Abstract Book 2010 - CIMT Annual Meeting

Abstractbook 2010 

8 th Annual Meeting

Dear colleagues, members and friends, 

On behalf of the organizing committee, we would like to welcome you to 

the 8th annual CIMT meeting in Mainz from May 26-28, 2010. The newly 

formed Scientific Program Committee has done an outstanding job this 

year putting together a comprehensive program which covers the whole 

range of innovative developments in immunotherapy. 

The scope of this year's meeting stretches from tumor immunosupression, 

regulatory issues, the potential of adjuvants and new adoptive cellular 

therapies to therapeutic vaccination. Because the gap between "bench and 

bedside" is still wide, we put focus on the question how translation can 

be successful. We will have the opportunity to share three days of plenary 

sessions, workshops and guided poster sessions. The expanded short 

talk sessions and the guided poster sessions are again main tracks in the 

program. An industry exhibition and satellite workshops accompany the 

conference. 

Your participation makes this meeting exciting. Welcome to Mainz! 

Ch. Huber (Mainz) 

P. Johnson (Southampton) 

U. Kalinke (Hannover) 

C. Melief (Leiden) 

H.G. Rammensee (Tuebingen) 

G. Schuler (Erlangen) 

P. Walden (Berlin) 

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The Association 

The association for Cancer Immunotherapy (CIMT) is a non profit organisation 

and was founded in the fall of 2002 as an information and 

education platform for immunological cancer therapy. Physicians and 

researchers from different fields of clinical and theoretical medicine 

from the universities of Berlin, Erlangen, Mainz and Tuebingen were the 

founders of the association. 

CIMT is located at the university medical center of the Johannes Gutenberg 

University Mainz. 

The association is financed by donations, congress fees and sponsoring. 

Our Goals 

The purpose of CIMT is to enhance the development of cancer immunotherapies 

by: 

* Establishing a communication platform in order to facilitate the com- 

munication between scientists of all faculties with interest in cancer 

immunotherapy. We improve knowledge exchange between industry 

based scientists, academic scientists, regulatory authorities and phy- 

sicians alike. Our scientific meetings, educational symposia and pu- 

blications serve this purpose. 

* Education of health-professionals with annual meetings, advanced 

education seminars and publication of textbooks. 

* Cooperation with partners in related consortia, regulatory agencies, 

journals, academic institutions and companies. 

* Initiation of working parties that actively accelerate the development 

in the field. 

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Who We Are

Managing Committee: 

Christoph Huber, chair 

Mainz, Germany 

Peter Johnson 

Southampton, UK 

Ulrich Kalinke 

Hannover, Germany 

Cornelis Melief 

Leiden, Netherlands 

Executive Director - Science: 

Sebastian Kreiter 


Scientific Secretary: 

Mustafa Diken 


Who We Are 

Hans Georg Rammensee 

Tuebingen, Germany 

Gerold Schuler 

Erlangen, Germany 

Peter Walden 

Berlin, Germany 

Executive Director - Translational Medicine: 

CIMT - Education: 

Cedrik Britten 


Torsten Seppmann 


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CIMT - Office: 

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Kristiane Preuss 

Isny, Germany 

Scientific Advisory Board: 

Philipp Beckhove 

Heidelberg, Germany 

Peter Brossart 

Bonn, Germany 

Ulrich Keilholz 

Berlin, Germany 

Christian Ottensmeier 


Giorgio Parmiani 

Milan, Italy 

Ugur Sahin 


Who We Are 

CIMT - Communications: 

Christine Castle 


Michael Schmitt 

Kiel, Germany 

Joachim Schultze 

Bonn, Germany 

Harpreet Singh 


Freda Stevenson 


Thomas Tüting 

Bonn, Germany

Visit our website 

www.cimt.eu 

find us on facebook 

Websites 

www.facebook.com/cimtmainz 

follow us on twitter 

www.twitter.com/C_IMT 

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Program Committee and Speakers 

2010 Program Committee Members: 

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Giorgio Parmiani 

Unit of immune-biotherapy of melanoma 

and solid tumors 

Fondazione Centro san Raffaele del Monte 

Tabor 

Milan, Italy 

Graham Pawelec 

Zentrum für Medizinische Forschung 

Sektion Transplantationsimmunologie/Immunhämatologie, 

Medizinische Klinik II 


Christian Ottensmeier 

Cancer Sciences Division 

Southampton University Hospitals 

Southampton, United Kingdom 

Axel Hoos 

Co-Chairman, CVC Executive Committee; 

Program leader in Immunology/Oncology 

at Bristol-Myers Squibb 

Bristol Myers Squibb Inc. 

Wallingford, USA 

Hansjoerg Schild 

Institute for Immunology 

JGU Universitätsmedizin Mainz 


Harpreet Singh 

Immatics 


Cecile Gouttefangeas 

Department of Immunology 

University of Tuebingen 


Dolores Schendel 

Institut für Molekulare Immunologie (IMI) 

Helmholtz Zentrum München 

München, Germany 

Freda Stevenson 

Molecular Immunology Group, Cancer 

Sciences Division 

School of Medicine, University of 

Southampton 

Southampton, United Kingdom 

Wolfgang Herr 

Innere Medizin 

JGU Universitätsmedizin Mainz 


Antoine Tesniere 

Institut Gustave Roussy 

INSERM Unit U848, „Apoptosis, Cancer & 

Immunity“ 

Paris, France 

Sebastian Kreiter 

Institute for Translational Oncology (TrOn) 

Johannes Gutenberg-Universität Mainz 


Cedrik Britten 

III. Medizinische Klinik und Poliklinik 

Johannes Gutenberg-Universität Mainz 


Sjoerd H. van der Burg 

Department for Clinical Oncology 

Leiden University Medical Center 

Leiden, Netherlands

2010 Speakers: 

G. J. Adema 

Radboud University Nijmegen Medical 

Centre 

Nijmegen Centre for Molecular Life Sciences 

(NCMLS) 

Nijmegen, Nethterlands 

Prof. Dr. Adema holds the chair in Molecular Immunology at 

the NCMLS Department of Tumor Immunology. His research 

mainly focuses on the molecular analysis of dendritic cells and 

regulatory T cells, and their function in the immune system in 

health and disease. The knowledge gathered in the fundamental 

immunological studies is translated into pre-clinical and clinical 

vaccination studies of cancer patients with autologous, 

antigen-loaded dendritic cell vaccines. 

James Allison 

Ludwig Center for Cancer Immunotherapy 

New York, USA 

James Allison has a longstanding interest the in the mechanisms 

of T cell activation and its regulation, as well as developing 

novel strategies for immunotherapy of cancer. He has made many 

significant contributions to our understanding T cell costimulation 

and inhibition, and conceived the notion of immunological 

checkpoint blockade for the therapy of cancer. He is currently 

the Chairman of the Immunology Program, Director of the Ludwig 

Center for Cancer Immunotherapy, David H. Koch Chair in 

Immunologic Studies, Attending Immunologist at the Memorial 

Sloan Kettering Cancer Center. He is also an Investigator of the 

Howard Hughes Medical Institute, a Member of the National 

academy of Sciences, and of the Institute of Medicine. 

Victor Appay 

INSERM 

Paris, France 

Dr. Appay leads the HIV pathogenesis and Immunosenescence 

group at the Institut National de la Santé et Recherche Médicale 

(INSERM). His main research interest is exploring the factors 

that govern the development and maintenance of effective 

CD8+T cell responses in HIV infection using basic immunology. 

The work of his team also focuses on the development with 

progressive HIV disease of premature immunosenescence, or 

immune ageing. 

John Barrett 

National Heart, Lung and Blood Institute 

National Institutes of Health 

Bethesda, USA 

John Barrett is Chief of the National Heart, Lung and Blood 

Institute‘s Stem Cell Allotransplantation Section of the Hemato- 

logy Branch, National Heart, Lung and Blood Institute, National 

Institutes of Health, in Bethesda, USA. His research is directed 

to defining the antigens involved in graft-versus-host disease, 

graft-versus-leukemia and graft versus tumor immunity and 

designing improved ways to reconstitute the immune system 

after stem cell transplantation using technologies to selectively 

eliminate harmful alloreactive T cells from the transplant while 

conserving graft-versus-leukemia reactivity. 

Philipp Beckhove 

Deutsches Krebsforschungszentrum 

Heidelberg, Germany 

PD. Dr. Philipp Beckhove leads an independent research group 

at the German Cancer Research Center. The objective of Dr. 

Beckhove’s research group „Translational Immunology“ is to 

gain new insights into the immune defence system of cancerous 

cells and to evolve the results from basic research through to 

clinical treatments. The pursuit of which is based on close interdisciplinary 

collaboration with the oncological departments of 

the university hospital. New therapeutic concepts developed by 

the research group are being “translated” into clinical application 

under the roof of the National Center for Tumor Diseases 

in Heidelberg (NCT). 

J. S. Bromberg 

Mount Sinai School of Medicine 

New York, USA 

Prof. Dr. Bromberg has great experience in starting, building, 

and reorganizing critical components of multiorgan transplant 

programs. In addition to his clinical transplantation practice, 

Prof. Bromberg is pursuing basic and clinical research involving 

immunology and transplantation. His current projects are 

focused on determination of key mechanisms in T cell homing 

and tolerance induction, investigation of signals important for 

induction and inhibition of Foxp3 expression in regulatory T 

cells (Treg) as well as interaction of Tregs with other cells in 

different disease models of autoimmunity. 

Federica Cavallo 

University of Torino 

Torino, Italy 

Federica Cavallo, PhD, is Associate Professor of Immunology 

(General Pathology) of the University of Torino. She is among 

the founders of the new Molecular Biotechnology Center (MBC) 

of the University of Torino, where her laboratory is now located. 

She has a successful experience in basic and translational cancer 

immunotherapy, and has a considerable expertise in transgenic 

mouse models of cancer and DNA vaccination. At present her 

lab is combining immune regulators of various kind and vaccines 

to prevent and cure autochthonous cancers in pre-clinical 

models. A major effort is devoted to grab new knowledge on 

tumor and microenvironment transcriptome from microarray 

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analysis and next generation sequencing data. These technologies 

are currently exploited to tease apart new mechanisms 

of carcinogenesis inhibition and to spot both miRNA and new 

oncoantigens to be exploited in a new generation of antitumor 

vaccines. 

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Helen X. Chen 

Investigational Drug Branch, Cancer 

Therapy Evaluation Program 

National Cancer Institute 

Bethesda, USA 

Dr. Helen Chen, M.D. is currently Associate Branch Chief of the 

Investigational Drug Branch of National Cancer Institute (NCI)’s 

Cancer Therapy Evaluation Program (CTEP). She is responsible 

for oversight of NCI-sponsored clinical trials involving antiangiogenesis 

therapies, EGFR/HER2 and IGF-1R targeting agents, 

and monoclonal antibody therapeutics. She is also involved in 

the development of NCI’s program for combination of molecular 

targeted investigational agents. She has authored more than 30 

publications and book chapters on these areas, and has been on 

scientific committees for AACR and AACR-EORTC-NCI Annual 

Meetings, the ASCO-NCI-EORTC Annual Meeting on Molecular 

Markers, and the International Symposium for Antiangiogenesis 

Therapies. Dr. Chen a diplomate of the American Board of 

Internal Medicine, and also serves as an attending physician in 

the NCI’s Phase I clinic. 

Mark M. Davis 

Stanford University School of Medicine 

Stanford, USA 

Professor Dr. Davis, Director of the Stanford Institute for Immunology, 

Transplantation and Infection (ITI), is a professor of 

microbiology and immunology and a Howard Hughes Medical 

Institute investigator. He is well known for identification in 

the 1980s of the elusive T-Cell receptor genes, which allow T 

lymphocytes to fight disease causing microbes, and he and his 

group have made many subsequent discoveries about this type 

of molecule and how it functions. 

Jolanda M. de Vries 

Nijmegen Centre for Molecular Life 

Sciences) 

Nijmegen, Netherlands 

Dr. Jolanda de Vries is Assistant Professor at the Department 

of Tumor Immunology at the Nijmegen Centre for Molecular 

Life Sciences. She was one of the pioneers to translate dendritic 

cell biology into potential clinical applications. The first clinical 

phase I/II studies in which patients were vaccinated with DCs 

loaded with tumor-specific peptides were initiated in 1997. She 

also developed a novel immuno-monitoring assay that is highly 

predictive for extended survival after vaccination with DCs (J 

Clin Oncology 2005). Her primary scientific interest continues 

along the line of DC-immunotherapy and in particular the 

migration and imaging of DC. For example, in-vivo imaging of 

ex-vivo labeled cells using MRI (Nature Biotechnology 2005). 

New opportunities for other cell-types (e.g. subsets of DCs) are 

now being developed. She recently completed the first plasmacytoid 

DC vaccination trial. 

M. V. Dhodapkar 

Yale University School of Medicine 

New Haven, USA 

Prof. Dr. Dhodapkar is the chief of Section of Hematology at Yale 

School of Medicine and the director of Department of Hematologic 

Malignancies at Yale Cancer Center. He is interested in 

studying the biology of the human immune system and how 

it interacts with growing tumors in patients. His main reseach 

goal is understanding and harnessing the properties of the 

immune system to detect, prevent and treat cancer, with a particular 

focus on multiple myeloma. 

Mary L. (Nora) Disis 

University of Washington 

School of Medicine 

Washington, USA 

Mary L. (Nora) Disis, M.D. is the Associate Dean for Translational 

Health Sciences in the University of Washington 

(UW) School of Medicine, Professor of Medicine and Adjunct 

Professor of Pathology and Obstetrics and Gynecology at UW 

and a Member of the Fred Hutchinson Cancer Research Center 

(FHCRC). Dr. Disis is an expert in breast and ovarian cancer 

immunology and translational research. Her research interest is 

in the discovery of new molecular targets in breast and ovarian 

cancer for the development of vaccine and cellular therapy for 

the treatment of those malignancies. 

Mark E. Dudley 


Bethesda, USA 

Dr. Mark E. Dudley is the head of the Cell Production Facility at 

the Surgery Branch, National Cancer Institute. The Cell Production 

Facility translates basic research discoveries into cell and 

gene therapies for patients with cancer. Current projects include 

closed system bioreactors, retroviral and lentiviral gene therapy 

vectors, and artificial antigen presenting cells. These systems 

are being developed in the context of ongoing clinical trials to 

treat patients with advanced melanoma and other cancers.


