC to ionising radiation (IR) activated ATM, phosphorylated CREB at the inhibitory S121 residue and attenuated IL-23 secretion. CREB S121 phosphorylation was ablated by ATMi without affecting the positively-acting CREB S133 residue. Consequently, upon ATM-inhibition, CREB activity in a luciferase reporter assay increased and this was prevented by a specific CREB-CBP interaction inhibitor (C-Ci). Moreover, ATMi-dependent IL-23 was ablated by C-Ci. In summary, ATM regulates IL-23 in DC and this is mediated by CREB. Radiotherpeutic doses of IR are similar to those in this study, raising the potential for modulation of these pathways in tumour-associated DC and macrophage. Ongoing studies are modeling the impact of IR on the tumour microenvironment and examing the role of tumour-associated stromal cells on the function of APC.1. Zhu et al 2008 Breast Cancer Res. 10: R952. Wang et al 2013 J. Immunol. 190: 3246
Speaker abstracts
The emerging role of regulatory T cells (Treg) in human cancer progression and immune therapies
Theresa Whiteside1
1 University of Pittsburgh, Pittsburgh, PA, USASome twenty years ago, the concepts of tumor-induced immunosuppression and tumor escape were finally acknowledged by the immunological community. Mechanisms responsible for tumor escape were unknown. In 1995, Sakaguchi and colleagues re-introduced suppressor T cells, now named Treg, as vital for immune regulation in health and disease. Subsequent studies showed that Treg accumulating in tumors and cancer patients’ blood exerted profound inhibitory effects on T effector cells and interfered with immunotherapies, including anti-tumor vaccines. Monitoring for Treg prior and after delivery of anti-tumor vaccines became critical for evaluation of therapeutic effects. Studies of Treg phenotypes and functions confirmed Treg diversity, plasticity and the ability to suppress by multiple molecular mechanisms. Today, the role Treg play in cancer progression and patient responses to therapy remains controversial. The perception that Treg accumulations predict poor cancer outcome is no longer true, as in some human cancers, the Treg frequency associates with improved prognosis. This dual role of Treg as “bad” or “good” is further complicated by the realization that inducible (i)Treg which operate suppressive pathways in tumors are phenotypically and functionally distinct from thymus-derived natural (n)Treg preventing autoimmunity. Intratumoral Treg are activated (CTLA-4+), strongly immuno-suppressive, co-express inhibitory receptors, including PD-1, TIM-3 or LAG-3 and may be resistant to oncological therapies, including ipilimumab or nivolumab therapies. In fact, some immunotherapies increase the frequency and functions of iTreg. The inhibitory ligands and factors, e.g., IDO or adenosine, present in the tumor microenvironment (TME) re-program iTreg to highly immunosuppressive long-lived cells resistant to Treg-depleting regimens, including anti-PD-1 antibodies. In TME, iTreg up-regulate PD-1 and PTEN phosphatase activity, thus inhibiting the mTOR pathway and driving iTreg differentiation and proliferation. Immunotherapies with checkpoint inhibitors may have to be combined with agents blocking immunosuppressive factors (adenosine, IDO, TGF-β, IL-10) to selectively silence iTreg without eliminating nTreg.
Sponsored Satellite: CTL
Direct detection of T and B memory lymphocytes by ELISPOT reveals HCMV exposure that serum antibodies fail to identify
Paul V. Lehmann1
1 Cellular Technology Limited (CTL), Ohio, USAIt is essential that donors are identified who have not been infected with HCMV in order to avoid infecting human recipients of transfusion products or of organs. In the present study, we tested the reliability of seronegativity identifying the lack of HCMV exposure of human subjects. Sixty six HCMV seronegative donors have been identified and their PBMC were tested in ELISPOT assays for the presence of HCMV-specific CD4, CD8 and B memory lymphocytes. Fifty seven percent of the HCMV seronegative donors displayed CD4 and CD8 T cell memory in addition to HCMV specific B cells providing three independent lines of evidence for having developed immunity to HCMV. Fifteen percent of the seronegative donors possessed CD4 and CD8 memory cells to HCMV, however, in the absence of memory B cells, and 16% had CD4 memory cells only. Only 12% of the seronegative donors showed neither T – or B- cell memory to HCMV qualifying as immunologically naïve to the virus. The data suggest that measurements of serum antibodies frequently fail to identify previous HCMV infection of humans that can be identified, however, by direct detection of HCMV-specific memory cells.
Speaker abstracts
Promoting cancer cell death to benefit immunotherapy
Thomas Sayers1
1 Leidos Biomedical Research Inc. , Frederick, Maryland, USACytotoxic T cells (CTL) and natural killer (NK) cells can destroy virally-infected cells and cancer cells. Two main molecular mechanisms have been described (1) the exocytosis of pre-formed lytic granules following contact with the target cells and (2) the secretion of members of the tumor necrosis factor family of proteins (death ligands) that induce the target cell to undergo apoptotic cell death. Interestingly these two lytic pathways are not functionally redundant in vivo. Since of the death ligands, only TRAIL or agonist antibodies to it death receptors (DR4 and DR5) could be administered to animals in the absence of substantial toxicities, the anticancer role of TRAIL was studied in more detail. TRAIL death receptor agonists did display substantial antitumor activity in a number of animal cancer models. However when TRAIL death receptor agonists were administered to cancer patients in the clinical trials no convincing anticancer responses were observed. Thus we began to search for “sensitizing agents”, i.e. compounds that in combination with TRAIL would enhance cancer cell death. We demonstrated that the proteasome inhibitor bortezomib was an excellent sensitizer of some cancer cells to TRAIL apoptosis both in vitro and in vivo. More recently we developed a high-throughput screening (HTS) procedure that identified the plant natural product withanolide E as a potent sensitizer of cancer cells to TRAIL-mediated apoptosis, despite the fact that as a single agent it had little direct effect on the cancer cells. Currently we are attempting to determine if more potent analogues of withanolide E can be identified and combined with TRAIL agonist antibodies for therapy, in addition to investigating the molecular mechanism that determines how active withanolides to sensitize cancer cells to death-ligand mediated apoptosis. Our overall aim is to identify compounds that further sensitize cancer cells to the cytotoxic effects of immunotherapy.
Conditions for successful therapeutic peptide vaccines
Cornelis Melief1, Marij Welters1, Tetje van der Sluis1, Sjoerd van der Burg1
1 Leiden University Medical Center, Leiden, NETHERLANDSTherapeutic cancer vaccines (TCV) have shown clinical activity by prolonging overall survival, not by inducing cancer regressions. Therefore efficacy of therapeutic cancer vaccines must be improved by the following approaches: 1) Choosing the right cancer associated antigens. The best antigens for targeting by TCV are mutant antigens in non-viral cancers and viral antigens in the case of virus-induced cancers. Although the most widely used antigens for TCV have been differentiation antigens or cancer testis antigens, the repertoire of T cells available against these antigens is usually blunted in the thymusby central immunological tolerance.2) Choosing the right vaccine platform. Concentrated delivery of both MHC class I and II epitopes is essential for two reasons. First, CD4+ and CD8 + T cells need to collaborate for optimal effector T cell induction and T cell memory formation. Second antigenic competition must be avoided. Appropriate TCV platforms are properly adjuvanted DNA, RNA and synthetic long peptide (SLP) vaccines.3) Choosing appropriate co-treatment. Adoptive T cell therapy of cancer is successful by combination with lymphodepleting chemotherapy or whole body irradiation and co-treatment with IL-2. Treatment with TCV has been successful in patients with premalignant lesions, but co-treatment is needed for efficacy in cancer patients because of the hostile cancer micro-environment. Standard chemotherapy depletes T cell-suppressive myeloid cells without affecting T cell numbers in patients with late stage cervical cancer. T cell function was drastically improved at the time of suppressive myeloid cell reduction, showing synergy with TCV in T cell response induction and in tumor eradication. Also, platinum-based chemotherapy allowed improved cancer cell apoptosis at low concentrations of TNF alpha produced by TCV-induced T cells. Other attractive co-treatments with TCV are checkpoint inhibitory MoAbs, MoAbs against immunosuppressive cytokines (TGFb. IL-10) and agonist MoAbs against selected members of the TNF Receptor family (CD27, CD40, CD134, CD137).
Speaker abstracts
Immunogenetics of cancer – from polymorphisms to clinic and beyond
Elissaveta Naumova1
1 University Hospital Alexandrovska, Medical University, Sofia, BULGARIAThe impact of the immune system on development of malignancies depends also on the genetic background of individuals. Polymorphisms in immune-related genes may influence the strength and type of anti-cancer immune response which in turn can be associated with the predisposition, prognosis and/or response to treatment in different cancers. Importance of immunogenetics in malignancies considering three highly polymorphic gene systems playing central role in modulating both the innate and adaptive immune response – HLA, cytokines and KIR will be reviewed in this presentation. Advances in recent years and current understanding along with our experience of the immune genetic profiles relevance as biomarkers for the risk of cancer development and application of these knowledge and achievements in clinic have been summarized. As the efficient immune response is a suppressor mechanism against emerging tumors, various immunotherapeutic approaches aim to boost patient’s immune reactivity. Use of immune-related genes as targets for new immunotherapies such as MHC class I Streptamer based technologies, immunomodulatory antibodies, gene modified cell lines, etc that could likely be efficient in the treatment of cancers in the era of the personalized medicine will be discussed.
Proffered Paper 3
Citrullinated vimentin, which is presented on MHC-II on tumor cells, is a novel rejection target for CD4 T cells
Victoria Brentville2, Rachael Metheringham2, Barbara Gunn2, Peter Symonds2, Ian Daniels2, Mohamed Gijon2, Wei Xue2, Lindy Durrant1,2
1 Nottingham University, Nottingham, UK, 2 Scancell Ltd, Nottingham, UKCD4 cells are potent effectors but CD4 responses to tumour associated antigens are attenuated. Cellular stress induces autophagy which leads to modification of proteins recognised by the immune system. In the absence of inflammation, immunity is regulated, whereas in its presence CD4 responses to modified self-antigens are stimulated. T cells commonly target modified self-antigens and have been shown to play a role in the pathophysiology of several autoimmune diseases. In this study the ability of these CD4 cells to target cancer has been explored. One modification is conversion of arginine to citrulline. Peptidylarginine deiminases (PADs), are a family of calcium dependent enzymes found in a variety of tissues, where their role is post translation citrullination of proteins. Immunisation with citrullinated vimentin peptides induced IFNγ and granzyme B secreting CD4 T cells in response to autophagic tumour targets. The induced CD4 responses to citrullinated self-peptide epitopes show minimal reactivity to the unmodified sequence. A single immunisation with modified peptide, up to 14 days after tumour implant, resulted in long term survival in 60-90% of animals with no associated toxicity. The anti-tumour responses were dependent upon CD4 but not on CD8 T cells. This study provides the first evidence that tumours can present citrullinated peptides that can be targets for CD4 cells. This presentation is dependent upon autophagy and PAD enzymes. The CD4 T cells release IFNγ and show direct cytotoxicity which results in potent anti-tumour responses in vivo. This approach is being fast tracked into the clinic in patient’s whose tumours express vimentin. This could be patients with mesenchymal tumours or in patients whose tumours undergo epithelial to mesenchymal transition as vimentin is one of the first proteins to be expressed in this transition
Speaker abstracts
Proffered Paper 4
T-cell receptor (TCR) repertoire profiling by deep sequencing as a biomarker for immunotherapy and a starting point for TCR gene therapy
Isabel Poschke3, Michael Flossdorf3, Marta Faryna2, Tana Omokoko1, Lena Appel3, Jessica Hassel4, Oliver Strobel4, Rolf Kiessling5, Rienk Offringa3,4
1 BioNTech Cell & Gene Therapies GmbH, Mainz, GERMANY, 2 BioNTech Diagnostics GmbH, Mainz, GERMANY, 3 German Cancer Research Center (DKFZ), Heidelberg, GERMANY, 4 Heidelberg University Hospital, Heidelberg, GERMANY, 5 Karolinska Institute, Stockholm, SWEDENCheckpoint inhibitors and adoptive transfer of tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical success in melanoma patients. We will make TIL-therapy available to melanoma patients in Heidelberg and are exploring its use in patients with pancreatic cancer, a devastating disease with a median survival of approximately two years even in patients eligible for surgery and adjuvant chemotherapy.While it is known that ≈50% of melanoma patients respond to TIL therapy, biomarkers for therapeutic success are lacking. The assumption that TIL are enriched for tumor-reactive T-cells, has been difficult to address experimentally and mechanisms governing the generation of the TIL-repertoire remain poorly understood. Next-generation sequencing (NGS) of T-cell receptors (TCR) allows us to study the composition of the TIL repertoire ex vivo, and during in vitro expansion. We find that the TIL TCR repertoire is i) distinct from the broad repertoire observed in blood; ii) usually dominated by large T-cell clones, possibly due to in situ expansion after tumor-antigen encounter; iii) likely heterogeneous throughout the tumor and iv) often not maintained during in vitro expansion, potentially leading to loss of important T-cell clones and a shift in tumor reactivity in the TILs available for patient treatment.TCR cloning based on TCR-α/β NGS data, though still technically challenging, provides us with a tool to ‘rescue’ TIL-TCRs with valuable reactivities that could be reintroduced to patients using transgenic T-cells. Furthermore, cloning of candidate TCRs will for the first time enable ex vivo studies on TIL tumor- and antigen-reactivity.Based on our initial data from melanoma and
pancreatic cancer TILs we will now investigate whether the TCR repertoire can serve as a predictive biomarker to identify patients likely to respond to immunotherapy and whether clonal maintenance from the original repertoire to the time of T-cell infusion is a quality biomarker for adoptive T-cell transfer.