D. I. Gabrilovich 

University of South Florida 

Tampa, USA 

Prof. Dr. Gabrilovich holds a chair in tumor immunology at the 

Department of Oncologic Sciences-University of South Florida. 

His research mainly focuses understanding the biology and 

mechanism of tumor-associated immunosuppression and exploring 

approaches to eliminate immunosuppression. His team is 

also working on development of novel effective cancer vaccines 

including genetically modified dendritic cells and the combination 

of apoptosis-inducing therapy and immunotherapy. 

John B.A.G. Haanen 

Netherlands Cancer Institute 

Antoni van Leeuwenhoek Hospital 


Prof. Dr. John B.A.G. Haanen, MD PhD is head of the Division 

of Medical Oncology at the Netherlands Cancer Institute/Antoni 

van Leeuwenhoek Hospital. His main research interest is immunotherapy 

of cancer and he was recently appointed professor of 

translational immunotherapy of cancer at the Leiden University. 

The main topics of his research are on the development of 

DNA-based vaccine strategies for the treatment of cancer and 

adoptive cellular therapy. In close collaboration with prof. Ton 

Schumacher he has developed a program for T cell receptor gene 

therapy. 

Mirjam Heemskerk 

Leiden University Medical Center 


Mirjam H. M. Heemskerk is currently Associate Professor and 

Head of the Laboratory of Experimental Hematology at the 

LUMC Department of Hematology which she joined to pioneer 

the retroviral introduction of antigen specific T cell receptors as 

a means to engineer leukemia specific immunity. Her research 

interests are characterisation of alloimmune responses and 

occurrence of delayed immune reconstitution after HLA mismatched 

stem cell transplantation, high throughput identification 

of hematopoiesis-restricted minor histocompatibility antigens 

by reverse immunology approaches and TCR gene therapy 

of hematological malignancies. A clinical trial is being set up to 

treat patients with hematological malignancies after allogeneic 

stem cell transplantation with TCR engineered T cells. 

Thomas Hünig 

Institute for Virology and Immunobiology 

University of Würzburg 

Würzburg, Germany 

Prof. Dr. Hünig holds a chair in Immunobiology at the Institute 

for Virology and Immunobiology. He was co-founder of the 

company TeGenero that developed the CD28-superagonist antibodyTGN1412. 

His main focus of research is regulation of T cell 

activation by CD28 and CTLA4. 

Ulrich Kalinke 

Twincore 

Hannover, Germany 

Prof. Dr. Kalinke is the Executive Director of TWINCORE 

Research Center in Hannover and former Head of the Department 

of Immunology at Paul-Ehrlich-Institut (Langen). His 

main research area is experimental infection focusing on the 

influence of interferons on adaptive immunity and how different 

pathogens prevent the release of interferons. 

Samir N. Khleif 


Bethesda, USA 

Prof. Dr. Khleif is head of the Cancer Vaccine Section, Vaccine 

Branch at the NCI and he also serves as a Special Assistant to the 

Commissioner of the Food and Drug Administration, leading 

the Critical Path Initiative for oncology. His reseach focuses on 

integrating translational basic laboratory research and clinical 

trials to understand the interaction between tumor cells and the 

immune system and to develop cancer vaccines. 

Andreas Mackensen 

University Erlangen 

Erlangen, Germany 

Professor Dr. Andreas Mackensen holds the chair position for 

Hematology/Oncology at the University Hospital of Erlangen. 

His main research area is the development of immunotherapeutic 

strategies for the treatment of malignant diseases. 

Raj K. Puri 

Division of Cellular and Gene Therapies 

U.S. FDA´s Center for Biologics Evaluation 

and Research 

Silver Spring, USA 

Raj K. Puri, M.D., Ph.D. is the Director of the Division of Cellular 

and Gene Therapies (DCGT) in the U.S. FDA’s Center for 

Biologics Evaluation and Research. He is also a Chief of Tumor 

Vaccines and Biotechnology Branch (TVBB). Dr. Puri oversees 

investigational new drug (INDs), investigational device exemptions 

(IDEs), and biological license applications (BLAs) for 

tumor vaccines, immunotherapy, cellular and gene therapy, tissue 

engineering, and xenotransplantation products and deve- 

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lopment of policies and guidance documents in these cutting 

edge areas of medical research. 

Dr Rezvani is a Clinical Senior Lecturer and Consultant Haematologist 

at Imperial College in London. She is the clinical lead in 

allogeneic stem cell transplantation and clinical director of the 

Jacie accredited GMP cellular facility. 

As a research fellow at the NIH from 2001-2008, her laboratory 

studies in immune responses to leukemia led to the initiation 

of 3 FDA approved clinical trials of vaccination in patients 

with myeloid malignancies. She received a highly competitive 

award from the Higher Education Funding Council for England 

(HEFCE) and started her position as a clinical senior lecturer 

at Imperial College in January 2008 where she has an active 

research laboratory program in transplantation and tumor 

immunology. 

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Katy Rezvani 

Imperial College London 

London, England 

Antoni Ribas 

University of California 

Los Angeles, USA 

Antoni Ribas, MD, is Associate Professor of Medicine and Surgery 

at University of California, Los Angeles. He practices oncology 

focused on new drug development for melanoma, and has 

an NIH-funded research laboratory focused also in melanoma. 

He also serves as Director of the Cell and Gene Therapy Core 

Facility, Assistant Director of the Tumor Immunology Program, 

Assistant Director of the Human Gene Medicine Program, General 

Clinical Research Center Advisory Board Member, and trains 

faculty at the Institute for Stem Cell Biology and Medicine. 

Dr. Ribas is a permanent committee member of the NCI grant 

review panels. Dr. Ribas‘ clinical and laboratory research involves 

the development of novel strategies for the treatment of cancer, 

including anti-CTLA4 antibodies, cancer vaccines, BRAF 

inhibitor and Mek inhibitor targeted therapy, nanoparticle delivery 

of siRNA, and gene therapy strategies. 

Martina Schuessler-Lenz 

Paul-Ehrlich Institut 

Langen, Germany 

Dr. Schuessler-Lenz is a Senior Clinical Assessor at the Division 

Medical Biotechnology of the Paul-Ehrlich Institut, and the 

alternate German member of the Committee for Advanced Therapies 

(CAT) of the European Medicines Agency. Her main area 

of expertise is the clinical development of Advanced Therapy 

Medicinal Products and of cancer immunotherapies. 

Protul A. Shrikant 

Roswell Park Cancer Insitute 

New York, USA 

Dr. Protul A. Shrikant completed a National Multiple Sclerosis 

Society Fellowship at the University of Minnesota, Center for 

Immunology (1999) and joined Roswell Park Cancer Institute in 

2000 as an Assistant Professor in the Department of Immunology 

(2000), where he currently is an Associate Professor with 

tenure and serves on the advisory council for the Centre for 

Immunotherapy. He also holds an adjunct appointment with the 

Departments of Microbiology and Immunology at State University 

of New York at Buffalo. His research is focussed on characterizing 

the molecular pathways by which instructions program 

CD8 T cell responses and exploit the insights for developing 

novel strategies for tumor immunity. 

Masato Tanaka 

RIKEN Research Center for Allergy 

and Immunology 

Yokohama, Japan 

Masato Tanaka, M.D. and Ph.D., graduated from Tokyo Medical 

and Dental University in 1988. I worked for Osaka Bioscience 

Institute and Osaka University Medical School, and engaged 

in research of the molecular mechanisms of apoptosis in laboratory 

of Prof. Shigekazu Nagata. I worked for Tularik Inc. in 

San Francisco, USA to investigate the molecular mechanisms 

of inflammatory reaction in 1997-1999. Since 2002, I became 

the team leader of Laboratory for Innate Cellular Immunity, 

RIKEN Research Center for Allergy and Immunology (RCAI), 

Yokohama Japan. In recent years, I focused on the physiological 

and pathological roles of apoptotic cell clearance by phagocytes, 

and strategic approach for immune regulation by dead 

cell clearance. 

David L. Urdal 

Dendreon Corporation 

Seattle, USA 

Dr. Urdal has been Chief Scientific Officer and a Director of 

Dendreon Corporation since he joined the company in July of 

1995. Dendreon (Nasdaq: DNDN) has been dedicated to targeting 

cancer and transforming lives through the discovery and 

development of novel products like Provenge®, an active immunotherapy 

for prostate cancer that has shown promise in Phase 

III clinical trials. 

From 1982 to 1995, he held various positions with Immunex 

Corporation, including President of Immunex Manufacturing 

Corporation, Vice President and Director of Development, and 

Head of the Departments of Biochemistry and Membrane Biochemistry. 

At Immunex he participated in the discovery, development 

and commercialization of hematopoietic growth factors, 

cytokines and cytokine receptor drugs, like Leukine® and 

Enbrel®. Dr. Urdal received an M.S. in Public Health and a Ph.D. 

in Biochemical Oncology from the University of Washington. He 

is inventor on 16 patents and author on 79 publications.


Weiping Zou 

Department of Surgery and Comprehensive 

Cancer Center 

University of Michigan School of 

Medicine 

Ann Arbor, USA 

Weiping Zou is a Professor of Surgery, Immunology and Biology 

and the director for Translational Research at the University of 

Michigan. His research interests are in tumor immunopathology 

and immunotherapy, with an emphasis on the cross-talk among 

immune cell subsets, stromal cells, tumor cells and tumor stem 

cells in the tumor microenvironment, and its impact on tumor 

immunity, tolerance and therapy. 

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Scientific Program CIMT 2010 Day 1 (May 26 th ) 

09:00-10:00 Welcome coffee 

14 

Welcome addresses 

10:00-10:05 C. Huber (CIMT Chairman, Mainz) 

10:05-10:10 G. Krausch (University President, Mainz) 

10:10-10:15 R. Urban (CSO, University Medicine Mainz) 

Session 1: Cellular therapies 

Chairpersons: M. Theobald (Mainz), S. Mielke (Würzburg) 

10:20-10:50 J. Barrett (Bethesda) 

Selective depletion of alloreactive T cells: promises and challenges 

10:50-11:20 M. Heemskerk (Leiden) 

Risks and benefits of TCR gene therapy 

11:20-11:50 A. Mackensen (Erlangen) 

11:50-13:30 Lunch break 

Industry satellite symposium I: 

Cellular therapy in solid tumors - New perspectives 

(organized by Miltenyi Biotec) 

12:15-12.40 J. de Vries (Radboud University Nijmegen Medical Center, Netherlands) 

Human plasmacytoid dendritic cells in cancer and cancer therapy; 

12:40-13.50 Philipp Beckhove (German Cancer Research Center Heidelberg, Germany) 

Regulation of T cell responses in cancer 

13.05-13.30 Mark E. Dudley (National Cancer Institute, NIH, Bethesda, MD, USA) 

Adoptive T cell therapy for melanoma 

11:50-13:00 CIMT members meeting 

Session 2: Tumor vaccination 

Chairperson: C. Ottensmeier (Southampton), C. Melief (Leiden) 

13:30-14:00 J.B. Haanen (Amsterdam) 

Therapeutic DNA vaccination by tattooing: from preclinical development to a first 

clinical trial 

14:00-14:30 K. Rezvani (London) 

The role of immunotherapy in the eradication of minimal residual disease in CML 

14:30-15:00 F. Cavallo (Torino) 

DNA Vaccination in Prevention and Cure of Autoch thonus Mammary Carcinoma in 

Her2-tg Mice – Implications for Clinical Testing 

15:00-15:30 D. Urdal (Dendreon, Seattle) 

Sipuleucel-T: Active cellular immunotherapy for castrate resistant prostate cancer 

15:30-15:50 Coffee break 

Short talk session I: Immune monitoring 

Chairpersons: C. Britten (Mainz), J. de Vries (Nijmegen) 

15:50-16:05 T. de Gruijl (Amsterdam) (# 021) 

Immune and clinical response monitoring in patients with metastatic hormone-refrac 

tory prostate cancer receiving combined Prostate GVAX and anti-CTLA4 immunotherapy

16:05-16:20 K. Laske (Tuebingen) (# 028) 

T-cell immunomonitoring in patients with renal cell carcinoma after multi-peptide 

vaccination 

16:20-16:35 M. Guidoboni (Meldola) (# 034) 

Changes of immune cell infiltrates induced by dendritic cell vaccination in melanoma 

patients’ tumor tissues: an immunohistochemical study 

16:35-16:50 S. Voland (Erlangen) (# 035) 

Characterisation and functional analysis of T-cell responses in melanoma patients 

vaccinated with peptide-loaded dentritic cells 

16:50-17:05 E. Aarntzen (Bemmel) (# 037) 

Skin-derived functional specific Tcells predict clinical outcome upon DC vaccination in 

stage III and IV melanoma patients 

Short talk session II: Therapeutic tumor vaccination 

Chairpersons: S. Grabbe (Mainz), P. thor Straten (Copenhagen) 

15:50-16:05 C Maccalli (Milan) (# 078) 

Definition of the immunological properties of cancer stem cells isolated from human 

glioblastoma 

16:05-16:20 I. Poschke (Stockholm) (# 079) 

Immature immunosuppressive CD14+HLA-DR-/low cells in melanoma patients are 

Stat3hi and over-express CD80, CD83 and DC-Sign 

16:20-16:35 E. Lion (Borgerhout) (# 081) 

Poly(I:C)-electroporated myeloid leukemic cell lines become highly susceptible to NK 

cell-mediated killing and phagocytosis by immature DC 

16:35-16:50 A. Bonertz (Heidelberg) (# 082) 

Antigen-specific Treg in colorectal carcinoma control T cell responses against a limited 

tumor antigen repertoire 

16:50-17:05 C. Lutz-Nicoladoni (Innsbruck) (# 087) 

Reinforcement of Cancer Immunotherapy by Adoptive Transfer of cblb-deficient 

Cytotoxic T Lymphocytes combined with a Dentritic Cell Vaccine 

Short talk session III: New targets & new leads 

T. Wölfel (Mainz), S. Kreiter (Mainz) 

15:50-16:05 R. Ashfield (Abington) (# 062) 

ImmTACs: bi-functional reagents for redirected tumour cell killing 

16:05-16:20 N. Cesares (Pamplona) (# 065) 

Suppression of Treg activity by a FOXP3 inhibitor peptide 

16:20-16:35 M. H. Andersen (Herlev) (# 069) 