Speaker abstracts
Fifteen years’ worth of lessons on the immunogenicity of tumors and tumor antigens; from melanoma to pancreatic cancer
Rienk Offringa1,2, Isabel Poschke1, Michael Volkmar1, Oliver Strobel2, Frank Bergmann3, Niels Halama4, Dirk Jäger4, Ugur Sahin5, Markus Büchler2
1 Div. Molecular Oncology of Gastrointestinal Tumors, German Cancer Research Center, Heidelberg; 2 European Pancreas Center, Surgery Clinic, University of Heidelberg; 3 Institute for Pathology, University of Heidelberg; 4 Dept. Clinical Oncology, National Center for Tumor diseases, Heidelberg; 5 BioNTech Inc., Mainz; GERMANYIn the late nineties, the prevailing view on anti-tumor T-cell immunity was that, at least in cancers without viral etiology, this response was primarily directed against over- and/or ectopically expressed tumor-associated self-antigens. By now, we have learned that the T-cell repertoire against the majority of these antigens is blunted due to their expression in the medullary thymic epithelial cells (mTECs), rendering cancer vaccines comprising these antigens ineffective. Furthermore, pre-clinical and clinical T-cell therapy experiments with genetically engineered T-cells targeting such antigens have revealed a lack of therapeutic index due to the destruction of vital somatic tissues expressing these self-antigens, thereby vividly demonstrating the biological need for central tolerance. Recently, T-cell recognition of neo-antigens encoded by somatic mutations was found to be the major driver of therapeutic T-cell responses in TIL therapy and checkpoint inhibition. This provided a plausible explanation for the high immunogenicity of melanomas and has also led the way towards successful immunotherapeutic intervention in other cancer types with high mutation rate, such as smoking-associated lung cancer. It also raises the question whether neo-antigenicity is sufficient to support T-cell therapeutic approaches in cancer types that display 5-10-fold lower somatic mutation rate, such as pancreatic cancerIn contrast to the general belief that pancreatic ductal adenocarcinoma is a poorly immunogenic tumor, we found cumulative evidence for an adaptive immune response in primary resected tumor biopsies. Immunohistochemistry reveals prominent T-cell infiltrates in the majority (~ 70%) of tumor biopsies, and these tumor-infiltrating lymphocytes (TILs) can be isolated and expanded ex vivo with similar efficiency as those isolated from melanoma. Furthermore, comparison of the T-cell receptor repertoire between TIL and PBMC isolates from patients points at the selective expansion of T-cell
subsets in the tumors*. Finally, in ~ 50% of tumor specimen, T-cell infiltration is accompanied by the presence of tertiary lymphoid structures that comprise areas rich in CD3+ T-cells and CD208+ dendritic cells as well as areas rich in B-cells and follicular dendritic cells.Based on these findings, we are currently analyzing the antigen-specificity of the T-cell response in human pancreatic cancer and are setting up clinical trials aimed at harnessing this response.* See abstract Isabel Poschke on TCR deep sequencing
Speaker abstracts
The continuing saga of understanding and improving anti-tumor immunity: From cancer vaccines to myeloid-derived suppressor cells
Suzanne Ostrand-Rosenberg 1
1 University of Maryland Baltimore County, Baltimore, USAAt the first PIVAC meeting in 2001 at Robinson College, Cambridge University, I had the opportunity to present our studies aimed at improving anti-tumor immunity by driving the activation of IFNγ-producing tumor-reactive CD4+ T cells. These studies had started over 10 years earlier and were based on the strategy that tumor cells could be genetically modified to express MHC class II molecules and the costimulatory molecule CD80 and thereby function as effective antigen presenting cells for novel tumor antigens. We tested these so-called “MHC II vaccines” in multiple mouse models. The strategy worked very well in vitro and in mice with established tumors of less than approximately 5-8 mm in diameter. Later studies expanded this vaccination strategy to human melanoma and breast cancer cells. In vitro experiments with patients’ PBMC showed not only impressive activation of IFNγ-producing tumor-reactive CD4+ T cells, but also significant CD8+ T cell-mediated lysis of wild type tumor cells. Mass spectrometry analyses demonstrated that the MHC II vaccines were effective because they presented novel peptides that were not presented by conventional APC. The vaccine cells presented novel peptides because they lacked the MHC II-associated invariant chain (Ii) and therefore trafficked intracellularly via a non-conventional pathway and picked up a different repertoire of peptides. Interestingly, subsequent in vivo mouse studies showed that the MHC class II vaccine cells were not the actual APC, but rather host dendritic cells were “cross-dressed” with MHC/peptide complexes from the tumor cells and that the DC were the actual APC. These reports were some of the first studies to demonstrate that DC not only acquired antigen but also acquired intact MHC I/ and II/peptide complexes directly from tumor cells. Although this vaccination strategy seemed promising and worked well in vitro with human tumors and PBMC, the approach did not work with all mouse tumors, leading us to be skeptical about its applicability. These failures also indicated to us that the immune system of individuals with advanced cancers were functionally impaired relative to tumor-free individuals. We first noticed in the late 1990’s that mice with mammary carcinoma had enormously enlarged spleens (lab jargon for the spleens was “jumbo spleen”). By the early 2000’s we realized that the enlargement was associated with excessive levels of circulating myeloid cells. Depletion of these myeloid cells
resulted in T cell-mediated rejection of long-established mammary carcinoma metastatic disease and elimination of tumor-promoting macrophages, and led us to conclude that the myeloid cells were potently immune suppressive. These myeloid cells subsequently were named “myeloid-derived suppressor cells” (MDSC) and have now been found by many investigators in virtually all cancer patients. Once we realized that MDSC were potent inhibitors of anti-tumor immunity, we focused on understanding how and why they accumulated in individuals with cancer. We identified various pro-inflammatory mediators as inducers of MDSC. This information led to the concept that chronic inflammation facilitates tumor development and progression by inducing MDSC and inhibiting anti-tumor immunity. We now believe that our MHC II vaccines, and most likely many other immunotherapy strategies that depend on activating the patient’s immune system, may not be effective in patients with advanced cancer because MDSC are inhibiting the development of anti-tumor immunity.
Speaker abstracts
Proffered Paper 5
xCT is a new cancer stem cell immunotherapeutic target for breast cancer
Stefania Lanzardo3, Laura Conti3, Ronald Rooke2, Roberto Ruiu3, Nathalie Accart-Gris4, Elisabetta Bolli3, Maddalena Arigoni3, Marco Macagno3, Giuseppina Barrera1, Stefania Pizzimenti1, Raffaele Adolfo Calogero3, Federica Cavallo3
1 Department of Clinical and Biological Sciences, University of Torino, Torino, ITALY, 2 Elsalys Biotech, Illkirch Graffenstaden, FRANCE, 3 Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, ITALY, 4 Novartis, Institute for medical research, Basel, SWITZERLANDThe several unsuccessful treatments in metastatic cancers might miss cancer stem cells (CSC), a sub-population of cells with a critical role in cancer. By contrast, a vaccine-elicited immune response against CSC might be particularly effective. The identification of oncoantigens (OA) expressed by CSC may provide new targets for effective anticancer vaccines.To this purpose, the transcription profile of the ErbB2+TUBO mouse mammary cancer cell line was compared with that of TUBO cells grown under specific conditions to generate CSC-enriched mammospheres. Integrating the data obtained with meta-analyses of seven independent human breast tumor data sets we identified new CSC mammary OA that were validated both in vitro and in vivo.Among the identified OA, we focused on xCT, a channel that supports glutathione synthesis regulating the intracellular redox balance. We observed that xCT expression increases in TUBO-derived mammospheres and in mammospheres obtained from triple negative breast cancer cells (i.e. 4T1 cells); its silencing, obtained with specific siRNA, or incubating mammospheres with its inhibitor sulfasalazine (2), significantly reduces TUBO cells ability to generate mammospheres. Anti-xCT vaccination impaired CSC ability to generate lung metastases derived from both an intravenous injection of TUBO-derived mammospheres and subcutaneous 4T1 tumors; furthermore, this effect was improved when combined with the cytotoxic drug doxorubicin. Moreover, the anti-xCT vaccination of mice bearing TUBO and 4T1 mammosphere-derived tumors reduced the
speed of tumor growth.In conclusion, this study provides a genomic characterization of mammary CSC and identifies a fresh target for new and potentially effective anticancer vaccines, thus providing a new tool for the design of combined therapeutic approaches that efficaciously target both CSC and more differentiated cells in breast cancers, leading to both cancer treatment and prevention of metastases and recurrence. BIBLIOGRAPHY1. Nagano O et al., Oncogene 2013.2. Chen RS et al., Oncogene 2009.
Speaker abstracts
Proffered Paper 6
Removed due to author’s request.
Proffered Paper 7
“Developing an awareness of the mechanism of Epithelial to Mesenchymal Transition (EMT) in prostate cancer; implications for immunotherapy”
Jayakumar Vadakekolathu1, David J Boocock1, Naomi Dunning-Foreman1, Robert C Rees1, John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UKAccumulating evidence suggests that cancer cells acquire invasive and migratory properties that are necessary for the invasion-metastasis cascade by reactivating a latent embryonic programme known as epithelial to mesenchymal transition (EMT). The role of EMT in prostate cancer have been reported using various cell models in the past. Moreover, androgen deprivation therapy a standard-of-care, first-line therapy for prostate cancer also reported to induce EMT through ZEB1 transcription factor in a negative feedback loop mechanism. Therefore, a detail understanding of EMT mechanisms is important to intervene the metastatic spread and dissemination of prostate cancer. Many of the causative mechanisms underlying EMT had been elucidated in in vitro cell models using artificial EMT induction such as TGF-β treatment and ectopic expression of selected transcription factors (TF). In this study we have investigated several prostate cancer cell lines (from metastatic and primary derived) and identified a cell line (OPCT-1) having naturally transitioning (EMT and possibly MET) cell populations. Several well characterised clonal progenies of this cell line with different EMT potential have been generated and characterised. Utilising this model, we have studied global transcriptional and proteome changes to understand the molecular mechanisms underlying EMT and also to identify novel targets for therapeutic interventions.
NK receptors and their ligands in cancer. Towards personalised NK cell based immunotherapy
Rafael Solana1, Raquel Tarazona2
1 IMIBIC - Hospital Universitario Reina Sofia - Universidad de Cordoba , Cordoba, SPAIN, 2 Immunology Unit. Dept.Physiology. University of Extremadura, Caceres, SPAINNatural killer (NK) cells are a key component of the innate immune response. Together with T cells, NK cells are an important part of the tumour surveillance system. The characterization of NK receptors and their ligands in cancer cells has opened new perspectives for NK cell-based immunotherapy of cancer. Ageing is associated to an increased incidence of cancer and to alterations of adaptive and innate immunity, a process referred as immunosenescence. It has been argued that the deterioration of the immune system associated to ageing affects cancer immunosurveillance, potentially contributing to cancer development in the elderly. Here we will present our work during the last 15 years on the characterization of the receptors and ligands involved in NK cell recognition of melanoma and acute myeloid blasts and the impact of ageing and persistent cytomegalovirus infections on NK cells from healthy elderly donors and cancer patients. Acute myeloid leukaemia (AML) patients show a decreased expression of several activating NK cell receptors and consequently a reduced ability to kill tumour cells. These alterations are more pronounced in elderly AML patients compared to younger patients. The contribution of age-associated NK cell immunosenescence to leukaemia immunosurveillance in these patients is discussed. In young cancer patients, abnormalities in NK cell phenotype and function have also been observed that resemble age-associated changes. Thus, cancer development may cause alterations that can be considered as premature immunosenescence. It has been postulated that NK cells get exhausted by chronic contact with tumour cells. The advances on the knowledge of NK cells-tumour interactions and on the effect of ageing and CMV infection on NK cells indicate that an effective personalised NK cell-based cancer immunotherapy should consider both age-associated alterations that influence the phenotype and function of NK cells and cancer-associated NK cell exhaustion.
Speaker abstracts
15 years of tinkering with T cells
Per thor Straten1
1 Center for Cancer Immune Therapy (CCIT), Copenhagen University Hospital Herlev, Herlev, Copenhagen region, DENMARKT cells are known to infiltrate tumors – so called tumor infiltrating lymphocytes - and the presence of clonally expanded T cells in tumors suggests expansion of T cells upon recognition of tumor associated antigens (TAA). We have shown that clonally expanded T cells are present in melanoma lesions, with a unique clonotype fingerprint in each lesion although some systemic involvement is evident as well. Using the reverse immunology approach, we have characterized tumor antigens from universal TAA with a focus on proteins that play an important role in the biology of cancer cells, e.g., survivin, Bcl-2, RhoC, and cyclin B1. Moreover, we have shown that these antigens are recognized by T cells among TIL as well as PBMC. Peptides from some of these tumor antigens have been tested in clinical trials in cancer patients, and shown to induce T cell reactivity and potential clinical activity, although the latter need to be demonstrated in larger clinical trials. More recently, we have returned to TILs in melanoma and exploited these in adoptive cell transfer (ACT) in the clinic. We and others have demonstrated induction of complete lasting responses in approx. 20 % of the patients. We have conducted a comprehensive monitoring and described characteristics of TIL cultures and the associated biological responses that correspond with clinical response. Data are accumulating that TILs may be of clinical relevance both as a prognostic and predictive marker. Thus, ways to transform a non-inflamed tumor to an inflamed tumor, heavily infiltrated by immune cells, is of huge interest. We recently demonstrated across several murine tumor models, that exercise leads to influx of immune cells in the tumors and an associated delay in tumor growth. As a consequence, exercise could represent a means by which infiltration of immune cells could be increased, which could be clinically relevant.
Poster abstracts
Poster abstracts
1Combination of mRNA hTERT transfected Dendritic Cells and repeated localized radiotherapy with Cyberknife shows an unexpected effect in a stage IV lung cancer patientIris Bigalke3, Kirsti Hønnåshagen3, Marianne Lundby3, Guri Solum3, Julitta Kasten1, Stein Sæbøe-Larssen3, Dolores Schendel2, Gunnar Kvalheim3
1 Helmholtz Zentrum München, München, GERMANY, 2 Medigene Immunotherapies AG, Martinsried, GERMANY, 3 Oslo University Hospital, Oslo, NORWAYRecently it has been described that radiotherapy (RT) gives regression of tumor growth at sites distant from primary field of irradiation (abscopal RT effects) based on various mechanisms through which RT actively induces anti-tumor immunity.We have previously shown that a new generation of fast DCs, using a maturation cocktail containing the TLR7/8 ligand R848, mount strong specific immune responses and prolong overall survival in different types of malignancies. Here we report our experience with DC vaccines and repeated localized RT in a poor risk metastatic lung cancer patient.In 7/2011 a 61-year-old woman was diagnosed with advanced adenocarcinoma in the lung with several bilateral metastatic lesions in lung and mediastinum and 2 brain metastases. Brain metastases were treated with Cyberknife and from September to November 2011 she was given several cycles of chemotherapy with only modest clinical effect. In 12/2011 we started vaccination with mRNA hTERT transfected DCs and the patient developed a stable disease. In 2/2013 DC vaccination was interrupted and RT given against the primary tumor due to bronchial obstruction. During RT the patient developed a pleurisy which was treated with high doses of corticosteroids. A new fast growing brain metastasis developed in the Pons in 5/2013 which was again treated with Cyberknife, corticosteroids were stopped. DC vaccination was continued and she was again brought into stable disease. In 4/2014 one lung metastasis slightly increased and was treated with Cyberknife while continuing DC vaccination. In 1/2015 a new frontal brain metastasis was diagnosed and again she had RT with Cyberknife. The patient continued with monthly
boosts of DC vaccines and in 6/2015 she still has a stable disease.Our findings suggest a synergistic clinical effect of combining DC vaccination and RT. More studies should follow to explore this combination.
2Adjuvant choice modulates self antigen specific CD4 responses generated by peptide vaccination Victoria Brentville2, Wei Xue2, Katherine Cook2, Peter Symonds2, Barbara Gunn2, Rachael Metheringham2, Lindy Durrant1,2
1 Nottingham University, Nottingham, UK, 2 Scancell Ltd, Nottingham, UKThe important role of tumour specific CD4 T cells in cancer therapy has been increasingly documented. However over coming self tolerance to induce efficient tumour specific CD4 T cell responses is an ongoing challenge for cancer vaccines. This study details the efficient induction of self epitope specific CD4 responses that can directly target tumour. Rapid generation of responses from a single peptide vaccination suggests these cells to be pre existing in the memory pool. Vaccination in combination with the TLR9 and TLR4 ligands CpG and MPLA results in maturation of responses to a Th1 phenotype also producing granzyme B. If immunisation with the same peptides is combined with Incomplete Freunds adjuvant, responses are skewed to a more regulatory phenotype producing IL10 and combination with a TLR7 agonist generates a mixed Th1/regulatory response. Th1 responses induced in the presence of CpG/MPLA provide efficient tumour protection whereas adjuvants inducing regulatory responses show no tumour protection. This is a phenomenon restricted to self epitope specific responses as adjuvant choice has little effect upon foreign epitope specific responses. These studies highlight the polarisation of self CD4 responses with use of different adjuvants and the potential impact of adjuvant choice in cancer vaccine design.
3Evaluation of novel metronomic chemotherapy and cancer vaccine combinatorial strategy.Maria Tagliamonte3, Annacarmen Petrizzo3, Maria Lina Tornesello3, Antonio Luciano2, Domenica Rea2, Antonio Barbieri2, Claudio Arra2,
Poster abstracts
Maria Napolitano4, Gennaro Ciliberto1, Luigi Aurisicchio6, Claudio Coscia5, Franco Maria Buonaguro3, Luigi Buonaguro3
1 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Pascale” - Scientific Direction, Naples, ITALY, 2 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Pascale” - Animal Facility, Naples, ITALY, 3 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Pascale” - Lab. Molecular Biology and Viral Oncology, Naples, ITALY, 4 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Pascale” - Laboratory of Clinical Immunology , Naples, ITALY, 5 Pantec Biosolutions AG, Ruggell, LIECHTENSTEIN, 6 Takis S.r.l., Rome, ITALYThe efficacy of cancer immunotherapy approaches can be significantly hampered by the tumor immune suppressive microenvironment. In the present study, a novel combinatorial strategy (i.e. metronomic chemotherapy plus vaccine) is evaluated in a mouse model in an aggressive therapeutic setting based on sub-cutaneous ectopic implantation of B16 melanoma cells. The chemotherapy is a multi-drug cocktail including taxanes and alkylating agents, administered in a daily metronomic fashion. The vaccine is a multi-peptide cocktail of universal tumor antigen hTERT epitopes adjuvanted in Montanide.Immunizations have been performed comparing a standard sub-cutaneous delivery with a laser-assisted epidermal immunogen delivery by P.L.E.A.S.E.Ò Professional (Pantec Biosolutions). The latter, indeed, creates evenly distributed micropores through to the epidermis and provides a great opportunity to efficiently deliver vaccine antigens to Langerhans cells (LC) in the epidermis.An interim analysis shows that the immunogenicity as well as the therapeutic effect on tumor growth of the vaccine is significantly increased by the combination with metronomic chemotherapy, and the laser-assisted epidermal immunogen delivery appears to be a very effective strategy as vaccine delivery. Complete description of both immunogenicity and therapeutic effects on tumor growth will be described.
4A Natural Polymer (NPX) as a new adjuvant for breast cancer vaccinationDebora Carpanese2, Isabella Monia Montagner3, Anna Dalla Pietà2, Gianfranco Pasut1, Anna Mero1, Paola Zanovello2,3, Antonio Rosato2,3
1 Department of Pharmaceutical and
Pharmacological Sciences, University of Padova, Padova, ITALY, 2 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, ITALY, 3 Istituto Oncologico Veneto IRCCS, Padova, ITALYIntroduction: The use of proteins as immunogens in cancer vaccination requires potent adjuvants to overcome their weak immunogenicity. As alum failed in cancer vaccination, many efforts have been done for the development of new adjuvants. Natural Polymers (NP) as TLR agonists are emerging as a new class of vaccine adjuvants, as they finely orchestrate the cross-talk between innate and adaptive immunity. We explored the potentiality of a NP (indicated as NPX for patent constraints) as a new carrier of immunogens to design more efficient cancer vaccines. To this aim, NPX was chemically linked to the extracellular domain of rat HER2/neu (rHER2/neu).Materials and Methods: Female BALB/c or BALB-neuT transgenic mice were immunized with rHER2/neu-NPX or with the same amount of protein emulsified in alum as control. Mouse sera and spleens were collected and antigen-specific humoral and cellular responses were characterized. Vaccinated BALB/c mice were challenged with rHER2-overexpressing TUBO cells to assess the protective or therapeutic activity of rHER2-NPX vaccination strategy.Results and conclusions: NPX disclosed an extremely efficient profile, since strong and long-lasting humoral and cellular immune responses were detected. NPX induced a fine balance between Th1 and Th2 responses, which turned out effective in both the prophylactic and therapeutic settings. Conversely, alum completely failed to confer any protection. Moreover, NPX vaccination was successful in breaking tolerance against rHER2/neu, and delayied the growth of spontaneous tumors in BALB-neuT mice.
5Induction of peptide-vaccine specific T cell responses coinciding with long term survival in a patient with metastatic cholangiocarcinoma.Anoop Chandran P5, Markus W. Löffler5,4, Karoline Laske5, Hans Bösmüller6, Irina Bonzheim6, Nico Trautwein5, Mathias Walzer5,3, Christopher Mohr3, Christopher Schroeder2, Franz J Hilke2, Huu-Phuc Nguyen2, Olaf Riess2, Karolin Thiel4, Silvio Nadalin4, Alfred Königsrainer4, Falko Fend6, Oliver Kohlbacher3, Cecile Gouttefangeas5, Stefan Stevanovic5,1,
Poster abstracts
Hans-Georg Rammensee5,1
1 German Cancer Consortium, DKFZ partner site Tübingen, Tübingen, GERMANY, 2 Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, GERMANY, 3 Applied Bioinformatics, Center for Bioinformatics, Quantitative Biology Center, and Dept. of Computer Science, University of Tübingen, Tübingen, GERMANY, 4 Department of General, Visceral and Transplant Surgery, University of Tübingen, Tübingen, GERMANY, 5 Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, GERMANY, 6 Institute of Pathology, University of Tübingen, Tübingen, GERMANYBackground: Cholangiocarcinomas are rare epithelial tumors arising from the liver bile ducts and are associated with a very poor prognosis (median survival about 28 months). Apart from surgery no other curative therapies are available. Immunotherapy looks promising considering recent clinical findings by Tran et al. (Science 2014). We report a case of a large unilocular cholangiocarcinoma diagnosed in 2010. Over the course of 3 years, the primary tumor, locally recurrent tumors on two occasions as well as a pulmonary metastasis were resected and analyzed.Vaccination: Based on previous knowledge and HLA-ligandomics, a vaccine cocktail containing 7 peptides (4 HLA-A*03 restricted and 3 MHC-class II specific) was administered 27 months after initial diagnosis (in accordance with §35 of the Declaration of Helsinki, Seoul 2008) with a tapering vaccination schedule and using montanide and imiquimod as adjuvants.Immunomonitoring and T cell responses: Histological examination of the pulmonary metastasis, excised 7 months after the start of peptide vaccinations, indicated higher lymphocyte infiltration, CD25 and perforin expression and lower FOXP3 expression compared to the earlier resected liver tumor tissues. Vaccination induced T cell responses against 3/7 vaccinated peptides.Clinical course: The tumor recurred twice in the liver after surgical resection and once in the lung shortly after the induction of peptide therapy. Currently, the patient is alive and well without detectable tumor manifestations, which is rare in a metastatic cholangiocarcinoma. No adverse effects were reported after vaccination except for local granulomas at the vaccination site. Having seen peptide specific T cell responses and subsequent tumor-free survival of the patient 33 months after start of therapy we consider our
multi-peptide vaccination a safe and promising approach for this hard to cure cancer.