Cellular Immune Responses Against Indoleamine 2,3-dioxygenase 

16:35-16:50 W. van Esch (Amsterdam) (# 072) 

T cell epitope discovery through HLA class I peptide ligand exchange and combinatori 

al coding 

16:50-17:05 N. Schaft (Erlangen) (# 074) 

Transfer of mRNA encoding chimeric antigen receptors specific for MCSP into CD4+ 

and CD8+ T cells 

Key Note Lecture 

Chairperson: C. Britten (Mainz) 

17:10-17:50 Mark M. Davis (Stanford) 

The coming golden age of human immunology 

19:30-23:00 Speakers dinner 

15


16 

Session 3: Combination Partners for Vaccination 

Chairpersons: A. Hoos (New York), G. Schuler (Erlangen) 

08:40-09:10 J. Allison (New York) 

Immune checkpoint blockade in cancer therapy: New insights and opportunities 

09:10-09:40 A. Ribas (Los Angeles) 

Immunosensitization: Targeted therapies to improve tumor immunotherapy 

09:40-10:20 H. Chen (Bethesda) 

CTEP/NCI’s model to facilitate clinical trials for combination between investigational 

agents 


Short talk session IV: CIP-Session 

Chairpersons: C. Ottensmeier (Southampton), M. Welters (Leiden) 

10:45-11:00 C. Britten (Mainz) 

Overview on CIP activities in 2009 

11:00-11:20 C. Gouttefangeas (Tuebingen) 

Update on the CIP HLA-multimer panels 

11:20-11:40 C. Ottensmeier (Southampton) 

Plans for the new ELISPOT panels (IVS T cell in UK and freezing medium in Mainz 

11:40-12:00 S. Singh (Leiden) 

A stable standard sample for harmonisation and validation of T cell assays 

12:00-12:20 S. Janetzki (New York) 

Influence of harmonization on assay performance – the CIC experience 

Short talk session V: Cellular therapy 

Chairpersons: M. Kalos (Philadelphia), R. G. Meyer (Mainz) 

10:50-11:05 N. Auphan-Anezin (Marseille) (# 045) 

Optimizing effector and memory properties of CD8 T cells for adoptive tumor 

immunotherapy 

11:05-11:20 D. Schendel (München) (# 048) 

High avidity T cell clones specific for tumor-associated antigens and how to find them 

11:20-11:35 D. Stolle (Mainz) (# 053) 

Generation of HCMV-/TAA-bispecific human T cells by either the genetic equipment of 

HCMV+ T cells with tumor-reactive TCRs or the combined retroviral transduction of 

bulk human T cells with HCMV-/ TAA-specific TCRs 

11:35-11:50 A. Bloetz (Mainz) (# 054) 

Allo-reactivity to HLA class II mismatch alleles in CD4+ T-cell subsets 

11:50-12:05 H. Echchannaoui (Mainz) (# 058) 

Evaluation of the safety of p53 TCR gene transfer in a humanized mouse model 

Short talk session VI: Tumor biology / interaction with 

the immune system 

Chairpersons: V. Umansky (Heidelberg), D. Schendel (Munich) 

10:50-11:05 M. Ramacher (Heidelberg) (# 091) 

Immunosuppression in transgenic mouse melanoma model induced by myeloid de 

rived suppressor cells

11:05-11:20 M. Koslowski (Mainz) (# 093) 

Tumor-associated genomic DNA hypomethylation effects positive autoregulation of HIF-1α 

11:20-11:35 E. Quaglino (Turin) (# 094) 

miRNAs expression profiles during ErbB2 driven mammary carcinogenesis 

11:35-11:50 C. Zimmer (Essen) (# 102) 

Impact of Endoplasmic reticulum aminopeptidases (ERAP) on T cell epitope genera 

tion in melanoma cells 

11:50-12:05 L. Hansmann (Regensburg) (# 104) 

Isolation of intact genomic DNA from FOXP3-stained and FACS-sorted regulatory T 

cells for epigenetic analysis 


Industry satellite symposium II 

12.30-14:00 (organized by Beckman Coulter) 

Dr. Andreas Boehmler / Dr. Michael Kapinsky 

Multilaser and Multicolour Flowcytometry -Upcoming challenges! 

New concepts in experimental setup and analysis 

Session 4: Regulation – how translation can be successful 

(organized by the CIMT RRP) 

Chairperson: T. Hinz (PEI), H. Singh (Tuebingen) 

14:00-14:15 U. Kalinke (Hannover) 

Regulatory challenges in translational research 

14:15-14:35 R. K. Puri (FDA-CBER, Rockville) 

Regulatory considerations for biologics for cancer therapy 

14:35-14:55 T. Hünig (Würzburg) 

Functional impairment of circulating T-cells questions validity of conventional PBMC 

testing: the case of the CD28 superagonist TGN1412 

14:55-15:15 M. Schuessler-Lenz (PEI, Langen) 

The role of the European Committee for Advanced Therapies in the Evaluation of Cancer 

Immunotherapies 

15:30-16:10 Panel Discussion 

Poster Session I 

Guided poster session (+ coffee & cookies) 

16:15-18.00 Therapeutic vaccination I, Chair: V. Lennerz (Mainz) 

Immune monitoring I, Chair: C. Bain (Lyon) 

Cellular therapy I, Chair: H. Voß (Mainz) 

New targets & new leads I, Chair: K. Thielemans (Brussels) 

Tumor biology & interaction with the immune system I, Chair: S. Pascolo (Zurich) 

Enhancing immunity & adjuvants, Chair: M. Radsak (Mainz) 

Social event 

19:30-23:30 Casual dinner in the “Rheingold” lounge 

17


18 

Poster Session II 

Guided poster session (+ pretzels) 

08:30-09:50 Therapeutic vaccination II, Chair: M. Schmitt (Rostock) 

Immune monitoring II, Chair: S. Groß (Erlangen) 

Cellular therapy II, Chair: R. Stripecke (Hannover) 

New targets & new leads II, Chair: V. Umansky (Heidelberg) 

Tumor biology & interaction with the immune system II, Chair: U. Hartwig (Mainz) 


Session 5: Chemo-Immunotherapy 

Chairperson: A. Tesnière (Paris), H.-G. Rammensee (Tuebingen) 

10:20-10:50 S. Khleif (Bethesda) 

Combination of immunotherapy and molecular targeted therapy: manipulating the 

inhibitory arm of the immune system 

10:50-11:20 M. Tanaka (Yokohama) 

Role of Sinus macrophages in the induction of anti-tumor immunity 

11:20-11:50 D. Gabrilovitch (Tampa) 

Mechanism of synergistic effect of immunotherapy and chemotherapy 

11:50-12:20 M. Dhodapkar (Yale) 

Immunity to pluripotency genes in spontaneous and therapy induced immunity 


Industry satellite symposium III 

(organized by Becton Dickinson) 

12:30 13:10 Dr. Andreas Weigert (Medizinische Fakultät der Goethe-Universität Frankfurt/ 

Main, Institut für Biochemie 1/Pathobiochemie) 

Characterization of tumor infiltrating immune cell populations by multicolor flow 

cytometry 

13:10-13:50 Dr. Marieke Essers (DKFZ Heidelberg, HI-STEM-Heidelberg Institute for 

Stem Cell Technologies and Experimental Medicine) 

Activation of dormant hematopoetic stem cells 

Session 6: Immune monitoring 

(organized by the CIMT Immunoguiding Program) 

Chairperson: Cécile Gouttefangeas (Tuebingen), S. H. van der Burg (Leiden) 

13:50-14:20 M.-L. (Nora) Disis (Washington) 

Immunomodulation of breast cancer 

14:20-14:50 V. Appay (Paris) 

Assessing qualitative attributes of HIV specific CD8+T cells: from phenotype to clonotype 

14:50-15:20 W. Zou (Ann Arbor) 

T cells subsets in the tumor microenvironment 

15:20-15:45 Coffee break

Session 7: Poised, Suppressed, Tolerized–Mechanisms of T-cell Immunity 

Chairpersons: H.J. Schild (Mainz), W. Herr (Mainz) 

15:45-16:15 J. S. Bromberg (New York) 

Induction and migration of regulatory T cells 

16:15-16:45 G. Adema (Nijmegen) 

Regulatory T cells in immunotherapy of cancer 

16:45-17:15 P. A. Shrikant (Buffalo) 

MenTORed CD8+ T cells for tumor immunity 

Closing words: Christoph Huber & Hansjörg Schild 

19

Sponsors, Partners & Industry exhibitors 

We gratefully acknowledge the recurring support that reaches us every 

year from our sponsors, partners and industry exhibitors. 

Sponsors 

Merck 

Roche 

Novartis 

Partners 

Deutsche Krebshilfe 

FZI Mainz 

20 

Wyeth 

Beckman Coulter 

Ganymed Pharmaceuticals 

Cancer Immunotherapy Consortium 

Becton Dickinson 

Miltenyi Biotec 

Immatics 

Cancer Immunology Immunotherapy (CII)

Industry exhibitors 

R&D Systems 

CTL Europe 

Millipore 

Bachem 

JPT Peptide 

Peprotech 

Thymed 

Caliper Life Sciences 

Bio Rad 

CellGenix 

Meda Pharma 

Invitrogen Deutschland 

Becton Dickinson 

Miltenyi Biotec 

Invatis 

Beckman Coulter 

Bio Sys GmbH 

Qiagen GmbH 

Charles River 

eBioscience Company 

Sanquin Reagents 

Immudex 

21

Poster Presentations 

22 

General Information 

Abstracts that have been selected for poster presentation have been divided into seven sessions: 

Therapeutic vaccination 

Immune monitoring 

Cellular therapy 

New targets & new leads 

Tumor biology & interaction with the immune system 

Enhancing immunity & adjuvants 

Posters will be discussed in a guided poster session. Presenting authors should be prepared 

to present and discuss their posters (about 5-10 minutes time).