6Potent anti-tumour immune responses to citrullinated enolaseKatherine Cook1, Ian Daniels1, Victoria Brentville1, Rachael Metheringham1, Wei Xue1, Peter Symonds1, Tracy Pitt1, Mohamed Gijon1, Lindy Durrant1,2
1 Scancell Ltd, Nottingham, UK, 2 University of Nottingham, Nottingham, UKCitrullination is a posttranslational modification which occurs as a result of cellular stress and leads to the generation of neoantigens. In rheumatoid arthritis this process generates potent immune responses. Our research suggests that the citrullination of peptides in rapidly growing tumour cells may be exploited by vaccines leading to tumour rejection.Alpha enolase is a glycolytic protein which is upregulated in many cancers and is citrullinated. In this study, screening citrullinated peptides from the enolase sequence detected significant IFNγ responses from a number of peptides. A single immunisation with Enolase 241-259cit peptide in HLA-DR4 transgenic mice induced IFNγ responses after 14 days (p=0.0124). Subsequent experiments showed that this response is CD4 mediate and also occurs in HLA-DP4 with but not in wild type C57Bl/6 mice. The IFNγ response develops rapidly and can be detected 2 days after immunisation.Immunoblots showed enolase expression in a number of tumour cell lines including the murine melanoma B16 and lewis lung carcinoma LLC2. Anti-tumour studies were performed with cell lines transfected with HLA-DR4 under a constitutive or IFNγ inducible promoter. Enolase 241-259cit peptide immunised mice showed increased survival for both B16-cDR4 (p=0.0001), B14 iDR4 (p=0.0001) and LLC2-cDR4 cell lines (p=0.0142). Increased survival was also seen when mice were rechallenged with tumour 38 days after immunisations.Assays on peripheral blood mononuclear cells from a small group of healthy donors suggest that the majority of humans are able to develop a citrulline specific CD4 proliferative response to Enolase 241-259cit peptide.This study identified a citrullinated Enolase peptide which induces a rapid, citrulline specific IFNγ response. This response has a therapeutic anti-tumour effect and a repertoire has been detected in humans. This provides further evidence that CD4 responses against modified
Poster abstracts
self-antigens may provide important targets for cancer vaccines.
7A clinical trial of a DNA vaccine (SCIB1) that targets dendritic cells in vivo in fully resected melanoma patients; a vaccine to prevent disease recurrence?L.G. Durrant2,7, C.H. Ottensmeier3, C. Mulatero4, P. Lorigan5, R. Plummer6, M. Cunnell7, R. Metheringham2, V. Brentville2, L. Machado2, I. Daniels2, D. Hannaman1, P.M. Patel71 Ichor Medical Systems, San Diego, CA, USA, 2 Scancell Ltd, Academic Dept of Clinical Oncology, Nottingham, UK, 3 Southampton University Hospital, Faculty of Medicine, Southampton, UK, 4 St James University Hospital, Leeds, UK, 5 University of Manchester, Institute of Cancer Sciences, Manchester, UK, 6 University of Newcastle-upon-Tyne, Northern Institute for Cancer Research, Newcastle, UK, 7 University of Nottingham, Academic Dept of Clinical Oncology, Nottingham, UKBackground:SCIB1 is a DNA vaccine encoding a human IgG1 antibody with CDRs that contain four epitopes from two melanoma antigens (three from gp100 and one from TRP2). The vaccine elicits potent anti-tumour responses by stimulating high frequency, high avidity T-cells via both direct and cross-presentation of antibody. A clinical study in stage III/IV melanoma patients, all with tumour present at study entry, showed that 2-8mg doses could induce T-cell responses in 7/9 patients with no associated toxicity. Encouragingly overall survival was 31 months. This study addresses the question as to whether SCIB1 can be used as an adjuvant therapy in fully resected melanoma patients to prevent further disease.Methods: Sixteen patients with fully resected stage III (n=9) or stage IV (n=7) melanoma were immunised with 4mg of SCIB1 by intramuscular electroporation at 3 weekly intervals and subsequently at 3 and 6 months. Patients could continue treatment for 5 years.Results:All 16 patients showed vaccine-epitope-specific T-cell responses (i.e. proliferation ex vivo and/or γIFN Elispot responses in-vitro). Twelve patients responded to all four epitopes, two patients to three epitopes, one to two epitopes and one to a single epitope. Five patients remain in the continuation phase - all show strong T-cell memory responses following boosting. At present, median survival time is 37 months
from trial entry and 41.5 months from diagnosis of metastases. Overall survival is 100% for both groups. Five patients relapsed at 1, 4, 14, 17 and 18 months but have shown no further recurrences at follow-up.Conclusion:These results show that a DNA vaccine encoding epitopes from melanoma antigens can induce measurable T-cell responses and, furthermore, it may confer protection from recurrence of melanoma with little associated toxicity. SCIB1 deserves further evaluation as an adjuvant therapy.
8TCR modifications that enhance proper chain pairing may also augment reactivity against naturally occurring mutant peptidesKendra Foley2, Timothy Spear2, Kauru Nagato1, David Murray2, Elizabeth Garrett-Mayer3, Michael I. Nishimura2
1 Chiba University, Chiba, JAPAN, 2 Loyola University Chicago, Maywood, IL, USA, 3 Medical University of South Carolina, Charleston, SC, USAUsing TCR gene modified T cells for adoptive cell transfer has been shown to have clinical success in treating melanoma and other malignancies. One of the challenges in using TCR gene modified T cells include proper expression of the introduced TCR and function. Mispairing between endogenous and introduced TCR chains allows for the potential of unanticipated off-target reactivity, autoimmunity, and impaired therapeutic efficacy against targeted antigens. One approach to improve TCR expression, pairing, and T cell function involves the modification of the introduced TCR genes to promote proper pairing. Some of these modifications include introducing a disulfide bridge in the alpha/beta constant regions, murine constant regions, codon optimization, TCR chain leucine zipper fusions, and a single chain TCR. We have previously cloned a novel TCR from a HLA-A2 restricted, HCV NS3:1406-1415-reactive T cell clone. We have developed a surface transduction marker, CD34t, that allows for measurement of TCR expression due to the 1:1 stoichiometric ratio between CD34t and TCR proteins. Our results revealed that the murinized Cβ2 TCR and the leucine zipper TCR have the highest levels of expression per transduced T cells when compared to the wild type TCR. It is evident that while some modifications have higher levels of TCR expression, this does not always result in increased function. Although, on a transduced
Poster abstracts
cell by cell basis, the murinized Cβ2 TCR and the leucine zipper TCR have the highest percentages of bifunctional cells. Our data has revealed proper chain pairing may have an impact on the T cell’s ability to be reactive against HCV NS3 mutant peptides. Our studies have given us a better understanding of how TCR modifications can impact expression and T cell function that may allow for optimization of adoptive T cell transfer to treat viral infections and malignancies.
9RNAdjuvant®, a novel, highly-potent RNA-based adjuvant, combines strong immunostimulatory capacities with a favorable safety profileRegina Heidenreich1, Johannes Lutz1, Aleksandra Kowalczyk1, Patrick Baumhof1, Birgit Scheel1, Söhnke Voss1, Karl-Josef Kallen1, Mariola Fotin-Mleczek1
1 CureVac GmbH, Tübingen, GERMANYPurified recombinant proteins and peptides, which are currently under development in various anti-cancer vaccination approaches, lack sufficient immunogenicity. Therefore, potent adjuvants are needed to induce strong and persistent anti-tumor immunity. However, currently only few adjuvants are licensed, most of which primarily enhance antibody, but not T cell responses.In our study, we demonstrate that the novel, well defined, and thoroughly characterized RNA-based adjuvant, RNAdjuvant®, significantly enhances anti-tumor immunity, and that even complete tumor rejection can be achieved. We found that in the syngeneic TC-1 tumor, a murine model of human HPV-induced cervical cancer, RNAdjuvant® in combination with E7 long peptide was able to induce functional memory responses that mediated complete tumor remission. This observation was striking, as adjuvantation with the commonly used immune enhancer poly(I:C) did not induce protective immunity.Our adjuvant mediates balanced and long-lasting immune responses including strongly enhanced T cell responses. It is composed of a single-stranded, non-coding and non-capped RNA of a defined sequence, complexed with a specific cationic peptide, is optimized for strong immunostimulatory properties and stabilized against degradation. Our adjuvant acts locally, promoting strong but transient up-regulation of anti-viral and pro-inflammatory cytokines, CXCR3-ligands and cytoplasmic RNA sensors at the injection site, avoiding any systemic cytokine release. These changes are followed by activation of immune cells in the draining lymph nodes. In
repeated dose toxicity studies carried out in mice and pigs no toxicity events were observed. In summary, our data suggest that RNAdjuvant®
represents a novel, highly efficacious adjuvant candidate that enhances anti-cancer responses in vaccination regimen by minimizing risk of inducing severe adverse effects. Phase I safety studies in humans is currently running. Our data suggest that RNAdjuvant® represents a novel breakthrough technology that could help to propel forward the field of cancer vaccines.
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racotumomab-alum cancer vaccine as switch maintenance therapy for advanced NSCLCMaurenis Hernandez3, Sailyn Alfonso2, Elia Neninger4, Eduardo Santiesteban6, Ramon Ortiz2, Yoana Flores8, Rosa Amador5, Soraida Acosta9, Loisel Bello1, Anet Valdes3, Carmen Viada3, Mayte Robaina7, Liset Sanchez3, Ana Maria Hernandez3, Amparo Macias3, Tania Crombet3
1 Antonio Luaces Iraola Hospital, Ciego de Avila, CUBA, 2 Celestino Hernandez Robau Hospital, Villa Clara, CUBA, 3 Center of Molecular Immunology, Havana, CUBA, 4 Hermanos Ameijeiras Hospital, Havana, CUBA, 5 III Congreso Hospital, Pinar del Rio, CUBA, 6 Jose Ramon Lopez Tabranes Hospital, Matanzas, CUBA, 7 National Coordinating Center for Clinical Trials, Havana, CUBA, 8 National Institute of Oncology, Havana, CUBA, 9 Saturnino Lora Hospital, Santiago de Cuba, CUBA Maintenance strategies increase progression free survival (PFS) and overall survival (OS) of advanced NSCLC patients. NeuGc-containing gangliosides are attractive targets for cancer Immunotherapy.Their expression has been reported in tumors but not in normal cells. Racotumomab-alum is an antiidiotype vaccine that induce immunological response against gangliosides, with a low toxicity profile.A phase II/III , placebo-controlled, double blind clinical trial was conducted to evaluate racotumomab-alum as switch maintenance therapy for 176 advanced NSCLC patients. Vaccination consisted of 5 doses, every 14 day (induction), followed by a maintenance every 28 days. Median OS (ITT) was 8.23 months in racotumomab-alum group and 6.17 months in placebo group (p= 0.036), with a 24 months OS rate of 19.4% in racotumomab-alum versus 7.7% in placebo . In those patients
Poster abstracts
who completed induction phase median OS was 10.97 months in racotumomab-alum group vs 6.27 months in placebo group (log rank test p= 0.0115), and the OS rate at 24 months was 24% and 6.2 % , respectively. Another ongoing phase III clinical trial is comparing racotumomab-alum (or nimotuzumab) vs docetaxel as switch maintenance therapy in one stratum of patients (n=233) (RPCEC00000179). Patients are randomized 2:2:1 to receive racotumomab-alum (n=93), nimotuzumab (anti-EGFR MoA) (n=93) or docetaxel(n=47), after first-line therapy. In the first planned interim analysis median OS and one-year survival rate were 11.2 months and 45.5% for racotumomab-group, 11.6 months and 48.4% for nimotuzumab group, vs 8.0 months and 26.5% for docetaxel group. Most frequent vaccine-related adverse events were injection-site reaction and flu-like symptoms, grade 1 or 2 according CTCAE v4.Racotumomab-alum vaccine has a clinical benefit as switch maintenance therapy for advanced NSCLC patients. Final analysis of ongoing trial will be reported in 2017.
11Efficient in vitro induction of T-cell responses specific for re-call and tumour-associated self-antigens using combinations of TLR agonists Kyriaki Ioannou1, Diana Dominguez1, Aniekan Etuk1, Linda D Barber1, Farzin Farzaneh1
1 Department of Haematological Medicine, King’s College London, London, UKPrevious studies have demonstrated that combinations of Toll-like receptor (TLR) agonists constitute substantially more effective adjuvants for the in-vivo induction of antigen-specific cellular immunity, than when used separately. Here we describe an in vitro screening strategy for identification of optimal TLR agonist combinations for induction of antigen-specific CD4+ and CD8+ T-cell responses. Following the invitro culture of human peripheral blood mononuclear cells with an Influenza A-derived MHC class I-restricted peptide, expansion of pentamer+, IFNγ-producing CD8+ T-cells, was examined. The combination of CpG (TLR9) and polyI:C (TLR3) substantially enhanced the fraction of pentamer+ and IFNγ-producing effector memory CD8+ T-cells specific for this re-call viral antigen. Furthermore, in-vitro expansion of both CD4+ and CD8+ T-cells specific for the tumour-associated self-antigen Wilm’s Tumour antigen (WT1) was successfully achieved using a library of overlapping 15aa-long
peptides, spanning the full sequence of WT1, in combination with CpG and polyI:C.This screening strategy is now being employed to identify combinations of TLR agonists and other adjuvants for more effective vaccination-mediated induction of cellular immunity against antigens that are associated with cancer and with persistent viral infections.