Guided Poster Sessions 

Session Date & time Chair Posters 

Therapeutic vaccination I 

Immune monitoring I 

Cellular therapy I 

New targets & new leads I 

Tumor biology & interaction 

with the immune system I 

Enhancing immunity & 

adjuvants 

Therapeutic vaccination II 

Immune monitoring II 

Cellular therapy II 

New targets & new leads II 


with the immune system II 

27.05.2010, 16:15 – 18:00 hrs 











V. Lennerz (Mainz) 

C. Bain (Lyon) 

H. Voß (Mainz) 

K. Thielemans (Brussels) 

S. Pascolo (Zurich) 

M. Radsak (Mainz) 

M. Schmitt (Rostock) 

S. Groß (Erlangen) 

R. Stripecke (Hannover) 

V. Umansky (Heidelberg) 

U. Hartwig (Mainz) 

001-012 

021-031 

L124 

040-050 

061-069 

078-092 

107-118 

L126 

013-020, 

L119-L122 

032-039 

051-060 

070-077, 

L123, L125 

093-106 

23

Abstract Nr. Title Poster session Oral short talk 

001 Bontkes 

002 Kotsiou 

003 Altvater 

004 Haenssle 

005 Vyth-Dreese 

006 Bernard 

007 Navarrete 

008 Schroeder 

009 Kuhn 

010 Mikyskova 

24 

Dendritic cells loaded with amplified mRNA isolated 

from leukemic stem cell like cells induce specific 

T-cells 

Antigen specific monoclonal antibodies directed 

against the HA-1 and HA-2 minor histocompatibility 

antigens 

Presentation of tumor-associated epitopes by activated 

human γδ T cells induces expansion of specific 

CD8+ T cells from naïve precursors 

Dendritic cells loaded with HSP70-expressing heatkilled 

melanoma cells efficiently activate autologous 

T cells 

MART-1 specific T cells after DNA tattoo vaccination 

of melanoma patients 

Investigating the impact of autophagy modulation 

on dendritic cell cancer vaccines 

Upfront Immunization With Autologous Recombinant 

Idiotype Fab Fragment Without Prior Cytoreduction 

in Indolent B-Cell Lymphoma 

In vitro characterization of an irradiated genetransfer 

medicinal product consisting of a prostate 

cancer derived cell line constitutively secreting 

interleukin-2 and interferon-gamma 

Phosphorothioate cap analogs increase stability and 

translational efficiency of RNA vaccines in immature 

dendritic cells and induce superior immune 

responses in vivo 

Interleukin 12 genetically-modified vaccines augment 

the effect of chemotherapy of human papilloma 

virus 16-associated tumours with gemcitabine 










Therapeutic vaccination I

Abstract Nr. Title 

011 Kuhs 

012 Gueckel 

013 Babiarova 

014 Abbas 

015 Vogt 

016 van Nuffel 

017 Schaft 

018 Haenssle 

019 Buhl 

020 Nielsen 

L119 Dutoit 

L120 Hilf 

Clinical Development of lentiviral vector-induced 

“SMART-DC” for melanoma immunotherapy 

targeted modification of HLA-A*02-restricted mucin- 

1-derived peptides results in induction of cytotoxic 

T cells cross-reactive with naturally presented 

peptides 

Development of different types of vaccine against 

WT1 tumor antigen 

Vaccine development through antigen targeting to 

mannose receptor 

Inhibition of tumor growth after subcutaneous 

injection of dendritic cells engineered to express 

angiogenesis-related flk-1 antigen in a murine HCC 

model 

T cell specificities after vaccination of melanoma 

patients with mRNA loaded DC 

Optimization of the T-cell stimulation capacity of a 

dendritic-cell-based melanoma vaccine 

Dendritic cells loaded with HSP70-expressing heatkilled 

melanoma cells efficiently activate autologous 

T cells 

Cryopreservation of high-concentrated peripheral 

blood mononuclear cells by controlled-rate freezer 

results in higher cell yields for dendritic cell-based 

immunotherapy 

The CDX-1307 vaccine: regulatory and clinical steps 

in developing a regimen 

Functional immune reactivity to antigens of the ovel 

IMA950 vaccine peptide set in glioma patients 

IMA950 - a novel multi-peptide cancer vaccine for 

treatment of glioblastoma 

Poster session 













Oral short talk 

25


L121 Pawlowski 

L122 van de Ven 

021 Santegoets 

022 Pfirschke 

023 Veron 

024 Zupke 

025 Savelyeva 

026 Tanriverdi- 

Akhisaroglu 

027 Low 

028 Laske 

029 Brix 

030 Brix 

26 

Synergy of the immunomodulators GM-CSF and 

Imiquimod for peptide-based therapeutic vaccines 

Exposure of CD34+ precursors to cytostatic 

anthraquinone-derivatives induces rapid dendritic 

cell differentiation 

Immune and clinical response monitoring in patients 

with metastatic hormone-refractory prostate 

cancer receiving combined Prostate GVAX and anti- 

CTLA4 immunotherapy 

The repertoire of melanoma antigens recognized by 

T cell responses in malignant melanoma patients 

Selection, validation and evaluation of a 30-Plex 

Luminex kit for immunomonitoring of a phase I 

clinical trial 

Nanoparticles for efficient labeling of dendritic cells 

to be used in cellular immunotherapy 

Vaccination and immune memory: modelling the 

bystander activation of CD4+ memory T cells in 

mice 

Spontaneous tumour antigen specific T cell responses 

occur frequently in patients with Carcinoma 

of Unknown Primary and are predominantly 

directed against EGFR, telomerase, MAGE-3 and P53 

Culturing PBMC in the presence of antigen results 

in a measurable expansion of memory T cells 

T-cell immunomonitoring in patients with renal cell 

carcinoma after multi-peptide vaccination 

Sensitive and reliable monitoring of cancer specific 

T cell responses 

MHC Dextramers: improved detection of antigenspecific 

T-cells in fluid samples and in situ 













26.05.2010, 

15:50 – 16:05 hrs 


16:05 – 16:20 hrs


L124 Zhang 

031 Gross 

032 Zelba 

033 Singh 

034 Guidoboni 

035 Voland 

036 Wagner 

037 Aarntzen 

038 Moodie 

039 Attig 

040 Kotsiou 

Goal of ELISPOT proficiency accomplished: ELI- 

SPOT assays provide reproducible results among 

different laboratories for T-cell immune monitoring 

— even in hands of ELISPOT novices 

Monitoring of vaccine-specific regulatory and helper 

CD4+ and cytolytic CD8+ T cells after vaccination 

with autologous antigen-loaded dendritic cells 

NY-ESO-1-specific T cells in long-term melanoma 

survivors 

A stable standard sample for harmonisation and 

validation of T cell assay Satwinder Kaur Singh, 

Changes of immune cell infiltrates induced by dendritic 

cell vaccination in melanoma patients‘ tumor 

tissues: an immunohistochemical study 

Characterisation and functional analysis of T-cell 

responses in melanoma patients vaccinated with 

peptide-loaded dentritic cells 

GPI-anchor negative T cells with impaired effector 

function persist after Alemtuzumab mediated T-cell 

depletion 

skin-derived functional specific Tcells predict clinical 

outcome upon DC vaccination in stage III and 

IV melanoma patients 

Response definition criteria for ELISPOT assays 

revisited 

Recommendations for HLA-peptide multimer 

staining experiments - Results from the second 

HLA-peptide multimer proficiency phase organized 

by the Cancer Immunotherapy Consortium 

Application of MHC class I single chain trimers for 

leukaemia immunotherapy 















16:05 – 16:20 hrs 


16:05 – 16:20 hrs 


16:05 – 16:20 hrs 

27


041 Wittmann 

042 Weigand 

043 Liang 

044 Kalos 

045 Grange 

046 Singh 

047 Blaudszun 

048 Schendel 

049 Albrecht 

050 Klobuch 

28 

Comparing genetically engineered T cells for chimeric 

TCR versus CD16 + Trastuzumab for adoptive 

immunotherapy against HER2 positive breast 

carcinomas 

Generation of CD4+ T cells with specificity for 

FMNL1 

Transfer of human T-cell receptor (TCR) containing 

murine chimeric constant betagamma-chain sequences 

reduces the risk of mixed heterodimers and 

enhanced the proliferation of transduced T cells 

Clearance of established leukemia in a mouse xenograft 

model by a single injection of primary T cells 

gene-modified to express chimeric antigen receptors 

that target CD19 

Optimizing effector and memory properties of CD8 

T cells for adoptive tumor immunotherapy 

Search for FLT3- ITD-reactive CD8+ T cells in the 

peripheral blood of healthy donors 

Adoptive transfer of ex vivo activated and antineoplastic 

drug loaded T lymphocytes retargeted to 

EpCAM expressing tumours 

High avidity T cell clones specific for tumor-associated 

antigens and how to find them 

IL-21 treated naive CD45RA+ T cells represent a reliable 

source for generating AML-reactive cytotoxic 

T lymphocytes with high proliferative potential and 

early differentiation phenotype 

T cell receptor RNA transfer into non-reactive 

human T lymphocytes turns them into potent CMVspecific 

effector cells 











27.05.2010 

10:50 – 11:05 hrs 


11:05 – 11:20 hrs


051 Distler 

052 Salguero 

053 Stolle 

054 Bloetz 

055 Stumpf 

056 Hoyer 

057 Foerster 

058 Echchannaoui 

059 Bourquin 

060 Schmitt 

Alloreactivity to HLA class I mismatch alleles mainly 

resides in CCR7+ naive and central memory CD8 

T cells 

Dramatic early expansion of CMV-reactive human 

T cells in vivo is supported by lentiviral vector-induced 

engineered dendritic cells 

Generation of HCMV-/TAA-bispecific human T cells 

by either the genetic equipment of HCMV+ T cells 

with tumor-reactive TCRs or the combined retroviral 

transduction of bulk human T cells with HCMV-/ 

TAA-specific TCRs 

Allo-reactivity to HLA class II mismatch alleles in 

CD4+ T-cell subsets 

Indirect presentation of HLA class II restricted minor 

histocompatibility antigens is a characteristic 

of HLA-DM resistant antigens 

Examination of T-helper cell / DC cross-talk by the 

transfection of T cells with RNA coding for TCRs 

Generation of multivirus-specific CD4+ and CD8+ 

T cells for adoptive immunotherapy 

Evaluation of the safety of p53 TCR gene transfer in 

a humanized mouse model 

Efficient eradication of subcutaneous but not of 

autochthonous gastric tumors by adoptive T cell 

transfer in a SV40 T antigen mouse model 

WT1 and RHAMM specific CD8+ T cells can be 

isolated and purified by streptamers for adoptive 

transfer 














11:20 – 11:35 hrs 


11:35 – 11:50 hrs 


11:50 – 12:05 hrs 

29


061 Buchwald 

062 Ashfield 

063 Woelfel 

064 Hornig 

065 Casares 

066 Dettmar 

067 Hirschhaeuser 

068 Guthmann 

069 Sørensen 

070 Andersen 

30 

The ubiquitin-conjugase-8 targets active fms-like 

tyrosine kinase-3 for proteasomal degradation 

ImmTACs: bi-functional reagents for redirected 

tumour cell killing 

Identification of acute myeloid leukemia-associated 

antigens recognized by allogeneic CD8+ T lymphocytes 

Combinatorial approach of a bispecific antibody 

and costimulatory antibody fusion proteins for 

targeted cancer immunotherapy 

Suppression of Treg activity by a FOXP3 inhibitor 

peptide 

Identification of clinically useful combinations of 

trifunctional antibodies with chemotherapy using 

different preclinical models 

Efficacy of catumaxomab in tumor spheroid killing 

is mediated by its trifunctional mode of action 

Cellular and humoral immune response to N-glycolyl-GM3 

elicited by racotumomab, an anti-idiotypic 

vaccine 

Cellular Immune Responses Against Indoleamine 

2,3-dioxygenase 

Identification of highly potent regulatory CD8+ 

T-cells specific for Heme Oxygenase-1 












15:50 – 16:05 hrs 


16:05 – 16:20 hrs 


16:20 – 16:35 hrs


071 Kleemann 

072 van Esch 

073 Suso 

074 Krug 

075 Lubojanski 

076 Staege 

077 Feger 

L123 Kyzirakos 

L125 Huijbers 

078 Maccalli 

079 Poschke 

080 Flores- 

Guzman 

Identification of T-cell defined varicella-zoster virus 

proteins 

T cell epitope discovery through HLA class I peptide 

ligand exchange and combinatorial coding 

Identification of novel CD4+ and CD8+ T cell 

epitopes from telomerase (hTERT) 

Transfer of mRNA encoding chimeric antigen receptors 

specific for MCSP into CD4+ and CD8+ T cells 

Identification of T cell-defined melanoma antigens 

Tumor antigens in Hodgkin Lymphoma 

Analysis of HPV L1 specific T-cell immunity 

Identification of novel EBV-specific MHC class-II 

epitopes 

Vaccination against the extra domain-B 

of fibronectin as a novel tumor therapy 

Definition of the immunological properties of cancer 

stem cells isolated from human glioblastoma 

Immature immunosuppressive CD14+HLA-DR-/low 

cells in melanoma patients are Stat3hi and overexpress 

CD80, CD83 and DC-Sign 

Potential cancer stem cells in ret transgenic mouse 

model of spontaneous melanoma 



















16:35 – 16:50 hrs 


16:50 – 17:05 hrs 


15:50 – 16:05 hrs 


16:05 – 16:20 hrs 

31


081 Lion 

082 Bonertz 

083 Ge 

084 Chen 

085 Dubrot 

086 Chachibaia- 

Saginashvili 

087 Lutz-Nicoladoni 

088 Schmid 

089 Sha 

090 Strothmeyer 

32 

Poly(I:C)-electroporated myeloid leukemic cell lines 

become highly susceptible to NK cell-mediated killing 

and phagocytosis by immature DC 

Antigen-specific Treg in colorectal carcinoma 

control T cell responses against a limited tumor 

antigen repertoire 

tumor-induced emigration of antigen-specific treg 

enables the bone marrow to foster spontaneous 

anti-tumor t-cell responses in breast cancer patients 

Disturbed NK cell function in CML patients at 

diagnosis does not recover under treatment with 

imatinib mesylate 

Treatment with anti-CD137 mAbs causes intense 

accumulations of liver T cells without selective antitumor 

immunotherapeutic effects in this organ 

Identification of clinically useful combinations of 

trifunctional antibodies with chemotherapy using 

different preclinical models 

Reinforcement of Cancer Immunotherapy by Adoptive 

Transfer of cblb-deficient Cytotoxic T Lymphocytes 

combined with a Dentritic Cell Vaccine 

Development of a novel transgenic mouse model for 

melanoma 

Influence of activation of human immune effector 

cells on the penetration into tumor spheroids 

Comparative Analysis of Predicted HLA Binding 

of Immunoglobulin Idiotype Sequences Indicates 

T Cell-Mediated Immunosurveillance in Follicular 

Lymphoma 






















16:20 – 16:35 hrs 


16:35 – 16:50 hrs 


16:50 – 17:05 hrs


091 Ramacher 

092 Tomsitz 

093 Koslowski 

094 Quaglino 

095 Lanzardo 

096 Halama 

097 Fraszczak 

098 Trad 

099 Hemmerling 

100 Koop 

Immunosuppression in transgenic mouse melanoma 

model induced by myeloid derived suppressor 

cells 

Establishment of a pre-clinical NOD/LtSz-scid 

IL2Rgammac null (NSG) mouse model to evaluate 

immunotherapeutic strategies for acute myeloid 

leukemia (AML) 

Tumor-associated genomic DNA hypomethylation 

effects positive autoregulation of HIF-1α 

A miRNAs expression profiles during ErbB2 driven 

mammary carcinogenesis 

Attenuation of PI3K/Akt-mediated tumorigenic 

signals through PTEN activation by DNA vaccineinduced 

anti-ErbB2 antibodies 

Natural Killer Cells are decreased in Human Colorectal 

Cancer Tissue and Liver Metastases despite 

High Levels of NK-Recruiting Chemokines 

Killer dendritic cells inhibit Treg differentiation 

Tumor infiltrating CD11c+ dendritic cells suppress 

T cell activation 

Human Langerhans cells reconstitute in skin xenografts 

Downregulation of cancer/testis antigen 45 (CT45) 

in human HT1080 cells analysed by proteomic 

screening 
























10:50 – 11:05 hrs 


11:05 – 11:20 hrs 


11:20 – 11:35 hrs 

33


101 Karakhanova 

102 Zimmer 

103 Heidemann 

Krogh 

104 Hansmann 

105 Lupp 

106 Castle 

107 Sevko 

108 Diaz-Valdés 

109 Bauer 

110 Femel 

34 

IFN-α up-regulates B7-H1 expression on dendritic 

cells and pancreatic tumor cells 

Impact of endoplasmic reticulum aminopeptidases 

(ERAP) on T cell epitope generation in melanoma 

cells 

the phospho-regulated calcium-binding cancer-testis 

antigen cabyr, interacts with glycolysis-regulating 

enzymes in lung cancer cells 

Isolation of intact genomic DNA from FOXP3stained 

and FACS-sorted regulatory T cells for 

epigenetic analysis 

Mechanisms of Treg-mediated suppression of graftversus-host 

disease 

DNA copy number, including telomeres and mitochondria, 

assayed using next-generation sequencing 

Paclitaxel in ultra-low doses reduces immunosuppression 

in ret transgenic tumor bearing mice 

Transforming growth beta 1 increases monocyte 

chemotactin protein-1 and interleukin-10 expression 

in A375 human melanoma cells enhancing 

monocyte migration and impairing dendritic cell 

function 

The TLR9 ligand CpG reduces the suppressive 

function of myeloid-derived suppressor cells via 

interferon alpha 

Identification of a potent, non-toxic adjuvant for use 

in therapeutic vaccines 














adjuvants 


adjuvants 


adjuvants 


adjuvants 


11:35 – 11:50 hrs 


11:50 – 12:05 hrs


111 Diekmann 

112 Reinis 

113 Kermer 

114 Lehmann 

115 Lladser 

116 Hotz 

117 Gonzalez- 

Carmona 

118 Trojandt 

L126 Klier 

mTOR inhibition by Rapamycin after intranodal 

RNA immunization improves the CD8+ memory 

T-cell response qualitatively and quantitatively 

DNA methyltransferase inhibitors as modulators of 

anti-tumour immunity: implications for their use as 

adjuvants for immunotherapy 

Antibody fusion proteins for cancer immunotherapy 

mimicking IL-15 trans presentation at the tumor site 

Modified vaccinia virus Ankara (MVA) - a safe vector 

virus and an adjuvant system for immunotherapy 

DAI (ZBP1/DLM-1) as a novel genetic adjuvant for 

cancer DNA vaccines that promotes CTL responses, 

overcomes tolerance and confers long-term tumor 

protection. 