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Generation of polyclonal and polyfunctional CD4+ Thelper1 cells against NY-ESO-1, WT1, MAGE-A3, Survivin and ROR-1 for adoptive T-cell immunotherapySimone Kayser5, Cristina Boß2, Christina Kyzirakos5,1, Stefan Stevanović4, Peter Lang5, Martin Röcken2, Rupert Handgretinger5
1 CeGaT GmbH, Tübingen, GERMANY, 2 Department of Dermatology, University Hospital, Tübingen, GERMANY, 3 Dr. von Haunersches Kinderspital, Ludwig-Maximilians-University Munich, Tübingen, GERMANY, 4 Eberhard Karls University, Department of Immunology, Tübingen, GERMANY, 5 University Children´s Hospital, Tübingen, GERMANYAdoptive T cell therapy (ACT) is a promising option to treat cancer. Th1 cells have been shown effective against viremia and cancer and are mediators of senescence in tumor cells. T cells against tumor associated antigens can be used for adoptive T-cell transfer approaches. According to these considerations, we developed a protocol according to good manufacturing practice (GMP) to generate Th1 cells against NY-ESO-1, WT1, MAGE-A3, Survivin and ROR-1. The protocol starts with a pre-sensitization of T cells with antigen. T cells were then enriched based on IFN-γ capture technique and expanded using autologous feeder and IL-2, IL-7 and IL-15. T-cell specificity and function was analyzed by flow cytometry. The T-cell lines showed high numbers of specific IFN-γ+ and TNF-α+ CD4+ T cells but no IL-10. This cytokine profile enables the induction of senescence in tumor cells. Both, CD4+ and CD8+ T cells had an effector memory phenotype and proliferated in response to the tumor antigens. Adding PD1 antibody to the T cells resulted in an improved proliferation in response to the antigens. Treatment of a WT1+ leukemia patient in remission with stem cell donor derived WT1 specific T cells in combination with WT1 vaccine was accompanied with WT1 CD4+ and CD8+ T cell responses and ongoing remission. Although tumor associated antigens are potential self antigens, it is possible
Poster abstracts
to induce a functional Th1 response in T cells from healthy donors making its application in allogeneic settings post stem cell transfer feasible. Further improvements could be achieved by combination of the therapy with antibodies to PD1.
13Antigen design for enhancement of the immunogenicity of alphavirus-based vaccines against hepatitis C virus infectionPeng Peng Ip1, Georgia Koutsoumpli1, Annemarie Boerma1, Toos Daemen1
1 Department of Medical Microbiology, University Medical Center Groningen, Groningen, NETHERLANDSInduction of broad-spectrum cellular immune responses against hepatitis C virus (HCV) class I and class II epitopes plays a pivotal role in HCV infection clearance. We have previously generated therapeutic HCV vaccines, based on a recombinant Semliki Forest virus (rSFV) vector expressing the entire or part of the HCV non-structural proteins (nsPs)1. In addition, inclusion of a series of helper T cell (Th) epitopes and endoplasmic reticulum (ER) targeting signals (sigHELP-KDEL) has been previously reported to enhance the immunogenicity of rSFV-based vaccines against human papillomavirus (HPV)2.Inclusion of the fusion protein sigHELP-KDEL into the rSFV vector encoding for the NS3/4A HCV protein (rSFV-sigHELP-NS3/4A-KDEL) was assessed in vivo for its potential to enhance the immunogenicity of NS3/4A HCV antigen, as compared to a standard rSFV-NS3/4A vaccine. Upon i.m immunization, both vaccines showed equal frequencies of NS3-specific CD8+ T cells in blood and spleen with similar effector capacity. Although, inclusion of the sigHELP-KDEL component resulted in higher production of the NS3/4A HCV protein it did not improve its stability. The latter in contrast to the stability of a fusion protein of E6 and E7 of HPV in the presence of the sigHELP-KDEL. Notably, rSFV immunization at a low dosage showed a minor difference between rSFV-NS3/4A and rSFV-sigHELP-NS3/4A-KDEL vaccines in terms of their cytolytic efficacy. Our studies suggest that the effect of a pan-HELPER cassette and ER targeting signal in a viral vector depends on CTL immunodominance and the nature of the target antigen. Activation of Th cells (CD4+) and induction of cytotoxic T lymphocytes (CTLs, CD8+) against subdominant epitopes in NS5A/5B upon sigHELP-KDEL immunization are ongoing.References:
1. Ip PP et al., Mol Ther. 2014 Apr;22(4):881-902. Ip PP et al., Gene Ther. 2015 Jul;22(7):560-7
14A pipeline for fast track identification of candidate neoantigens from cancer exome sequencing dataChristina Kyzirakos2, Christopher Mohr1,3, Sorin Armeanu-Ebinger2, Magdalena Feldhahn2, Mathias Walzer1, Moritz Menzel2, Dirk Hadaschik2, Sven Nahnsen3, Oliver Kohlbacher1,3, Saskia Biskup2
1 Applied Bioinformatics Group, University of Tübingen, Tübingen, GERMANY, 2 CeGaT GmbH, Tübingen, GERMANY, 3 Quantitative Biology Center (QBiC), University of Tübingen, Tübingen, GERMANYVirtually every tumor harbors somatic mutations. These mutations can lead to mutation-derived neoantigenic peptides presented on the MHC molecules of the patient’s tumor. T cells are able to recognize those alterations and may in turn kill the transformed cells. The importance of such neoantigens in an effective T cell-derived tumor defense has recently been demonstrated in various immunotherapeutic contexts like immune checkpoint inhibition, TIL therapy and vaccination. The quality and quantity of those neoantigens was shown to be of high prognostic and therapeutic value. To provide a practicable method for the identification of these highly individual tumor-specific neoantigens, we have established an optimized workflow combining exome and transcriptome sequencing or database derived expression data, HLA typing and peptide epitope prediction. The workflow is applicable to FFPE as well as fresh frozen tumor samples.Workflow: FFPE samples are revised and macrodissected by pathologists to maximize the tumor content of the sample. Exome sequencing of a tumor and normal tissue sample is performed and tumor-specific somatic single nucleotide variants and the patient’s HLA-type are determined. Transcriptome analysis can be performed in parallel using fresh frozen or RNA-stabilized tumor samples. Neoantigenic epitopes are predicted in silico using SYFPEITHI, NetMHC and NetMHCpan. Resulting peptide sequences are ranked using expression data, peptide motifs, allele frequencies, potential relevance of the respective variant for tumor development and biochemical features.Results: Datasets from FFPE and fresh frozen tumor samples were generated. For both sample types, a set of highly promising peptides
Poster abstracts
was compiled. The whole workflow can be accomplished in only three weeks.Conclusion: The established workflow provides an efficient and time saving method for the identification of tumor-specific mutations and putative patient-individual neoantigens for multiple purposes including the development of cancer-specific vaccines, adoptive T-cell transfer, prediction of clinical utility of immune checkpoint inhibitors and immunomonitoring.
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Removed due to author’s request.
16How T-cell resistance evolves in the course of melanoma progressionFang Zhao1, Antje Sucker1, Soldano Ferrone2, Volker Lennerz3, Thomas Wölfel3, Dirk Schadendorf1, Klaus Griewank1, Annette Paschen1
1 Department of Dermatology, University Hospital, University Duisburg-Essen and German Cancer Consortium (DKTK), Essen, GERMANY, 2 Department of Surgery, Massachusetts General Hospital, Boston, USA, 3 University Medical Center of the Johannes Gutenberg University Mainz, III. Medizinische Klinik and German Cancer Consortium (DKTK), Mainz, GERMANYCD8+ T lymphocytes (CTL) can exert potent in vivo cytotoxicity against autologous melanoma cells. However, T-cell activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here we studied the immunogenicity of multiple consecutive metastases obtained in stage IV disease from three melanoma patients.Tissue samples were collected over a period of one to seven years. Immunohistochemistry analyses revealed a heterogeneous expression of HLA class I molecules between consecutive patient metastases. Melanoma cell lines established from the distinct lesions showed corresponding stable HLA phenotypes. T cell-resistant HLA class I-negative melanoma cells developed in the course of progressive disease in each patient. This phenotype was caused by the lack of B2M expression that originated from an aberration in one chromosome 15q associated with the loss of one B2M allele and an inactivating mutation in the second B2M gene. By SNP array analyses we found the chromosome 15q aberration to be present already in early HLA class I-positive melanoma cell lines, pointing to the initial genetic alteration that predisposed to
total HLA class I loss.Based on these data we suggest screening of tumor samples for genetic alterations with potential strong impact on immunogenicity for further improvement of melanoma immunotherapy.
17iVacALL: A personalized peptide-vaccination design platform for pediatric acute lymphoblastic leukemia patients based on patient-individual tumor-specific variantsArmin Rabsteyn4, Christina Kyzirakos4, Christopher Schröder3, Marc Sturm3, Christopher Mohr1, Mathias Walzer1, Ulrike Pflückhahn4, Michael Walter3, Magdalena Feldhahn1, Karoline Laske2, Michael Bonin3, Martin Ebinger4, Stefan Stevanovic2, Peter Bauer3, Oliver Kohlbacher1, Cecile Gouttefangeas2, Hans-Georg Rammensee2, Rupert Handgretinger4, Peter Lang4
1 Institute for Applied Bioinformatics, University of Tübingen, Tübingen, Baden-Württemberg, GERMANY, 2 Institute for Cell Biology, Department of Immunology, University of Tübingen, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Tübingen, Tübingen, Baden-Württemberg, GERMANY, 3 Institute for Human Genetics, University of Tübingen, Tübingen, Baden-Württemberg, GERMANY, 4 University Children’s Hospital Tübingen, Department of General Paediatrics, Oncology/Haematology, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Tübingen, Tübingen, Baden-Württemberg, GERMANYAcute lymphoblastic leukemia is the most common pediatric malignancy. Standard chemotherapy is a successful treatment in 80% of patients, only about 20% develop a relapse, however these patients have a dismal prognosis. Prevention of relapse after first-line chemotherapy or SCT is therefore mandatory. Accumulation of somatic mutations is one characteristic feature of malignant cells. These single nucleotide variants can lead to altered amino acid sequences of the translated proteins, which in turn can be presented as antigenic peptides on HLA molecules of malignant cells. Mutated peptides represent ideal T cell targets being true neoantigens, specific for the tumor, and should not have been subject to central tolerance selection mechanisms. A peptide vaccination composed of mutated T cell epitopes specific for individual patient
tumors is therefore a promising approach to prevent relapse in high-risk patients. For this purpose we detect nonsynonymous mutations by whole exome and transcriptome sequencing of patient leukemic blasts and normal tissue. HLA binding peptides harboring the altered amino acids are subsequently predicted by algorithms SYFPEITHI, NetMHC and NetMHCpan.Whole exome sequencing was performed for 20 sample pairs. ALL-specific SNVs and InDels were identified using a comparative bioinformatics pipeline. The determined variants were further validated by deep sequencing with an average of 12 (+/-8) validated mutations per patient. For all patients, MHC I & II epitopes could be predicted successfully.We applied the platform for some patients based on compassionate need and designed individual peptide vaccines. The vaccination schedule provides 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvant. Response to the vaccination was monitored by detection of peptide-specific T cells occurring over time in patient peripheral blood. Monitoring was performed for each vaccination time point. In 3 patients we could detect a developing CD4+ T cell response against the vaccinated mutated MHC II binding peptides.
18Prothymosin alpha(100-109) induces TH1-type immune responses in vivoPinelopi Samara1, Kyriaki Ioannou1, Nadia Kavrohorianou3, Sylva Haralambous3, Hubert Kalbacher2, Wolfgang Voelter2, Ourania Tsitsilonis1
1 Department of Animal & Human Physiology, Faculty of Biology, University of Athens, Athens, GREECE, 2 Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, GERMANY, 3 Laboratory of Transgenic Technology, Hellenic Pasteur Institute, Athens, GREECEThe carboxy-terminal decapeptide proTα(100-109) constitutes the immunoreactive fragment of prothymosin alpha (proTα). ProTα(100-109) and proTα ligate TLR-4, promote the maturation of DCs and prime tumor reactive T cell functions in vitro. In this study, we assessed the in vitro toxicity of proTα(100-109) and proTα, and whether they induce analogous immune responses in vivo. PBMCs and cancer cells incubated with proTα(100-109) or proTα were analyzed by MTT, Annexin/PI and cell cycle. C57BL/6 mice were subcutaneously inoculated with B16.F1 melanoma cells and intraperitoneally treated with 2 cycles of GM-CSF,
proTα(100-109) or proTα, and a B16.F1-specific peptide extract (AWE). Tumor growth and animal survival were monitored. Splenocytes and serum were tested for cytotoxicity by 51Cr-release assay and cytokine production by CBA, respectively. Tumors were also analyzed. ProTα(100-109) and proTα were not cytotoxic to PBMCs or cancer cells, and did not promote cell-cycle progression. As previously shown in vitro, in the presence of cancer antigens, both peptides retarded tumor growth and prolonged animal survival by 25 days compared to controls. Assessment of ex vivo cytotoxicity of mouse splenocytes verified the induction of B16-reactive and non-specific responses, mediated by activated cytotoxic T and NK lymphocytes, respectively. Sera from proTα(100-109) or proTα-treated animals contained high IFN-γ, marginal IL-4, while their tumors were infiltrated by more T cells compared to controls. Our results suggest that proTα and proTα(100-109), when co-administered with tumor antigens, can induce in vivo antigen-reactive TH1-biased immune responses. As they are not toxic to normal cells, neither promote cancer cell proliferation, their eventual therapeutic use as adjuvants to orchestrate antitumor-reactive immune responses in cancer patients, should be considered.Acknowledgments: IKY FELLOWSHIPS OF EXCELLENCE FOR POSTGRADUATE STUDIES IN GREECE-SIEMENS PROGRAM; IKYDA 165/2010; FP7 REGPOT-CT-2011-284460 (ΙΝsPiRE)
19Immunemonitoring of a successful patient-individualized vaccine for tumor therapy in metastasized pancreatic cancerKatja Sonntag2, Moritz Menzel1, Saskia Biskup1, Rupert Handgretinger2, Karin Schilbach2
1 CEGAT Biotech Company, Tübingen, GERMANY, 2 Childrens Hospital University Tübingen, Tübingen, GERMANYNew sequencing approaches fuel the hope for patient-individualized vaccines as a common anticancer agent. Here we describe a 62-year-old patient`s T-cell response towards 4 peptides that he had received as a person-individualized tumor vaccine after Gemcitabin and 10/28 cycles Folfirinox that were unable to control his fully metastasized pancreatic cancer. Comparing the exome of a liver metastasis with germline-sequences from healthy tissue identified two unique mutated tumor antigens for which two peptides were designed each. Peptide pairs spanned the respective mutated sequence yet
Poster abstracts
differed in their 5` and 3`end.To clarify whether vaccination successfully established a T-cell answer the patient`s T-cells response towards vaccine peptides was determined 16 and 20 months after initial vaccination and boosting. To this end patient`s MNCs were isolated from peripheral blood, stimulated with vaccine peptide(s), mock or no peptide (controls) in a short-term-culture to enrich a potentially established epitope-specific T-cell share. Day 10, T-cells responding to respective peptide with IFN-g production were sorted and analyzed for coreceptor expression and the complexity and composition of their T-cell-receptor repertoire.This strategy identified 7 and 13 unique T-cell clones responsive to a particular vaccine peptide. Peptides of one of the peptide pairs were able to stimulate unique sequences each, while demonstrating also stimulatory effects on T-cell clones specific and responsive to the “related” peptide. CDR3-regions indicate persistence of specific T-cell clones over time and even more exiting persistence of CDR3-sequences since identical CDR3-regions emerged involving rearrangements different V-,D-,J-segments. Moreover germline-encoded TCRs were detected. Molecular analysis correlates with state of disease, the patient is tumor free. Thus, the molecular signature of the vaccine-established T-cell response enables us to track the immune response in the patient even months/years later and demonstrates that patient-individualized vaccines have a tremendous therapeutic potential.
20Ex vivo expansion and activation of natural killer cells from sarcoma patients for autologous adoptive immunotherapySabine Schleicher4, Frank Traub3, Torsten Kluba1, Hans-Georg Kopp2, Dario Campana5, Rupert Handgretinger4, Matthias Pfeiffer4
1 Clinic for Orthopaedics and Orthopaedic Surgery, Municipal Hospital Dresden, Dresden, GERMANY, 2 Department of Internal Medicine II, University Hospital Tuebingen, Tuebingen, GERMANY, 3 Department of Orthopaedic Surgery, University Hospital Tuebingen, Tuebingen, GERMANY, 4 Department of Pediatric Hematology and Oncology, University of Tuebingen, Tuebingen, GERMANY, 5 Department of Pediatrics, National University of Singapore, Singapore, SINGAPOREBackground: Therapeutic outcomes for high-grade soft tissue and bone sarcomas remain
poor, which is largely due to severe chemoresistance of this aggressive tumor type. Therefore, alternative approaches are needed addressing novel targets that are different from current multimodal chemotherapy.Natural killer (NK) cells are a major component of the innate immune system which exert direct cytotoxicity against malignant or otherwise stressed cells. Interest in NK cell-based immunotherapy has increased since new protocols for the purification and expansion of large numbers of highly cytotoxic cells have become available.Methods: Peripheral blood mononuclear cells (PBMC) from patients with different sarcoma subtypes were co-cultured with irradiated, genetically engineered K562-mb15-41BBL cells in the presence of IL-2 for 14 days. Expansion efficiency, NK receptor repertoire and the expression of NK cell ligands on autologous sarcoma cell lines were investigated by flow cytometry. Cytolytic activity of NK cells was tested using the EuTDA cytotoxicity assay based on time-resolved fluorometry.Results: Ex vivo expansion of PBMC from sarcoma patients allowed the generation of large numbers of NK cells exhibiting enhanced activation characteristics and high cytolytic activity against autologous tumor cell lines, yet retaining tolerance towards normal body cells. Moreover, this method has been adapted to clinical-grade conditions, producing a sufficient quantitiy of highly cytotoxic NK cells for immunotherapy.Discussion: Based on these data, adoptive transfer of ex vivo expanded and activated autologous NK cells is a new treatment modality that warrants clinical testing in sarcoma patients.
21Randomized, open-label Phase III study to evaluate the adjuvant vaccination with tumor RNA-loaded autologous Dendritic Cells versus observation of patients with resected monosomy 3 uveal melanomaBeatrice Schuler-Thurner1, Jan Doerrie1, Niels Schaft1, Stefanie Gross1, Michael Erdmann1, Stefan Schliep1, Andreas Baur1, Gerold Schuler1
1 Universitätsklinikum Erlangen, Erlangen, GERMANYAccumulating evidence suggests that T cells recognizing mutated cancer antigens are crucial for successful cancer immunotherapy. They seem to represent the common mechanistic pathway to control tumors and prolong overall survival in active antigen-specific (vaccination) as well as passive antigen-non-specific
Poster abstracts
(checkpoint blockade) therapies. Dendritic Cells (DC) besides mutated antigens are the other key to develop better T-based vaccines. Following the discovery of DC maturation and the development of methods to generate mature human DC from monocytes, we have over the years systematically developed the adoptive transfer of these monocyte-derived DC for cancer vaccination loaded with antigens in form of peptides or RNA transfection. In lately secluded trials high immunogenicity and long-term clinical benefit correlating with certain biomarkers in blood (including simple ones such as eosinophilia) became evident. Our recent work has focused on the induction of T cell responses against passenger and driver mutations. Following preclinical work and evidence for clinical efficacy in metastatic disease we have designed a randomized phase III (NCT01983748) trial in high risk (monosomy 3) uveal melanoma using RNA-transfected DC to vaccinate against the total antigenic repertoire of patient´s individual tumors to retard or prevent metastases after resection of the primary tumor in the eye. A total of 200 patients will be randomized into arm A (DC vaccination) or arm B (observation as standard of care). Twenty million mature, monocyte-derived DC loaded with autologous tumor RNA are administered respectively at 8 vaccination time points over two years. Objectives are 1) to prolong DFS, 2) to prolong OS and 3) to induce and measure immune responses. The trial has recently been started and is currently performed in cooperation with Departments of Ophthalmology at 8 University Hospitals in Germany (Erlangen, Essen, Hamburg Eppendorf, Homburg/Saar, Köln, Lübeck, Tübingen, and Würzburg).
22DNA damage repair protein as a regulator of IL-23 dependent immune responses in dendritic cellsSabaria Shah2, Qunwei Wang2, Ania Malecka2, Poulam Patel2, Jane Goodall1, Andrew Jackson2
1 University of Cambridge, Cambridge, UK, 2 University of Nottingham, Nottingham, UKDNA-PK is a protein kinase of paramount importance regarding its role in repairing DNA double stand breaks. We observed that DNA-PK plays an important regulatory factor for IL-23 production by dendritic cells (DC) which might well be explained by alternate functions of DNA-PK in DC other than DNA repair. Understanding the alternate roles of DNA-PK in DC is important
because of their central role in cross-priming, and naïve T cell differentiation through different cytokine production.Increased IL-23 secretion was observed in Toll-like receptor 4 (TLR4) activated human monocyte derived-DC (moDC), freshly isolated myeloid DC (BDCA-1+), mouse bone marrow derived dendritic cells and macrophages following inhibition of DNA-PK using the highly selective antagonist NU7441. We also demonstrated increased IL-23p19 transcription in the monocytic tumour cell line U937. Interestingly, inhibition of DNA-PK also led to increased IL-6, IL-1β and IL-10. Although, TLR4 triggered DC produced higher level of IL-23 upon DNA-PK inhibition, there was negligible effect with TLR1/2, TLR3, TLR5, Dectin-1 and Dectin-1/TLR2 agonists. When naïve CD4+ T-cells were activated DNA-PK inhibited DC supernatant, they preferentially primed Th17 responses.We tested the hypothesis that increased IL-23 was IL-1β dependent, however blocking IL-1β receptor with IL-1RA did not affect DNA-PK regulated IL-23. Furthermore we demonstrated prolonged activation of NFKb following DNA-PK inhibition, however the role of this is currently under investigation. This study identifies a novel immune-regulatory role for DNA-repair systems in DC resulting in the regulation of a series of Th17 associated cytokines. As Th17 cells play important roles in autoimmune responses and tumour progression these pathways have the potential to impact on new cancer immunotherapies.
23Can the peripheral immune profile in breast cancer patients be used to predict tumour antigen T cell responses?Christoforos Haritos1, Nicole Janssen2, Christopher Shipp2, Lisa Speigl2, Jithendra Kini Bailur2, Sotirios P. Fortis1, Sonia A. Perez1, Constantin N. Baxevanis1, Graham Pawelec2
1 Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, GREECE, 2 Tübingen University Hospital, Tübingen, GERMANYIt is becoming increasingly clear that the immune profile of breast cancer patients is informative for patient prognosis. Specifically, immune cells with suppressor or effector function have been shown to be valuable in the prediction of patient course on an individual basis. Our previous study has shown that patients with in vitro HER2-reactive peripheral CD8+ T cells, high levels of circulating plasmacytoid
Poster abstracts
dendritic cells (pDCs) but low ratios of Myeloid-Derived Suppressor Cells (MDSCs):pDCs and Regulatory T cells (Tregs):pDCs experience a survival advantage. In the present study we have tested T cell responses to additional tumour associated antigens along with an expanded panel of leukocyte phenotypes including myeloid cells (10 different phenotypes including monocytes, MDSCs, pDCs and monocyte-derived dendritic cells (mDCs)), T cells (8 phenotypes including Tregs) and NK cells in a cohort of 50 prospectively enrolled breast cancer patients with early stage invasive ductal carcinoma. Additionally, we have performed an in vitro expansion of T cells responding to peptides derived from the tumour-associated antigens Survivin and MUC-1, as well as HER-2 to validate our previous results, using influenza peptides as a positive biological control. To characterise the type of the T cell response to these antigens, responding CD4+ and CD8+ cells were assessed for IFNγ, TNF, IL-2, IL-5, IL-10 and IL-17 production per cell by intracytoplasmic flow cytometry, producing 6-cytokine T cell polyfunctionality data. Thus, this study sought to assess the relationship between the distribution of peripheral blood myeloid cells, T cells and NK cells and the presence or absence of in vitro T cell responses to tumour-associated antigens in breast cancer. Ongoing analyses will reveal associations between the levels of different leukocyte populations with T cell responses to tumour-associated antigens. Subsequent clinical follow-up will prospectively reveal associations of these factors with patient clinical course.