A rational protocol of repeated TLR7 stimulation circumvents 

induction of TLR tolerance and translates 

into efficient anti-tumor therapy 

Combining subcutaneous inoculation of AFPexpressing 

DC with intraperitoneal injection of IL-12expressing 

DC increases survival in an established 

orthotopic HCC model 

Effects of the anti-cancer agent topotecan on human 

monocyte-derived dendritic cells 

Combined bacterial antibody therapy for colorectal 

carcinoma 



adjuvants 


adjuvants 


adjuvants 


adjuvants 


adjuvants 


adjuvants 


adjuvants 


adjuvants 


adjuvants 


35

36 

Therapeutic vaccination

001 Bontkes | Therapeutic vaccination 

Dendritic cells loaded with amplified mRNA isolated from 

leukemic stem cell like cells induce specific T-cells 

Hetty J. Bontkes, Jurjen M. Ruben, Willemijn A. van den Ancker, Theresia M. Westers, Gert 

J. Ossenkoppele, and Arjan A. van de Loosdrecht 

Department of Hematology , Cancer Center Amsterdam, V-ICI, VU University Medical Center, 

Amsterdam, The Netherlands 

It is thought that recurrent acute myeloid leukemia 

(AML) originates from chemotherapy resistant quiescent 

leukemic stem- or leukemia initiating cells 

(LSC). The application of immunotherapeutic approaches 

to eradicate LSC remaining after first line 

chemotherapy may contribute to improved disease 

outcome. Vaccination strategies have used dendritic 

cells (DC) ex vivo pulsed with tumor-derived 

whole lysates as modalities to present a broad range 

of tumor antigens to T cells to stimulate effective 

anti-tumor T-cell immunity in vivo. It is likely that 

certain proteins expressed by LSC have a distinct 

antigenicity as compared to more mature AML 

blasts and thus provide targets for specific T-cells. 

Even without identification of specific antigens, 

LSC can be a useful source of tumor antigens in DC 

vaccination-based immunotherapy. CD34+CD38- 

LSC can be identified using malignant stem cell 

associated cell surface markers including C-type 

lectin-like molecule-1 (CLL-1) and lineage markers 

such as CD7 and CD56. However, the low frequency 

of these cells precludes the use of LSC derived apoptotic 

cells or lysates for DC loading. Alternatively, 

mRNA isolated from LSC can be amplified and 

subsequently transfected into DC. We have made 

use of the HLA-A2 positive CD38- AML derived cell 

line MUTZ-3 which contains a subpopulation of 

CD34+CLL1+ cells which resembles the phenotype 

of a putative LSC. CLL1+CD34+ stem cell like cells 

as well as CLL1+CD34- CLL1-CD34+ and CLL1- 

CD34-cells were isolated by FACS sorting and total 

RNA was isolated. mRNA was converted to cDNA 

and amplified by PCR using the SMART system. 

Subsequently, mRNA was in vitro transcribed 

from the amplified cDNA. Amplification of CLL1 

and survivin transcripts was confirmed by RT-PCR. 

Mature monocyte derived DC (MoDC) were generated 

from HLA-A2 positive healthy donor blood and 

transfected with amplified CLL1+CD34+ derived 

mRNA and used to stimulate autologous CD8β+ 

T-cells. After three weekly re-stimulations with 

CLL1+CD34+ mRNA transfected DC, specificity of 

the T-cells was analyzed either by IFNγ production 

upon stimulation with autologous immature MoDC 

transfected with GFP mRNA, mRNA amplified from 

total MUTZ-3 cells or sorted subpopulations, or by 

cytotoxicity towards MUTZ-3 subpopulations. The 

generated T-cells specifically produced IFNγ in response 

to MUTZ-3(subpopulation) mRNA loaded 

iDC but not in response to GFP mRNA transfected 

DC. Furthermore, the generated CTL killed the 

CLL1+CD34+ and CLL1+CD34- populations more 

efficiently compared to the CLL1-CD34+ populations. 

We show that MoDC transfected with RNA amplified 

from one MUTZ-3 sub-population resembling 

the phenotype of LCS cells are capable of inducing 

T-cells which recognize both cells transfected with 

mRNA from the LSC resembling MUTZ-3 subset as 

well as the more mature subsets. The efficacy and 

feasibility of this approach is currently tested in an 

autologous AML setting in vitro. 

37

002 Kotsiou | Therapeutic vaccination 

Antigen specific monoclonal antibodies directed against the 

HA-1 and HA-2 minor histocompatibility antigens 

Eleni Kotsiou 1,2 , Jonathan Silk 3 , Vincenzo Cerundolo 3 , Julian Dyson 2 , and Keith G. Gould 1 

1 

Department of Immunology, Wright-Fleming Institute, St Mary‘s Campus, Imperial College, 

London W2 1PG, UK 

2 

Immunobiology Section, Commonwealth Building, Hammersmith Hospital, Imperial College, 

London W12 0NN, UK 

3 

Tumour Immunology Group, Weatherall Institute of Molecular Medicine, University of Oxford, 

Oxford OX3 9DS, UK 

38 

Monoclonal antibodies with the specificity of a T 

cell receptor (TCR) (also known as TCR mimic antibodies) 

can be used for the targeting of tumor or 

viral epitopes and are potentially very useful in the 

clinical setting. 

The HA-1 and HA-2 minor histocompatibility (H) 

antigens are restricted by human leukocyte antigen 

(HLA) A2 and expressed only on normal and malignant 

haematopoietic cells. We have produced 

soluble (lacking cytoplasmic and transmembrane 

regions) HA-1 and HA-2 HLA-A2 single chain 

trimers (SCTs) where the peptide, β2-microglobulin 

and major histocompatibility complex (MHC) heavy 

chain are covalently linked together via glycine/ 

serine rich linkers. We then used the soluble SCT 

proteins to immunize HHD (HLA-A2 transgenic) 

mice for the production of antigen specific monoclonal 

antibodies by taking advantage of the ‘humanized’ 

immune system of the transgenic mice 

which will recognize the HLA-A2 molecule as ‘self’ 

but the peptide epitope as foreign. 

The screening of the serum of the immunized mice 

(after three rounds of immunization) revealed the 

presence of antibodies preferentially reacting with 

the HA-1 and HA-2 epitopes. The screening of hybridoma 

clones produced after fusion of spleen cells 

from innunized mice with myeloma cells showed 

that, rather than recognising the peptide, the majority 

of the monoclonal antibodies were directed 

against other immunogenic epitopes present in the 

sequence of the HLA-A2 SCTs. 

The development of new SCT fusion proteins which 

can potentially improve the immunization strategy 

will be described.

003 Altvater | Therapeutic vaccination 

Presentation of tumor-associated epitopes by activated human 

γδ T cells induces expansion of specific CD8+ T cells from 

naïve precursors 

Bianca Altvater 1 , Silke Landmeier 1 , Sibylle Pscherer 1 , Anna Hansmeier 1 , Barbara Savoldo 2 , 

H. Juergens 1 , Claudia Rossig 1 

1 University Children´s Hospital Münster, Department of Pediatric Hematology and Oncology, 

Albert-Schweitzer-Str. 33, 48149 Münster 

2 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, U.S.A 

Efficient antigen presentation is an important 

prerequisite for the induction of therapeutic T-cell 

immunity against viral and tumor antigens. In an 

Epstein Barr virus (EBV) model, we previously demonstrated 

that bisphosphonate-activated γδ T cells 

are potent stimulators of functional EBV-specific T 

cell responses in both normal donors and Hodgkin 

lymphoma patients. Here, we extended these observations 

by investigating the capacity of activated γδ 

T cells to induce primary T-cell responses against 

the tumor-associated, poorly immunogeneic antigens 

PRAME and STEAP-1 from the naive T-cell 

repertoire. 

Peripheral blood-derived γδ T cells were expanded 

from three individual HLA-A2 positive healthy 

donors by stimulation with the aminobisphosphonate 

zoledronic acid (1 µg/ml) in the presence of 

rhIL-2 (100 U/ml) and rhIL-15 (10 ng/ml). To investigate 

the ability of activated γδ T cells to stimulate 

expansion of PRAME- and STEAP-1 specific 

cytotoxic T cells (CTL) in vitro, purified autologous 

CD8+ T cells were cocultured with either dendritic 

cells (DCs) or activated γδ T cells pulsed with 

pools of HLA-A2 restricted peptides derived from 

either STEAP-1 or PRAME, followed by two stimulations 

with peptide-pulsed K-562 cells, genemodified 

to express human HLA-A2, CD80, CD40L, 

and OX40L (K-562-APCs). ELISPOT analysis demonstrated 

efficient and equivalent induction of 

specific CD8+ T-cell responses by both DCs and 

γδ T-APCs. Both types of CTLs had a predominant 

effector memory phenotype (CCR7-CD45R0+). 

Even though antigen-experienced precursor CTLs 

against the tumor-associated antigens are absent in 

the majority of healthy donors, these experiments 

do not formally prove that the expanded T cells are 

a consequence of T-cell priming. To demonstrate 

the origin of CTLs from antigen-naïve T cells, we 

performed additional coculture experiments using 

CD45RA+ T cells, purified from the CD8+ T-cell 

population by magnetic cell selection, as responder 

cells. Again, ELISPOT analysis after primary stimulation 

with PRAME peptide-pulsed autologous 

DCs or γδ T-APCs demonstrated specific and potent 

CTL reactivity against both pooled and individual 

PRAME peptides. Importantly, γδ T-APC induced 

CTLs were not inferior to those generated using the 

DC gold standard. 

Thus, γδ T cells represent a promising source of 

highly efficient professional APC for antigen-specific 

immunotherapy of cancer. In current experiments, 

we are establishing the capacity of γδ T 

cells endogenously expressing STEAP-1 or PRAME 

full-length antigen in inducing functional primary 

CTL responses. 

39

004 Haenssle | Therapeutic vaccination 

Dendritic cells loaded with HSP70-expressing heat-killed melanoma 

cells efficiently activate autologous T cells 

Holger A. Haenssle 1 , Susanne Knudsen 1 , Anke Schardt 2 , Timo Buhl 1 , Lars Boeckmann 1 , 

Michael P. Schön 1 

1 Department of Dermatology and Venereology, Georg August University, Göttingen, Germany 

2 Max Planck Institute for Experimental Medicine, Göttingen, Germany 

40 

There is considerable interest to develop strategies 

that enhance cross-presentation pathways of dendritic 

cells (DCs) to elicit a strong cytotoxic T cell 

activation for cancer vaccination protocols. 

In order to best exploit the enhanced cross-presentation 

of antigens bound to heat shock protein 70 

(HSP70), we analyzed melanoma cell preparations 

for their HSP70 expression. For generation of heatnecrotic 

cell material melanoma cells were kept at 

42°C for 90 min and then heated to 56°C for a final 

30 min. Apoptotic Annexin V + /Propidium iodide– 

melanoma cells were generated by irradiation with 

1.0 J/cm2 UV-B. Western blotting revealed strong 

upregulation of HSP70 after heat-killing in contrast 

to UV-B irradiation. The uptake of fluorescently 

labeled heat-killed necrotic vs. apoptotic melanoma 

cells by DCs at various levels of maturation was assessed. 

Immature DCs (iDCs) internalized HSP70expressing 

necrotic material to a much higher extent 

(61% ± 7%) than apoptotic material from UV-Birradiated 

cells (48% ± 5%). Maturation-inducing 

cytokines did not affect the uptake when added 

simultaneously with the tumor cell preparations. 

Loading DCs with heat-necrotic or apoptotic melanoma 

cells slightly reduced CD83 expression while 

leaving CD208 (DC-LAMP) expression unchanged. 

As determined by IFN-γ-detecting ELISPOT assays, 

iDCs loaded with HSP70-expressing heat-killed 

melanoma cells activated autologous T cells most 

effectively when used without any further maturation, 

whereas DCs loaded with apoptotic material 

required maturation. 

In conclusion, HSP70-expressing melanoma cells 

could be generated by heat-killing. Loading immature 

DCs with heat-killed melanoma cells resulted 

in a superior priming of autologous T cells 

in vitro.

005 Vyth-Dreese | Therapeutic vaccination 

MART-1 specific T cells after DNA tattoo vaccination of 

melanoma patients 

Florry Vyth-Dreese 1 , Martin van der Maas 1 , Willeke van de Kasteele 1 , Trees Dellemijn 1 , 

Sandra Adriaansz 2 , Henk Mallo 2 , Bastiaan Nuijen 3 , Christian Ottensmeier 4 , Lindsey Low 4 , 

Christian Blank 1,2 , Ton Schumacher 1 and John Haanen 1,2 

1 Division of Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, 

1066 CX Amsterdam, The Netherlands 

2 Medical Oncology Department, The Netherlands Cancer Institute-Antoni van Leeuwenhoek 

Hospital, 1066 CX Amsterdam, The Netherlands 

3 Pharmacy, Slotervaart Hospital, 1066 EC Amsterdam, The Netherlands 

4 Cancer Sciences Division, University of Southampton, Southampton SO16 6YD, United Kingdom 

Recently, we developed a novel, rapid and potent 

intradermal DNA vaccination method, called DNA 

tattooing. Previous studies in experimental systems 

showed a strong increase in vaccine-specific T cells 

compared to intramuscular vaccination, parallelled 

by robust anti-tumor T cell responses and rejection 

of established subcutaneous tumors. These 

results prompted us to initiate a phase I clinical 

study with an inhouse manufactured GMP-grade 

DNA vaccine, pDermatt. Here we report data from 

the immunomonitoring of our first phase I clinical 

trial of DNA tattoo vaccination of stage IV melanoma 

patients using this DNA vaccine encoding a 

tetanus toxin fragment c-modified MART-1 epitope 

fusion protein. 

Sofar, six patients were treated with DNA tattoo 

vaccination on day 0, 3 and 6 as a prime and on day 

28, 31 and 34 as a boost vaccination. Per cohort of 

3 patients the vaccine dose was doubled from 0.5 

mg to 1 mg DNA per tattoo. Flow cytometry and 

EliSpot assays were performed to examine the presence 

and specificity of T cells in peripheral blood 

samples, taken before therapy and at week 2, 4, 6 

and 8. Biopsies were taken from the vaccination site 

at week 2 and 6 and examined for the presence of 

immune cell subsets and specific T cells by immunohistochemistry 

and flow cytometry. 