24Mapping the melanoma microenvironment: do immunological features predict prognosis?Nicole Janssen1, Christopher Shipp1, Benjamin Weide1, Graham Pawelec1
1 Tübingen University Hospital, Tübingen, GERMANYIn cancer patients, interactions between the immune system and the tumour can result in cancer control, or have a tumour-stimulating effect. Recent evidence suggests that the immunological characteristics within the tumour are a major prognostic factor. Studies supporting this notion have shown a correlation between high relative levels of tumour-infiltrating lymphocytes and improved clinical outcome in several cancer types. In contrast, pro-tumour effects can be mediated through the suppression
of anti-tumour immunity or stimulation of cancer cells by secreted products. The impact of many immunological features of the tumour microenvironment such as the local presence of cytokines has not been well defined, but their importance for clinical outcome is supported by a recent publication showing a negative correlation between IL-6 expression in tumour tissue and disease-specific survival in ovarian cancer. Here, we have measured levels of myeloid cells and soluble factors in the tumour mass to build a more detailed picture of how immunological features of the tumour might relate to patient clinical outcome. Our results show that PGE2 was expressed in 60% of metastatic melanoma deposits (n=41), while IL-6 and GM-CSF were expressed to varying degrees in the tumour of every patient (n=64 and 47, respectively). The relative levels of these three factors did not relate to patient survival. Tumour-infiltrating CD14+ cells co-expressed CD16 at higher frequencies than in blood, but the presence of these CD14+ CD16+/- cells was also not predictive of patient outcome in this study. In addition to cellular components and soluble factors of the tumour microenvironment, we plan to measure levels of senescent and proliferating tumour cells, T-cells, transcription factors, fibroblasts and MHC molecules in order to paint a more detailed picture of the tumour microenvironment. Our results will shed light on whether immunological features are predictive of survival in metastatic melanoma.
25DNA immunization against Aurora kinase A and the effect of anti-CD25 treatmentIva Kaštánková1,2, Ingrid Poláková1, Martina Dušková1, Michal Šmahel1,2
1 Department of Immunology, Institute of Hematology and Blood Transfusion, Prague, CZECH REPUBLIC, 2 Faculty of Science, Charles University , Prague, CZECH REPUBLICAurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies. Since we used this protein as a target of DNA immunization, we attempted to increase its immunogenicity by the addition of the PADRE helper epitope and to decrease its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified a nonapeptide representing an H-2Kb epitope. In an ELISPOT assay we showed that mutagenesis of the kinase domain did not alter the immunogenicity of the DNA vaccine and the
Poster abstracts
PADRE epitope enhanced the efficacy of DNA immunization by a gene gun but the AURKA-specific immunity was still low. Therefore, we combined DNA immunization with anti-CD25 treatment that depletes regulatory T cells (Tregs) and thus could increase immune responses against the AURKA self-antigen. The antibody against CD25 was applied either two days before the first immunization or on days of three immunizations given at one-week interval. Anti-CD25 treatment did not enhance the PADRE-specific response of splenocytes, but it markedly augmented the response against AURKA when three doses of anti-CD25 were applied. The anti-tumor effect of this combined immunotherapy was tested by therapeutic treatment of tumors induced with TC-1 cells producing the E6 and E7 oncoproteins from human papillomavirus 16, but neither one nor three doses of the anti-CD25 antibody supported the DNA immunization against AURKA. When the combination with DNA immunization against the E7 antigen was tested, only one inoculation of anti-CD25 significantly reduced tumor growth. Moreover, single anti-CD25 administration in three doses accelerated tumor growth. Our results suggest that Treg modulation should be carefully evaluated to achieve the optimal anti-tumor effect.This project was supported by grants CZ.1.05/1.1.00/02.0109 and NT13862-4/2012 from the Czech Ministry of Health.
26Using multi-dimensional flow cytometry to characterize polyfunctional and cross-reactive T cell responses against naturally occurring mutant HCV antigens and HCV+ hepatocellular carcinomaTimothy Spear1, Patricia Simms1, Yuan Wang3, Lance Hellman3, Brian Baker3, Hugo Rosen2, Michael Nishimura1
1 Loyola University Chicago, Maywood, IL, USA, 2 University of Colorado, Denver, CO, USA, 3 University of Notre Dame, Notre Dame, IN, USAWhile adoptive cell transfer (ACT) has been gaining clinical success, this methodology offers potential to address biologic questions concerning a T cell’s response against tumor or viral antigen. Using a high affinity, HLA-A2-restricted HCV NS3:1406-1415-reactive TCR, we have characterized cross-reactive recognition of wildtype and various naturally occurring mutant epitopes. Rather than using one dominant pro-inflammatory cytokine to characterize reactivity, which is the standard in the field, we
characterized polyfunctional potential on a per-cell basis by simultaneously analyzing 7 different parameters, including CD107a, IFNγ, TNFα, IL-2, IL-4, IL-17A, and IL-22 using flow cytometry. Out of the 128 possible combinations of polyfunctionality, multivariate analysis in Pestle/SPICE yielded distinct “cytokine signatures” among CD4+ or CD8+ transduced T cells, often differing between donor PBL. This complex analysis also highlighted novel populations of T cells simultaneously producing both type 1 and type 2 cytokines that would not otherwise be identified using standard reactivity methods.Interestingly, while cytokine patterns differed among mutant epitope stimulation, these changes didn’t necessarily correlate with TCR-pMHC KD or t1/2 measurements, suggesting affinity is not the only important factor governing recognition. Furthermore, while CD8 is necessary for certain mutant recognition, both affinity- enhancing and lck-binding signaling components are required. Additionally, comparing peptide vs. tumor stimulations highlighted a preferential loss of higher-level polyfunctional cells when stimulated by hepatocellular carcinoma cells engineered to express naturally processed NS3 and its variants.These multi-dimensional analyses emphasize the vast heterogeneity of T cells in a given culture or between donors as well as phenotypes that cross traditional boundaries set to “type” a T cell, suggesting the field has oversimplified T cell function. Further comprehensive analysis of T cells’ polyfunctional potential will help shed light on T cells’ biologic capacity and what may be the “best” T cell to use for ACT.
27Natural killer cell interaction with melanoma cells: receptors, immunoedition and immunoescape Federico Sierra3, Francisco Matias Soto3, Sara Morgado3, Beatriz Sánchez-Correa3, Juan J. Gordillo3, Carmen Campos2, Alejandra Pera2, Esther Duran1, Javier G Casado3, Rafael Solana2, Raquel Tarazona3
1 Histoloy and Pathology Unit. University of Extremadura, Caceres, SPAIN, 2 IMIBIC - Hospital Universitario Reina Sofia - Universidad de Cordoba, Cordoba, SPAIN, 3 Immunology Unit. Dept.Physiology. University of Extremadura, Caceres, SPAINIntroduction: Natural killer (NK) cells are early responders of the innate immune system against tumor cells. NK cell activation depends on a complex balance mediated by activating and
Poster abstracts
inhibitory receptor signaling. Melanoma cells have developed mechanisms to avoid NK cell lysis such as shedding of ligands for activating receptors or the down-regulation of activating receptors on NK cells from melanoma patients leading to hypo-responsive NK cells. The aim of this study was to investigate the interactions between melanoma cells and NK cells in vitro and discuss their contribution to melanoma escape.Methods: Melanoma cell lines obtained from the OISTER project were grown on 24 well plates until reaching confluence. Then, resting PBLs or IL-2 stimulated PBLs (lymphokine activated killer, LAK) were added to the wells (ratio 1:1) and incubated for 24h. The expression of NK cell receptors and their ligands on melanoma cells after in vitro co-culture was analyzed by multiparametric flow cytometry.Results: MHC class I expression on melanoma cells increased after co-culture with PBLs or LAK cells. An increased expression of CD155 (ligand of DNAM-1 and TACTILE) was found on WM793, WM1205LU and MaMel56 cell lines. A decreased expression of ULBP3 and ULBP2 (ligands of NKG2D) was observed on MaMel56 and MaMel60 respectively. DNAM-1, NKp30 and TACTILE expression was reduced on NK cells after co-culture with MaMel56 and DNAM-1 and NKp46 decreased after co-culture with MaMel45 cells. IRNE cells induced a decrease of NKp30 and NKp46 and WM793 a reduction in the expression of NKp30 and DNAM-1.Conclusions: NK cell interaction with melanoma down-regulates the expression of several activating receptors and up-regulates MHC class I on melanoma favoring immunoescape from NK cells. The relevance of the decreased expression of NKG2D ligands and the increased expression of CD155 observed on melanoma cells after co-culture requires further analysis.
28A therapeutic Her2-Neu vaccine alone or in combination with anti-Her2 mAb inhibits tumor growth in HLA-A2 transgenic miceThi TRAN5, Mariana DINIZ5, Estelle DRANSART4, Alain GEY1, Sylvie GODEFROY2, Yu-Chun LONE3, Ludger JOHANNES4, Eric TARTOUR5,1
1 Hopital Européen Georges Pompidou. Service d’Immunologie Biologique, Paris, FRANCE, 2 Immunotarget, Paris, FRANCE, 3 INSERM U1014, Hôpital Paul Brousse , Villejuif, FRANCE, 4 Institut Curie. INSERM U1143-CNRS UMR3666, Paris, FRANCE, 5 Université
Paris Descartes-INSERM U970-PARCC, paris, FRANCEHer2/neu is a validated target in cancer as various antibodies against this antigen demonstrated their clinical efficacy in patients with breast and gastric cancers. The Her2369-
377 peptide(E75) has been shown to be immunogenic in human and an E75 peptide vaccine is currently tested in a phase III clinical trial. We have developed a vector, the non-toxic B-subunit of Shiga toxin which targets antigens to dendritic cells and favours cross-presentation of exogenous antigens to CD8 Tcells. We coupled the E75 peptide to STxB and showed that immunization of humanized HLA-A2 transgenic mice with STxB-E75 elicits higher levels of anti-Her2-neu CD8 Tcells detected by tetramer analysis than the use of the non vectorized E75 peptide. The specific CD8+ Tcells induced by STxB-E75 were more functional, cytotoxic in vivo and of higher avidity than those elicited by the E75 peptide. We demonstrated than STxB-E75 is more efficient to inhibit the growth of human Her2-HLA-A2-B16 tumor in HLA-A2 mice than the E75 peptide in both a prophylactic or a therapeutic setting, and its efficiency is abrogated when CD8 is depleted in vivo. We then observed that the anti-Her2 CD8 Tcells elicited in HLA-A2 mice recognize human breast tumor cell lines and HLA-A2 B16 clones expressing high and also low levels of hHer2/neu. Next, we assess the efficiency of STxB-E75 on HLA-A2-B16 tumors expressing low levels of hHer2/neu in a therapeutic setting, in combination or not with anti-Her2 mAb(4D5). We showed a synergy between the combination of anti-Her2 mAb and STxB-E75 in the inhibition of low expressing Her2/neu tumor growth compared to the use of the antibody or the vaccine alone. The body of experiments presented in this work clearly demonstrated that the STxB-E75 vaccine is a potent candidate to be tested in the clinics for Her2 expressing tumor.
29HPV prophylactic/therapeutic DNA vaccines: chimeric E7/L2 antigens fused to signal sequences of a plant protein. Francesca Paolini2, Silvia Massa1, Gianfranca Curzio2, Olivia Demurtas1, Rosella Franconi1, Aldo Venuti21 ENEA, Rome, Lazio, ITALY, 2 Regina Elena National Cancer Institute, Rome, Lazio, ITALYIn previous studies, we showed that the signal sequence of the polygalacturonase inhibiting protein (PGIPss) from Phaseolus vulgaris can
Poster abstracts
drive HPV16-E7 proteins to the plant secretory compartment improving the production of HPV antigens in tobacco plants (Franconi et al., 2006). In order to ameliorate the efficacy of DNA vaccines for HPV therapy, one useful strategy is to fuse viral genes with sequences with adjuvant activity. Thus, we explored the ability of this PGIPss to drive antigens into the secretory pathway of mammalian cells in order to improve DNA vaccines that, in particular, suffer from poor IgG induction. HPV16 E7-, chimeric E7/L2- and L2-derived antigens were N-terminally fused to PGIPss and cloned into pVAX vector. HEK293 cells were transfected with double CsCl-purified DNA vaccine preparations. Immunofluorescence for PGIPss-antigens and immunoblotting of intracellular and secreted proteins were made. By these procedures, PGIPss-HPV fusion antigens were detected both in culture medium and inside transfected cells whereas same antigens without PGIPss were found only inside the cells. In addition, fusion constructs seems to be located within the cell secretory pathway. For in vivo studies, C57BL6 mice were treated with same constructs by i.m. injection with/without electroporation. Production of specific IgG and IFN-gamma producing splenocytes were analyzed by ELISA and ELISPOT, respectively. DNA electroporation of fusion constructs elicited the production of anti-HPV16-L2 antibodies. IgG titer was much higher in animals injected with PGIPss-L2 that in those injected with L2 alone. Moreover, a chimeric construct, PGIPss-L2-E7, was able to induce specific cellular immunity that in turn produced anti-tumor effects in C57BL6 mice. In conclusion, for the first time we documented that a signal sequence of a plant protein works in mammalian cells. This achievement allowed us to produce improved prophylactic/therapeutic chimeric DNA vaccines against HPV.