To enable flow cytometric analysis for specific T 

cells, biopsies were taken from the vaccination 

site and cultured in vitro for 2 weeks in high dose 

IL-2 (6000 IU/ml). In 5/6 patients, compared to 0/6 

normal skin biopsies obtained from breast cancer 

operations, this resulted in outgrowth of MART- 

1-specific CD8 T lymphocytes, with 3/5 patient 

samples showing 30-50% MART-1 specific CD8 T 

cells. Highest numbers were obtained from week 

6 biopsies with a preferential increase in MART-1 

modified specific T cells. 

Virtually no therapy induced increases were observed 

in percentages of MART-1 specific peripheral 

blood CD8 T cells. EliSpot analysis revealed therapy 

induced IFNγ responses to MART-1 modified and 

wild type peptides in 2/3 patients tested in cohort 

1. These responses followed similar, although 

delayed, kinetics compared to tetanus fragment c 

responses which served as internal controls. 

Examination of immune cells at patient vaccination 

sites showed similar numbers of CD8 T cells, compared 

to normal skin, located in the subepidermal 

or dermal, but not epidermal areas. An increase 

was found for activated DC expressing CD80. From 

these data it is concluded that DNA tattoo vaccination 

induced MART-1 specific CD8 T cell migration 

to the vaccination site. 

41

006 Bernard | Therapeutic vaccination 

Investigating the impact of autophagy modulation on dendritic 

cell cancer vaccines 

Dannie Bernard 1 , Julian J. Lum 2 , Yonghong Wan 1 and Jonathan L. Bramson 1 

1 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada 

2 Deeley Research Center, BC Cancer Agency, Vancouver Island, British Columbia, V8R 6V5, Canada 

42 

Autophagy has been linked to extended survival 

when cells are faced with cellular stress. Ex-vivo 

derived dendritic cells (DCs) undergo substantial 

stress upon antigen loading and in-vivo delivery 

and the importance of autophagy in protecting 

DCs from these stresses is poorly understood. 

We have employed 2 strategies to investigate the 

impact of autophagy on DC vaccines. In the first 

case, we employed rapamycin as an inducer of autophagy 

in an effort to precondition the DCs prior 

to infection with recombinant adenovirus (Ad) 

and recombinant vesicular stomatitis virus (VSV). 

Kinetic analyses showed that rapamycin was effective 

at inhibiting its target mTOR within 5 hours of 

treatment and the inhibition was maintained for at 

least 24 hours post-transduction. Preconditioning 

of DCs with rapamycin did not affect infectivity, 

as measured by GFP expression, nor did it affect 

maturation, defined by MHC II, CD40, CD86, IL-12 

and TNF-a expression. However, we noted a 10-fold 

reduction in type I interferon secretion following 

VSV transduction, but not Ad transduction. We 

are currently conducting in-vivo experiments with 

rapamycin-preconditioned DC vaccines and results 

will be discussed at the meeting. In the second 

case, we have investigated the role of basal autophagy 

in the context of DC vaccination. We have 

crossed ATG5fl/fl mice with CD11c-Cre mice to generate 

conditional knockout mice in which DCs are 

autophagy-deficient. Results obtained thus far from 

in-vitro phenotyping experiments of virally trans- 

duced DCs indicate that autophagy deficiency does 

not significantly impact maturation of the cells. 

The importance of autophagy following delivery of 

DC vaccines still remains to be determined. This 

work was supported by grants from CIHR, OCRN 

and TFF.

007 Navarrete | Therapeutic vaccination 

Upfront Immunization With Autologous Recombinant Idiotype 

Fab Fragment Without Prior Cytoreduction in Indolent B-Cell 

Lymphoma 

Marcelo Navarrete 1 , Kristina Heining-Mikesch 1 , Frank Schüler 2 , Cristina Bertinetti-Lapatki 1 , 

Gabriele Ihorst 3 , Andrea Keppler-Hafkemeyer 1 , Gottfried Dölken 2 , Hendrik Veelken 1 

1 Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany 

2 Department of Hematology and Oncology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany 

3 Department of Medical Biometry and Statistics, University Medical Center Freiburg, Freiburg, Germany 

The monoclonal immunoglobulin ("Idiotype", Id) 

of B-cell lymphomas constitutes a tumor-specific 

antigen. Id immunization of follicular lymphoma 

(FL) patients as remission maintenance after conventional 

chemotherapy induces anti-Id antibodies 

that are associated with improved PFS. To ease 

the challenge of manufacturing Id vaccines on a 

patient-individual basis, we have developed the 

expression of Id protein as Fab fragment (FabId) 

in E. coli based on validated Ig clonality criteria 

(Bertinetti et al., EJH 2006). In a phase I trial, we 

have demonstrated the feasibility and tolerability 

of intradermal injection of FabId with the adjuvant 

MF59 in conjunction with subcutaneous GM-CSF 

(Bertinetti et al., Cancer Res 2006). 

Since the effects of upfront Id vaccination in lymphoma 

patients without prior chemotherapy are 

unknown, we conducted a phase II trial designed 

to demonstrate the immunological efficacy of FabId 

vaccination as upfront intervention for asymptomatic 

lymphoma patients and for remission consolidation. 

Given the strong but indirect evidence for 

Id-directed, T cell-mediated immuno-surveillance 

in FL (see poster by Strothmeyer et al.), we investigated 

vaccination-induced T cell responses. 21 

patients with untreated indolent B-cell lymphoma 

and 20 patients in remission after cytoreductive 

therapy received 6 intradermal injections of FabId 

with subcutaneous sargramostim. Circulating 

antigen-reactive T cells were measured by IFNγ 

ELISpot that was validated by blinded interlabo- 

ratory testing (Britten et al., CII 2008). Humoral 

immune responses were assessed by ELISA. 

FabId vaccination induced immune responses in 

76% of untreated and in 80% of remission patients. 

Poisson regression analyses and epitope mapping 

demonstrated specificity of T cell responses for 

individual Id features. In untreated patients, cellular 

immunity was associated with superior PFS 

(p=0.002) and with durable remissions in 6 of 15 

FL (p=0.04). In remission maintenance, anti-Id antibodies 

correlated with PFS (p=0.02). Prior rituximab 

and low B-cell counts were associated with 

failure to develop anti-Id antibodies. FabId immunization 

effectively induces immune responses in 

both pretreated and untreated lymphoma patients. 

Similar to other Id vaccines, humoral immunity is 

associated with control of residual lymphoma. In 

contrast, cellular anti-Id immunity may be effective 

against untreated lymphoma. Sustained remissions 

in patients with vaccination-induced cellular immunity 

suggest clinical benefit and warrant randomized 

trials comparing this vaccine with expectant 

management as upfront strategy for asymptomatic 

follicular lymphoma. 

43

008 Schroeder | Therapeutic vaccination 

In vitro characterization of an irradiated gene-transfer medicinal 

product consisting of a prostate cancer derived cell line 

constitutively secreting interleukin-2 and interferon-gamma 

Petra Schroeder 1 , Carsten Lindemann 1 , Uwe Irmer 1 , Henning Weigt 2 , Bernd Eisele 2 , Klaus Kuehlcke 1 

1 EUFETS GmbH, Vollmersbachstraße 66, 55743 Idar-Oberstein, Germany 

2 Vakzine Projekt Management GmbH, Mellendorfer Straße 9, 30625 Hannover, Germany 

44 

In a first in man Phase I/II study, a prostate cancer 

cell line constitutively secreting the pro-inflammatory 

cytokines interferon-gamma (IFN-γ) and interleukin-2 

(IL-2) has shown anti-tumor response 

(median PSA doubling time prolonged from 63 days 

to 114 days, p = 0.0035; Brill et al., J Gene Med 

2007 and Hum Gene Ther 2009). Further drug development 

requires thorough characterization of 

the tumour vaccine. We designed a non-clinical 

program regarding aspects of safety, stability and 

the proof of principle of the tumour vaccine adopting 

an in vitro approach based on human cells 

to rectify the limitations of homologous animal 

models. A clinical scale manufacturing process 

including centralized irradiation was developed 

in parallel. Considering safety and stability of the 

tumour vaccine we tested the viability, colony formation, 

phenotype (surface marker expression of 

the prostate specific antigens PSA, PSMA, EpCAM), 

the release and bioactivity of IL-2 and IFN-γ, vector 

integrity and the release of replication competent 

retrovirus besides the generic tests stipulated for 

such products. The absence of tumourigenicity, 

e.g. outgrowth of potentially remaining replication 

competent cells, of the finally irradiated cells was 

controlled by the assessment of proliferating cells 

in long term cultures and proliferation assays. In 

order to show the induction of an immunological 

reaction which is considered as a prerequisite to 

induce an anti-tumour response we evaluated the 

allogenic response towards the irradiated cells, 

analysed the deposition of complement and the 

uptake of cellular particles by phagocytes. The stability 

and safety as well as the functionality and biologic 

activity of the cellular product could be reliably 

demonstrated. Results of phenotyping, colony 

formation, bioassays and the proof of principle will 

be presented.

009 Kuhn | Therapeutic vaccination 

Phosphorothioate cap analogs increase stability and translational 

efficiency of RNA vaccines in immature dendritic cells and 

induce superior immune responses in vivo 

Andreas N. Kuhn 1 , Mustafa Diken 1 , Sebastian Kreiter 1 , Abderraouf Selmi 1 , Joanna Kowalska 2 , 

Jacek Jemielity 2 , Edward Darzynkiewicz 2 , Christoph Huber 1 , Özlem Türeci 1 , and Ugur Sahin 1 

1 Department of Internal Medicine III, Division of Translational and Experimental Oncology, 

University Medicine Mainz, Germany 

2 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Poland 

Vaccination with in vitro transcribed RNA coding 

for tumor antigens is considered a promising approach 

for cancer immunotherapy and has already 

entered human clinical testing. 

One of the basic objectives for development of RNA 

as a vaccine is the optimization of immunobioavailability 

of the encoded antigen in vivo. In previous 

work, we have developed plasmid templates 

for in vitro transcription of RNA-encoded antigens 

with modified 3‘ structures (3‘ UTR and poly(A)tail) 

stabilizing the RNA and optimizing its translational 

performance in dendritic cells (DCs), the 

major antigen-presenting cells (1). Moreover, we 

achieved significant leverage of MHC class I and II 

presentation of the antigen in DCs by introducing 

routing signals (2). By combining these measures 

we were able to profoundly improve properties of 

RNA-encoded vaccines. 

Yet another important structural element of in vitro 

transcribed RNA is the 5‘ cap structure. Capping of 

in vitro synthesized RNA is achieved by transcribing 

the DNA template in the presence of the cap 

dinucleotide m7GpppG as structural homolog of the 

endogenous cap structure. Recently, several synthetic 

cap analogs have been described with modifications 

that influence stability and translational 

characteristics of the respective RNA species. 

Up to now, such modified cap analogs have been 

primarily employed to study RNA metabolism. 

Their impact, however, on bioavailability of the 

encoded protein in antigen presenting cells such 

as DCs and eventually on induction of potent T-cell 

responses in vivo has never been investigated. Therefore 

we have preclinically evaluated novel cap 

analogs for immunopharmacological improvement 

of vaccines based on antigen-encoding RNA. We 

demonstrate that RNAs capped with the D1 diastereoisomer 

of m27,2‘-OGppSpG (beta-S-ARCA) have 

increased stability and translational efficiency in 

immature but not mature DCs. Accordingly, in vivo 

delivery of the antigen as beta-S-ARCA(D1)-capped 

RNA species led to increased protein expression 

and enhanced priming and expansion of naïve antigen-specific 

T-cells in mice. We expect that our 

findings pave the way for inauguration of modified 

cap analogs into RNA vaccine development and 

will likely contribute to a better clinical outcome. 

(1) Holtkamp et al. (2006) Blood 108: 4009-17 

(2) Kreiter et al. (2008) J Immunol 180: 309-18 

45

010 Mikyskova | Therapeutic vaccination 

Interleukin 12 genetically-modified vaccines augment the effect 

of chemotherapy of human papilloma virus 16-associated 

tumours with gemcitabine 

Romana Mikyšková, Jana Šímová, Marie Indrová, Jan Bubeník, Jana Bieblová, Milan Reiniš 

Department of Tumour Immunology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 

Prague, Czech Republic 

46 

Experiments were designed to examine whether 

local administration of genetically-modified interleukin 

12 (IL-12)-producing vaccine can enhance 

the chemotherapy of human papilloma virus (HPV) 

16-associated tumour with gemcitabine, a cytostatic 

agent that is known to reduce myeloid derived 

suppressor cells. Major histocompatibility class I 

(MHC I) positive non-metastasizing murine carcinoma 

TC-1 model was used to examine the effect 

of local IL-12 gene therapy in the minimal residual 

tumour disease induced by intraperitoneal cytoreductive 

therapy with gemcitabine. Mice with 

established 0.2-0.3 cm in diameter TC-1 tumours 

were treated with gemcitabine and, subsequently, 

with irradiated, IL-12-producing tumour cells. 

It was found that local administration of IL-12producing 

vaccine enhanced the effect of cytoreductive 

therapy with gemcitabine in TC-1 (MHC I 

positive) tumours. To investigate the antitumour 

and antimetastatic effect of IL-12 plus gemcitabine 

combination in another clinically relevant setting, 

surgical minimal residual tumour disease of MHC 

I-deficient MK16 tumours was used. Mice with established 

1 cm in diameter MK16 tumours were 

operated and subsequently treated with gemcitabine 

and/or IL-12-producing vaccine. Combination of 

the IL-12 genetically-modified tumour vaccine with 

gemcitabine treatment after surgery significantly 

reduced the percentage and size of MK16 tumour 

recurrences as well as the percentage and number 

of lung metastases, as compared to the group of 

operated-only mice and groups treated with gemcitabine 

or IL-12-producing vaccine alone. The therapeutic 

effectiveness exerted by the gemcitabine plus 

IL-12 combination in the MK16 tumour model was 

demonstrated by the detection of interferon gamma 

(IFNγ) production by spleen cells using ELISPOT 

and ELISA test. The highest production of IFNγ was 

found in the group treated with gemcitabine plus 

IL-12-producing cells. The percentage of myeloid 

derived suppressor cells (CD11b+/Gr-1+ cells) 

was analyzed three days after administration of 

the IL-12-producing vaccine. The lowest percentage 

of CD11b+/Gr-1+ was found in the group treated 

with the combination of gemcitabine plus IL-12producing 

cells. Taken together, we conclude that 

IL-12 augments the effectiveness of chemotherapy 

of HPV16-associated tumours with gemcitabine 

and increases the anti-tumour immune response. 