30Peptide Libraries for the Comprehensive Coverage of the Tumor Mutanome for Immune Monitoring and Immuno TherapyPaul von Hoegen1, Tobias Knaute1, Ulf Reimer1, Holger Wenschuh1
1 JPT Peptide Technologies GmbH, Berlin, GERMANYMany cancers are associated with functional mutations which now can be identified using new DNA sequencing methods. Sequence information is used for the generation of personalized cancer vaccines with promising clinical benefit. Tumor-specific protein-coding mutations
(truly individualized epitopes) are selected for immunotherapy. The use of actual sequence data of an individual patient is the optimal basis for treatment and immune monitoring. As a fast tool with broad applicability even across patient cohorts peptide libraries could be an application in cases where the individual sequence is not (yet) available.Pools of antigen peptides are frequently applied for T-cell stimulation in T-cell assays and for immune stimulation for treatment as they are well suited for presenting sequence diversity. However, libraries used today normally consist of overlapping peptides of a wild-type antigen sequence. The concept was already extended for the presentation of sequence diversity in antigens of HIV where sequence diversity between individual viruses poses many challenges. For the ENV protein an increase in sequence coverage from 10 % to 34 % was achieved. Due to the limited number of peptides a complex filtering of sequence data has to be applied to enrich the library with peptides carrying different tumor-specific mutations.Our aim is to generate peptide libraries for specific antigens which represent a generic cancer mutanome applicable to a large number of patients before or as alternative to truly individualized approaches. A library can contain the full length sequence of the antigen enriched by peptides representing overlapping scans for relevant mutations. Alternatively, known or predicted epitopes can be selected and be enriched with the mutated epitopes. Overlapping libraries allow application to multiple HLAs. We have built a pipeline for the efficient generation of such libraries and have the technologies to generate them at GxP level.
31Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosisRebekka Wehner5, Marieta Toma7, Anja Kloß5, Susanne Füssel3, Barbara Seliger4, Elfriede Noessner6, Knut Schäkel2, Manfred Wirth3, Gustavo Baretton7, Marc Schmitz5,1
1 Center for Regenerative Therapies Dresden, Dresden, GERMANY, 2 Department of Dermatology, University Hospital, Heidelberg, GERMANY, 3 Department of Urology, University Hospital of Dresden, Dresden, GERMANY, 4 Institute for Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), GERMANY, 5 Institute of Immunology, Medical Faculty, Dresden University of Technology,
Poster abstracts
Dresden, GERMANY, 6 Institute of Molecular Immunology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, GERMANY, 7 Institute of Pathology, University Hospital of Dresden, Dresden, GERMANYDendritic cells (DCs) are essential for the induction and maintenance of antitumor immunity. Previously, we reported that 6-sulfo LacNAc+ (slan) DCs (formerly termed M-DC8+ DCs), a myeloid human blood DC subset, produce various proinflammatory cytokines, display cytotoxic activity and efficiently stimulate natural killer (NK) cells and T lymphocytes. More recently, it has been demonstrated that slanDCs accumulate in metastatic tumor-draining lymph nodes from cancer patients. Therefore, slanDCs may contribute to antitumor immunity.Here, we analysed slanDCs in 263 clear cell renal cell carcinoma (ccRCC) and 227 tumor-free tissue samples and we found increased frequencies of slanDCs in the tumor tissues. slanDCs were also detectable in the majority of metastatic lymph nodes and distant metastases. In addition, we associated the frequency of slanDCs in ccRCC tissues with important clinico-pathological characteristics. Remarkably, a higher density of slanDCs was significantly correlated with a reduced progression-free, tumor-specific or overall survival of ccRCC patients. Freshly prepared ccRCC-infiltrating slanDCs expressed HLA-DR and low densities of CD86, but no CD40, CD80, CD83 or CCR7. Whereas proinflammatory cytokines were not found, a proportion of slanDCs expressed the anti-inflammatory cytokine IL-10. Further studies revealed that primary ccRCC cells efficiently impaired slanDC-induced T-cell and NK-cell activation.In conclusion, higher slanDC numbers in ccRCC tissues are associated with poor prognosis. The tolerogenic phenotype of slanDCs leading to an insufficient activation of antitumor immunity may represent a novel immune escape mechanism of ccRCC. These observations may have implications for the design of therapeutic strategies that harness tumor-directed functional properties of DCs.
32SCIB2 targets NY-ESO-1 epitopes to antigen presenting cells via CD64 to induce potent anti-tumour immunity which is enhanced by Treg depletion or checkpoint blockade Wei Xue2, Rachael Metheringham2, Vicky Pudney2, Barbara Gunn2, Peter Symonds2, Lindy
Durrant1,2
1 Nottingham University, Nottingham, UK, 2 Scancell, Nottingham, UKDue to its immunogenicity, restricted normal expression and widespread tumour expression, NY-ESO-1 is an ideal target for tumour therapy. In this study, four NY-ESO-1 sequences that encode the majority of class I and class II MHC epitopes have been incorporated into a huIgG1 antibody DNA vaccine (SCIB2). The vaccine allows in vivo targeting of dendritic cells via the high affinity FcγR1 receptor. When tested in HLA transgenic mice, SCIB2 elicited higher frequency and avidity T cell responses than peptide vaccines. It was the only vaccine that stimulated cytotoxic CD8 responses with the ability to kill naturally processed and presented NY-ESO-1 antigen on tumour cells. SCIB2 also efficiently induced NY-ESO-1 specific cytotoxic CD4 cells expressing granzyme B. These SCIB2 induced T cell responses resulted in control of established B16-HHDII-NY-ESO-1 tumours, allowing long term survival in 35% of mice. As vaccine induced T cell responses can be restricted by an immunosuppressive tumour environment, the combination of SCIB2 and Treg depletion or CTLA-4 or PD-1 blockade on anti-tumour immune responses was assessed. Treg depletion and PD-1 had no effect on the T cell response but CTLA-4 blockade was shown to further enhance the cytotoxicity of the SCIB2 induced CD8 response. In combination with SCIB2 and either Treg depletion or anti-CTLA-4 or PD-1 blockade long term survival from established tumours was enhanced to 64%, 67% and 100% respectively. This study showed that targeting NY-ESO-1 epitopes to APCs resulted in a potent cancer immunotherapy which was further enhanced by Treg depletion or checkpoint blockade.
33SCIB1 DNA vaccination synergises with PD-1 blockade to induce efficient tumour therapy of poorly immunogenic tumourWei Xue2, Vicky Pudney2, H. Yagita, Peter Symonds2, Katherine Cook2, Rachael Metheringham2, Lindy Durrant2,1
1 Nottingham University, Nottingham, UK, 2 Scancell, Nottingham, UKRecent studies have suggested that patients who respond to check point inhibition share responses to a set of neoeptiopes. Unfortunately, the majority of cancer patients do not express these neoepitopes and may not benefit from checkpoint blockade alone. Cancer vaccines
Poster abstracts
can overcome this limitation by stimulating high avidity T cell responses to tumour associated antigens. We investigated the anti tumour effect of a DNA vaccine encoding a human IgG1 antibody engineered to express melanoma specific T cell epitopes (SCIB1) and checkpoint inhibitor (PD-1 blockade). The number and function of tumour infiltrating T cells from each treatment were also investigated. In this study we show that these effective cancer vaccines may further benefit from checkpoint inhibition. Vaccination with SCIB1 induced high frequency and avidity tumour specific CD4 and CD8 responses in HLA-DR4 transgenic mice. These high avidity responses were not modulated in the presence of established tumour but resulted in increased survival and tumour regression in both animal models (45% long term survival) and in melanoma patients. PD-1 blockade resulted in a similar anti-tumour response to vaccine with 50% of mice showing long term survival. SCIB1 vaccination was associated with increased CD8 T cell infiltrate on tumours as well as enhanced proliferation of CD8 T cells within the tumour but was also associated with in vivo up regulation of PD-L1 on the tumour. The combination of PD-1 blockade with vaccination further enhanced both CD4 and CD8 T cell infiltrate and induced potent anti-tumour responses and 85% survival in an established tumour model. These results highlight the benefit of combining PD-1 blockade with vaccines and in particular with SCIB1.
Poster abstracts
Participants
Firstname Surname Country Email
Gosse Adema NETHERLANDS
Maya Caroline Andre SWITZERLAND
Tobias Baechle LIECHTENSTEIN [email protected]
Costas Baxevanis GREECE
Elodie Belnoue SWITZERLAND [email protected]
Grete Berntsen NORWAY [email protected]
Iris Bigalke NORWAY [email protected]
Anne-Mette Bjerregaard DENMARK [email protected]
Victoria Brentville UK [email protected]
Luigi Buonaguro ITALY
Debora Carpanese ITALY [email protected]
Esteban Celis USA
Anoop Chandran GERMANY [email protected]
Raja Choudhury AUSTRALIA [email protected]
Katherine Cook UK [email protected]
Pierre Coulie BELGIUM
Ian Daniels UK [email protected]
Steffen Dettling GERMANY [email protected]
Lindy Durrant UK [email protected]
Karine Dzhandzhugazyan DENMARK [email protected]
Attila Edelmann GERMANY [email protected]
Knut Elbers GERMANY [email protected]
Masashi Emoto JAPAN [email protected]
Nicole Ettischer GERMANY [email protected]
Farzin Farzaneh UK
Carl Figdor NETHERLANDS
Erica Fleming USA [email protected]
Kendra Foley USA [email protected]
Mariola Fotin-Mleczek GERMANY [email protected]
Federico Garrido SPAIN
Gustav Gaudernack NORWAY
Cecile Gouttefangeas GERMANY [email protected]
Vikcotia Graf GERMANY [email protected]
Dirk Hadaschik GERMANY [email protected]
Regina Heidenreich GERMANY [email protected]
Maurenis Hernandez Perez CUBA [email protected]
Norbert Hilf GERMANY [email protected]
Madeleine Hipp GERMANY [email protected]
Henoch Hong GERMANY [email protected]
Krisztian Horvath GERMANY [email protected]
Else Marit Inderberg-Suso NORWAY [email protected]
Kyriaki Ioannou UK [email protected]
Andrew Jackson UK [email protected]
Nicole Janssen GERMANY [email protected]
Francine Jotereau FRANCE
Simone Kayser GERMANY [email protected]
Rolf Kiessling SWEDEN
Alexei Kirkine DENMARK [email protected]
Sven Koch GERMANY [email protected]
Georgia Koutsoumpli NETHERLANDS [email protected]
Aleksandra Kowalczyk GERMANY [email protected]
Sabrina Kuttruff-Coqui GERMANY [email protected]
Christina Kyzirakos GERMANY [email protected]
Sandra Lazzaro GERMANY [email protected]
Paul Lehmann USA
Markus Löffler GERMANY [email protected]
Alejandro Madrigal UK
Firstname Surname Country Email
Alexander Martens GERMANY [email protected]
Cornelis Melief NETHERLANDS
Rachael Metheringham UK [email protected]
Isabella Monia Montagner ITALY [email protected]
Marit Renée Myhre NORWAY [email protected]
Elissaveta Naumova BULGARIA
Michael Nishimura USA [email protected]
Bianca Nußbaum GERMANY [email protected]
Rienk Offringa GERMANY
Suzanne Ostrand-Rosenberg USA
Annette Paschen GERMANY [email protected]
Graham Pawelec GERMANY
Monika Pawelec GERMANY [email protected]
Isabel Poschke GERMANY [email protected]
Armin Rabsteyn GERMANY [email protected]
Judith Ramage UK [email protected]
Hans-Georg Rammensee GERMANY
Carmen Rapp GERMANY [email protected]
Saskia Rösch GERMANY [email protected]
Roberto Ruiu ITALY [email protected]
Sunil Kumar Saini DENMARK [email protected]
Pinelopi Samara GREECE [email protected]
Thomas Sayers USA
Karin Schilbach GERMANY [email protected]
Sabine Schleicher GERMANY [email protected]
Marc Schmitz GERMANY [email protected]
Silke Schnell GERMANY [email protected]
Gerold Schuler GERMANY [email protected]
Beatrice Schuler-Thurner GERMANY [email protected]
Sabaria Shah UK [email protected]
Christopher Shipp GERMANY [email protected]
Harpreet Singh GERMANY [email protected]
Michal Smahel CZECH REPUBLIC [email protected]
Rafael Solana SPAIN
Guri Solum NORWAY [email protected]
Timothy Spear USA [email protected]
Lisa Speigl GERMANY
Slavica Stevanovic-Heck GERMANY [email protected]
Raquel Tarazona SPAIN [email protected]
Per thor Straten DENMARK
Thi Diem Tran FRANCE [email protected]
Roman Trenz GERMANY
Ourania Tsitsilonis GREECE [email protected]
Jayakumar Vadakekolathu UK [email protected]
Aldo Venuti ITALY [email protected]
Paul von Hoegen GERMANY [email protected]
Rebekka Wehner GERMANY [email protected]
Theresa Whiteside USA
Kilian Wistuba-Hamprecht GERMANY [email protected]
Wei Xue UK
Henning Zelba GERMANY [email protected]
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