This work was supported by grants No. 301/09/1024 and No. 

301/07/1410 from the Grant Agency of the Czech Republic and by 

grant of the Clinigene project EU-FP6-NOE No. 018933.

011 Kuhs | Therapeutic vaccination 

Clinical Development of lentiviral vector-induced „SMART-DC“ 

for melanoma immunotherapy 

Sandra Kuhs 1 , Ralf Gutzmer 2 , Bala Sai Sundarasetty 1 , Sylvia Borchers 1 , Gustavo Salguero 1 , 

Arnold Ganser 1 , Rainer Blasczyk 3 , Henk Garritsen 4 , Thomas Woelfel 5 , Farzin Farzaneh 6 and 

Renata Stripecke 1 

1 Dept. of Hematology, Hemostasis, Oncology and Stem Cell Transplantation 

2 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Poland 

3 Dept. of Transfusion Medicine, Hannover Medical School 

4 Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH 

5 Hematology and Oncology, University of Mainz, Germany 

6 Dept. of Hematological Medicine, Kings College, London, UK 

Clinical ex vivo production of dendritic cells (DC) 

is costly, time-consuming and difficult for largescale 

clinical trials. In addition, ex vivo grown DCs 

are not highly viable once re-infused in the body 

and antigen-presentation and bio-distribution are 

sub-optimal. We developed a novel technology for 

production of DC consisting of lentiviral vector 

(LV) transduction of growth factors and full-length 

antigens into monocytes that results into induction 

of “SMART-DCs” (Self-differentiated Myeloidderived 

Antigen-presenting-cells Reactive against 

Tumors). This concept has been extensively tested 

and validated in pre-clinical melanoma mouse 

models for safety and for induction of protective 

immunity. Here, self-inactivating lentiviral vectors 

containing interspacing 2A elements and a nonencoding 

Wpre were produced in a bicistronic (coexpressing 

simultaneously human GM-CSF and 

IL-4) or tricistronic design (expressing in addition 

the melanoma-associated antigens MART-1 or 

TRP-2). Co-expression of all the transgenes was validated 

in 293T cells transduced with these vectors. 

Overnight transduction of fresh or cryopreserved 

CD14+ monocytes resulted in persistent autocrine 

production of GM-CSF (average 7 ng/ml) and IL-4 

(average 0.8 ng/ml), inducing the self-differentiation 

of long-lived (up to 3 weeks) human SMART- 

DCs, which fully recapitulated the immunophenotype 

of conventional DCs. For future GMP process 

development, we evaluated monocyte transduction 

in a closed GMP-grade bag system. We were able 

to demonstrate that SMART-DCs were produced 

more effectively in bags than in culture flasks. Monocytes 

co-transduced with the bicistronic vector 

plus a vector expressing full length MART-1 were 

recognized by T cell clones specific for MART-1 

in an HLA-restricted manner, assessed by IFN-γ- 

ELISPOT-Assay. PBMCs that were primed/boosted 

in vitro with autologous SMART-DCs expressing 

MART-1 and assayed by IFN-γ-ELISPOT-Assay 

(using peptide-pulsed T2 cells as target cells) demonstrated 

the induction of MART-1-specific T cell 

responses. Pre-clinical validation of SMART-DCs 

engineered with tricistronic vectors for MART-1 

or TRP2 expression, for the assessment of their 

potency to stimulate and expand autologous T cells 

obtained from melanoma patients is underway. A 

pre-GMP lentiviral production for generation of 

GMP compliant vector will follow, for development 

of a phase I immunotherapy clinical trial for melanoma 

patients. 

47

012 Gueckel | Therapeutic vaccination 

Targeted modification of HLA-A*02-restricted mucin-1-derived 

peptides results in induction of cytotoxic T cells cross-reactive 

with naturally presented peptides 

Brigitte Gückel 1 , Simone Kayser 1 , Helen Hörzer 1 , Janet Peper 1 , Despina Rudolf 2 , Dorothee 

Wernet 3 , Stefan Stevanovic 2 , Dagmar Sigurdardottir 2 

1 Department of Obstetrics and Gynecology, University of Tuebingen, 72076 Tuebingen, Germany 

2 Department of Immunology, Institute for Cell Biology, University of Tuebingen, 72076 Tuebingen, Germany 

3 Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tuebingen, 72076 Tuebingen, 

Germany 

48 

The characterization of T-cell epitopes derived 

from tumor associated antigens fostered peptidebased 

cancer vaccines. Well characterized mucin 

(MUC)-1 derived HLA-A*02-restricted epitopes 

have been used for vaccinations in renal cell 

cancer, breast and ovarian cancer; however, the clinical 

efficacy had not yet met expectations. Altered 

peptide ligands (APLs) that might have enhanced 

HLA-binding and thus, improved immunogenicity 

are under investigation. 

In this study, we tested the CD8+ T-cell responses 

against two MUC-1 epitopes. The (TαL) substitution 

at the anchor position 2 in peptide M1.1 (MUC-1950- 

958) induced a 10-fold increase in peptide-MHC 

binding, as shown by T2-binding assay. Replacement 

of (LαK) at position 1 in the M1.2 peptide 

(MUC-112-20) increased its solubility in aqueous 

solution while not affecting its MHC-binding. 

To compare the CD8+ T-cell response against 

MUC-1 peptides and their APLs, peripheral blood 

lymphocytes of healthy donors were stimulated 

with peptide-pulsed antigen-presenting cells for 

several rounds. The frequency of peptide-reactive 

CD8+ T cells was determined by intracellular IFNγ-staining. 

While T cells specific for the natural 

peptides and the M1.2-APL were rarely detected, 

many donors produced significant frequencies of 

CD8+ T cells reactive with the M1.1-APL. M1.2- 

APL-specific CD8+ T cells were simultaneously 

recognized by HLA-A*02 tetramers containing the 

natural and the modified peptide, which suggests 

that the modification in the anchor peptide did not 

significantly affect T-cell recognition. Similarly, 

M1.1-APL-specific T cells efficiently lysed T2 cells 

loaded with either the natural or the modified 

peptide. Interestingly, a similar cross-reactivity was 

seen with rare M1.2-specific T cells. More importantly, 

M1.1-APL-specific T cells lysed the MUC-1 

highly expressing tumour cell line MCF-7 which 

presents the natural M1.1 epitope. 

In summary, CD8+ T cells induced against both 

MUC-1-APLs are able to cross-react with naturally 

presented parental epitopes and, in the case of 

M1.1-APL, the ease of induction of peptide-specific 

T cells makes this peptide a good candidate for vaccination 

strategies.

013 Babiarova | Therapeutic vaccination 

Development of different types of vaccine against WT1 

tumor antigen 

Katarina Babiarova, Luda Kutinova, Eva Brabcova, Jitka Krystofova, Petr Hainz and Sarka 

Nemeckova 

Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague 2, U Nemocnice 1, 

CZ-128 20, Czech Republic 

The Wilms’ tumor gene 1 (WT1) encodes a tran- 

scription factor, initially identified as a suppressor 

in the etiology of Wilms’ tumor. It is expressed at 

high levels in a variety of human cancer including 

leukemia and various types of solid tumors. WT1 

protein has been reported to serve as target antigen 

for tumor-specific immune responses. 

The aims of this study were to prepare experimental 

vaccines of different type against WT1 tumor 

antigen and to evaluate their potency in immunotherapy 

of tumors in mouse model. We prepared 

DNA vaccine pBSC/WT1-GUS based on the fusion 

gene β-glucoronidase from E.coli (GUS) which 

was shown previously to have superior anti-tumor 

effect. We also constructed the viral vaccine P13- 

WT1-GUS based on the recombinant vaccinia virus 

(rVACV) strain P13 with the expression of fusion 

protein WT1-GUS under the control of either earlylate 

(H5) or synthetic early-late (E/L) VACV promoters. 

Every vaccine contains a fragment of murine 

WT1 gene coding for a peptide with the length of 

155aa (94.-249.aa) containing several CTL epitopes. 

We also used peptide vaccines derived from WT1 

with motifs predicted to bind to Db murine MHC 

I. The efficacy of these vaccines was tested in the 

immunization experiments in mice. The ELISPOT- 

IFNγ assay and measurement of the intracellular 

IFNγ was used to detect T cell immune response 

specific for WT1 peptides. Highest response was 

elicited by the RMFPNAPYL peptide. The immunization 

of mice either with pBSC/WT1-GUS or P13- 

WT1-GUS in combination with WT1 peptide vaccine 

P(126-135) as a booster revealed a low WT1 peptide 

specific T cell immune response in about 50% of 

immunized mice. Similar results were found after 

the immunization of mice using combination of all 

three types of vaccines. Anti-tumor effect of immunization 

with the different combination of our 

three vaccines were determined in mouse model of 

WT-1 positive tumors induced by the TRAMP-C2 

cells. We observed that priming with rVACV based 

vaccine combined with booster with RMFPNAPYL 

peptide in incomplete Freund adjuvant resulted in 

enhancement of the growth of TRAMP-C2 cells in 

mice. However immunization with one vaccine 

only (P13-WT1-GUS) was able to decelerate the 

growth of TRAMP-C2 tumors. 

This work was supported by grants NS 10660-3/2009 from IGA 

MZ ?R and 78608 from GAUK. 

49

014 Abbas | Therapeutic vaccination 

Vaccine development through antigen targeting to mannose 

receptor 

Zaigham Abbas 1 , Richard Pleass 2 , Giuseppe Mantovani 3 , Lindy Durrant 1 and Luisa Martinez-Pomares 1 

1 School of Molecular Medical Sciences, University of Nottingham 

2 School of Biology, University of Nottingham 

3 School of Pharmacy, University of Nottingham 

50 

Dendritic cells (DC) are unique antigen presenting 

cells which play a major role on the antigen presentation 

and initiation of the immune response 

by regulating B- and T- cell activation. Antigen 

targeting to DC receptors is an effective, safe and 

specific method for the vaccine development. MR 

is an endocytic receptor expressed by subpopulations 

of DC. Antigen targeting through MR leads 

to enhanced antigen uptake and presentation to T 

cells which makes it a favourite receptor for the development 

of vaccines against diseases that require 

T-cell immunity such cancer and viral infections. 

We have used two approaches to target antigen to 

MR; (i) MR-specific chimeric antibodies carrying 

several model antigens and (ii) antigens conjugated 

to novel glycopolymers. The binding efficiency of 

the chimeric antibodies has been assessed by using 

ELISA and BIACORE and the glycopolymers have 

been tested for their interaction with MR. These 

glycopolymers will be coupled to a mutated form 

of the model antigen ovalbumin (OVA) lacking Nglycosylation 

sites as native OVA is an excellent MR 

ligand. These novel reagents are currently being 

tested for their ability to induce T-cell activation 

in vitro using co-cultures of antigen presenting 

cells and T cells and in-vivo. Our results will 

greatly benefit the development of antigen delivery 

methods suitable for robust T cell activation.

015 Vogt | Therapeutic vaccination 

Inhibition of tumor growth after subcutaneous injection of dendritic 

cells engineered to express angiogenesis-related flk-1 antigen 

in a murine HCC model 

Annabelle Vogt 1 , Georges Decker 1 , Esther Raskopf 1 , Volker Schmitz 1 , Tilman Sauerbruch 1 , 

Wolfgang H. Caselmann 2 and Maria A. González-Carmona 1 

1 Department of Internal Medicine I, University of Bonn, Bonn, Germany 

2 Bavarian State Ministry of the Environment and Public Health, Munich, Germany 

Background: Dendritic cells (DC) are professio- 

nal antigen presenting cells able to prime T-cells 

against tumor associated antigens (TAA) such 

as AFP. However, tumor cells downregulate the 

expression of TAA inducing immune tolerance. 

VEGFR-2 (flk-1) is a receptor expressed by proliferative 

endothelial cells, which are mainly involved 

in the tumorangiogenesis. Its ligand VEGF has been 

shown to stimulate endothelial cell mitogenesis and 

cell migration. The aim of this study was to induce 

a more effective immune response by targeting the 

angiogenesis-associated antigen, flk-1 in vivo. Therefore, 

DC were engineered to express soluble flk-1 

using an adenoviral vector (Ad) encoding for flk-1. 

Methods: Ad-flk1 encoding for murine flk-1 was 

constructed using the AdEasy system. As control, 

Ad-LacZ was used. DC were obtained from the bone 

marrow of C3H-mice and adenovirally transduced 

on day 6. To study the effect of the vaccination with 

flk-1-expressing DC in the angiogenesis, C3H-mice 

were immunized by subcutaneous (s.c.) injection 

of 106 flk- or LacZ-expressing DC. One week later, 

500µl of Matrigel enriched with VEGF was injected 

s.c. in the abdomen. Density of newly formed vascular 

vessels was evaluated by CD31-staining. To 

evaluate antitumoral effects, 106 Hepa129-cells 

were injected s.c. into the right flank of C3H-mice 

to induce a tumor. Mice were treated twice with 106 

flk1- or AdLacZ-transduced DC. 

Results: A significant inhibition regarding the formation 

of vascular vessels could be assessed one 

week after the vaccination with flk-1 expressing 

DC (3,5+/-1,6 vascular vessels in the group of mice 

treated with flk-1 DC vs. 31,75+/-8 in the group 

treated with LacZ-DC, p

016 van Nuffel | Therapeutic vaccination 

T cell specificities after vaccination of melanoma patients with 

mRNA loaded DC 

An MT Van Nuffel 1 , Daphné Benteyn 1 , Sofie Wilgenhof 1,2 , Jurgen Corthals 1 , Carlo Heirman 1 , Bart Neyns 2 , 

Kris Thielemans 1 and Aude Bonehill 1 

1 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium 

2 Department of Medical Oncology, Universitair Ziekenhuis Brussel, Brussels, Belgium 

52 

The incidence of melanoma doubles every 10 years. 

The use of full-length tumor associated antigens 

(TAAs) to load dendritic cells (DCs) is proposed 

to improve DC based immunotherapy because 

this should allow vaccination irrespective of the 

patient’s HLA-type and stimulation of a broad spectrum 

of TAA-recognizing T cells. 

Our objective was to investigate the in vivo stimulatory 

potency of DCs loaded by electroporation with 

defined, full-length TAA-encoding mRNA. Defined 

mRNA was chosen as antigen carrier because it can 

be modified to enhance the antigen presentation by 

DCs to both CD8+ and CD4+ T cells. The exact T 

cell specificities of vaccine stimulated T cells were 

analyzed. 

Histologically confirmed AJCC stage IV melanoma 

patients included in a phase I clinical study 

donated a skin biopsy one week after the last of 

four biweekly DC-vaccines. Vaccination occurred 

with an equal mix of TriMix-DC (i.e. DCs matured 

using constitutive active TLR4, CD40L and CD70 

mRNA) co-electroporated with mRNA encoding 

gp100, tyrosinase, Mage-C2 or Mage-A3 fused to 

the HLA-class II targeting sequence of DC-Lamp. 

Skin biopsies were taken 72h after induction of the 

delayed type hypersensitivity (DTH) response by 

intradermal injection of a small amount of vaccine 

DCs. DTH-infiltrating lymphocytes (DIL) were cultured 

during 2,5 weeks in the presence of 100 IU/ 

ml IL-2. The DIL were re-stimulated overnight by 

autologous EBV-B cells electroporated with TAA 

mRNA. Activated T cells were detected by both 

CD137 upregulation in flow cytometry and cytokine 

secretion (IFN-γ and TNF-α) using a cytokine 

bead assay. Once T cells specific for a vaccine 

antigen were found, the recognized epitope was determined 

using autologous EBV-B cells loaded with 

overlapping peptides covering the TAAs. The HLA 

restriction was assessed by allogeneic EBV-B cells 

expressing relevant HLA-types and vaccine TAA. 

This way, we found CD8+ and CD4+ T cells 

specific for the previously unidentified epitopes 

RTCQCSGNF and YPPLHEWVLREG, derived 

from the tumor antigens tyrosinase and Mage-A3, 

respectively. These epitopes were presented respectively 

in the less common HLA-B57 and the 

common HLA-DQB1*05. In vivo stimulation of the 

CD8+ T cells by the TriMix-DCs was confirmed by 

the analysis of blood samples obtained pre- and 

postvaccination. 

In conclusion, vaccination of melanoma patients 

with mRNA loaded DCs leads to in vivo stimulation 

of CD8+ and CD4+ T cells recognizing previously 

unknown epitopes presented in both common and 

less common HLA-types. This finding underscores 

the broadness of the induced T cell response and 

thereby the strength of using mRNA loaded DCs. 

On the other hand, it shows that DC-therapy is no 

longer solely suitable for a selected population with 

a certain HLA-type but can be beneficial for all patients.

017 Schaft | Therapeutic vaccination 

Optimization of the T-cell stimulation capacity of a dendriticcell-based 

melanoma vaccine 

Niels Schaft 1 ,*, Jan Dörrie 1 ,*, Tanja Schunder 1 , Christian Wohn 1 , Verena Wellner 1 , Stefanie 

Baumann 1 and Gerold Schuler 1 

1 Department of Dermatology, University Hospital Erlangen, Erlangen, Germany 

* Contributed equally 

Tumor-antigen-loaded dendritic cells (DC) are pro- 

mising tools as therapeutic vaccine for the treat- 

ment of malignant melanoma. Although immune 

responses, such as induction of tumor-antigen-specific 

T cells, were observed in many studies with 

DC vaccines, these did not correlate with clinical 

responses. Electroporation of monocyte-derived 

DC with RNA, to load them with antigen, or to manipulate 

their function, has become a widely used 

method, also in clinical applications. Our aim in 

this study was to optimize the T-cell stimulation capacity 

of DC. Therefore, we electroporated an optimized 

CD40L-encoding RNA (optCD40L) alone, or 

a combination of (non-optimized) CD40L-, CD70-, 

and constitutively active TLR4 (caTLR4)-encoding 

RNAs (TriMix, as described by K. Thielemans et 

al.) into mature or immature DC, respectively. In 

addition, these DC were electroporated with a RNA 

encoding a tumor antigen (Ag). We chose MelanA 

as a model-Ag, for its well-characterized and highly 

immunogenic HLA-A2-presented peptide EAAGI- 

GILTV. The optCD40L transfection in mature DC 

resulted in enhanced expression of the maturation 

markers CD25, CD40, CCR7, CD70, and OX40L, 

and in secretion of the pro-inflammatory cytokines 

IL-12p70, IL-6, IL-8, and TNF-alpha. Simultaneously, 

the DC retained their capacity to migrate in a 

CCR7-dependent way. The DC‘s capacity to expand 

autologous T cells was analyzed by in vitro stimulation 

and subsequent HLA-A2-MelanA tetramerstaining 

in combination with phenotyping (CCR7/ 

CD45RA) for T-cell function. The capability of the 

DC to induce expansion of T cells in a second and 

third round of stimulation was improved by the 

transfection of optCD40L RNA. The TriMix transfection 

in immature DC resulted in maturation of 

these DC, as shown by up-regulation of maturation 

markers, and a secretion of IL-12p70. However, the 

T-cell expansion capacity was less efficient compared 

to cytokine-cocktail-matured, MelanA-RNAelectroporated 

DC. 

We believe that these studies point the way to improved 

DC that will induce better and longer lasting 

immune responses in the vaccination against 

cancer. 

53

018 Haenssle | Therapeutic vaccination 

Dendritic cells loaded with HSP70-expressing heat-killed melanoma 

cells efficiently activate autologous T cells 

Holger A. Haenssle 1 , Susanne Knudsen 1 , Anke Schardt 2 , Timo Buhl 1 , Lars Boeckmann 1 , 

Michael P. Schön 1 

1 Department of Dermatology and Venereology, Georg August University, Göttingen, Germany 

2 Max Planck Institute for Experimental Medicine, Göttingen, Germany 

54 

There is considerable interest to develop strategies 

that enhance cross-presentation pathways of dendritic 

cells (DCs) to elicit a strong cytotoxic T cell 

activation for cancer vaccination protocols. 

In order to best exploit the enhanced cross-presentation 

of antigens bound to heat shock protein 

70 (HSP70), we analyzed melanoma cell preparations 

for their HSP70 expression. For generation of 

heat-necrotic cell material melanoma cells were 

kept at 42°C for 90 min and then heated to 56°C 

for a final 30 min. Apoptotic Annexin V /Propidium 

iodide- melanoma cells were generated by 

irradiation with 1.0 J/cm2 UV-B. Western blotting 

revealed strong upregulation of HSP70 after heatkilling 

in contrast to UV-B irradiation. The uptake 

of fluorescently labeled heat-killed necrotic vs. 

apoptotic melanoma cells by DCs at various levels 

of maturation was assessed. Immature DCs (iDCs) 

internalized HSP70-expressing necrotic material to 

a much higher extent (61% ± 7%) than apoptotic 

material from UV-B-irradiated cells (48% ± 5%). 

Maturation-inducing cytokines did not affect the 

uptake when added simultaneously with the tumor 

cell preparations. Loading DCs with heat-necrotic 

or apoptotic melanoma cells slightly reduced CD83 

expression while leaving CD208 (DC-LAMP) expression 

unchanged. As determined by IFN- -detecting 

ELISPOT assays, iDCs loaded with HSP70-expressing 

heat-killed melanoma cells activated autologous 

T cells most effectively when used without 

any further maturation, whereas DCs loaded with 

apoptotic material required maturation. In conclusion, 

HSP70-expressing melanoma cells could be 

generated by heat-killing. Loading immature DCs 

with heat-killed melanoma cells resulted in a superior 

priming of autologous T cells in vitro.

019 Buhl | Therapeutic vaccination 

Cryopreservation of high-concentrated peripheral blood mononuclear 

cells by controlled-rate freezer results in higher cell 

yields for dendritic cell-based immunotherapy 

Timo Buhl 1 , Anke Schardt 1 , Tobias Legler 2 , Michael P. Schön 1 , Holger A. Hänßle 1 

1 Department of Dermatology and 

2 Department of Transfusion Medicine of Georg August University, Göttingen, Germany 

Cryopreservation of immature or mature dendritic 

cells (DC) has been described as a suitable method 

to achieve large numbers of DC for immunotherapeutic 

trials against cancer. Recently it was shown 

that cryopreservation of peripheral blood mononuclear 

cells (PBMC) with subsequent differentiation 

into DC is superior compared to cryopreservation 

of immature or mature DC in terms of resulting DC 

quantity and immuno-stimulating capacity. The 

aim of our study was to establish an optimized 

protocol for the cryopreservation of highly-concentrated 

PBMC for DC-based immunotherapy. Cryopreserved 

cell preparations were analyzed regarding 

recovery, viability, phenotype, and functional 

properties. Results were compared to fresh DC 

generated instantly from the same donor. In contrast 

to PBMC frozen by standard isopropyl alcohol 

freezing containers, PBMC cryopreservation in an 

automated controlled-rate freezer (CRF) resulted 

in significantly higher cell yields of immature and 

mature DC after thawing and subsequent differentiation 

to DC. The CRF is connected to liquid nitrogen, 

allowing the freezing program to exactly 

control and adjust the temperature of the freezing 

chamber. Therefore, the crystallization heat at the 

freezing point of the sample can be compensated 

by a sharp temperature decrease in the freezing 

chamber. Immature DC yields and total protein 

content after using CRF were comparable to freshly 

generated DC and exceeded results of standard isopropyl 

alcohol freezing by approximately 50%. DC 

generated after both freezing protocols of PBMC revealed 

no relevant phenotypic or functional differences. 

Our analyses included phenotypic markers, 

allogenic T cell stimulatory assays, viability tests 

and microarray cytokine profiles. The latter showed 

that few cytokines were differently secreted during 

DC maturation depending on the method of cryopreservation. 

The majority of cytokine levels was 

not altered in correlation with the method of cryopreservation. 

In summary, automated controlled 

rate freezing of PBMC represents a much improved 

freezing method capable of increasing DC yields for 

cancer immunotherapy. 

55

020 Nielsen | Therapeutic vaccination 

The CDX-1307 vaccine: regulatory and clinical steps in 

developing a regimen 

Michaela Nielsen, Larry Thomas, Laura Vitale, Li-Zhen He, Venky Ramakrishna, Jennifer 

Green, Thomas Davis 

Celldex Therapeutics, Inc., Needham, Massachusetts 02494, USA 

56 

CDX-1307 is a dendritic cell (DC)-targeting cancer 

vaccine, consisting of a monoclonal antibody 

against the mannose receptor, CD206, fused to the 

β-chain of human chorionic gonadotropin (hCGβ). 

hCGβ is frequently expressed by common epithelial 

cancers, may facilitate cancer progression, 

and correlates with poor prognosis. The antibody 

portion of the vaccine targets it to CD206 on DCs, 

where the molecule is internalized and processed 

into peptides that are presented on the DC surface. 

The CDX-1307 vaccine regimen was developed in 

a step-wise approach. Without a suitable animal 

model available, the initial Phase I study was a 

dose-escalation with intradermal administration of 

CDX-1307 in patients with incurable cancer. Safety 

of the vaccine was established by demonstrating 

tolerability in cohorts of 6 patients at 0.3, 1.0, and 

2.5 mg respectively, given every two weeks for a 

total of 4 doses. 

Pre-clinical data and clinical experience demonstrated 

that inclusion of adjuvants leads to optimal 

immune responses. The cytokine GM-CSF, which 

mobilizes and activates antigen-presenting cells 

(APCs) and up-regulates CD206 on APCs, was thus 

proposed to be added to the study regimen. After a 

toxicology study with CDX-1307 + GM-CSF in hMRtransgenic 

mice showed no noteworthy adverse reactions, 

combination treatment with CDX-1307 and 

GM-CSF was approved and initiated. 

Further studies indicated that immune responses 

are significantly enhanced by toll-like receptor 

(TLR) agonists. Two such agents were proposed 

to be added to the CDX-1307 + GM-CSF regimen 

in additional cohorts: the TLR7/8 antagonist Resiquimod 

(3M) or the TLR3 antagonist Hiltonol® 

(Poly-ICLC). Both cohorts were approved based on 

previous human experience with the antagonists, 

which were proposed to be used in a concentration 

below their established maximum tolerated doses 

(MTD). 

Our pre-clinical studies showed that combining 

TLR agonists enhances the immune response and 

anti-tumor activity of cancer vaccines. Therefore, 

a regimen that combines GM-CSF, Resiquimod and 

Hiltonol® (Poly-ICLC) was proposed. This regimen 

was approved after review of toxicology data from 

a rabbit local reactogenicity study with various 

combinations of the three adjuvants. One particular 

challenge of the combination regimen is the 

local administration of four different agents, one 

of which is topically administered (Resiquimod), 

while the others are injected. Most rabbits that received 

the three adjuvant combination developed 

minor skin reactogenicity, with only one animal 

developing some slowly resolving subcutaneous 

edema. 

The clinical data demonstrated that the combination 

of the TLR agonists increased the frequency and 

severity of local reactions, but they were limited to 

grade 1 and 2 and resolved. In fact no dose limiting 

toxicities were reported, while robust anti-hCG-β 

humoral and T cell responses were reported. Evidence 

of clinical impact was observed in a subset of 

patients, and was noted to be associated with generation 

of anti-hCG-β responses. These data provide 

support for further evaluation of the CDX-1307 

vaccine regimen in patients with hCG-β-expressing 

tumors. A Phase II study in patients with newly diagnosed, 

resectable hCGβ-positive bladder cancer 

involving the full combination regimen has been 

accepted by the FDA and is pending initiation.

L119 Dutoit | Therapeutic vaccination 

Functional immune reactivity to antigens of the novel 

IMA950 vaccine peptide set in glioma patients 

Valérie Dutoit 1 , Norbert Hilf 2 , Steffen Walter 2 , Toni Weinschenk 2 , Philippe Beckhove 3 , Christel 

Herold-Mende 4 , Paul Walker 1 , Harpreet Singh 2 and Pierre-Yves Dietrich 1 

1 Laboratory of tumor immunology, Oncology department, Geneva University Hospital, Geneva, Switzerland 

2 immatics biotechnologies GmbH, Tuebingen, Germany 

3 Translational Immunology Unit, The German Cancer Research Center, Heidelberg, Germany 

4 Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Germany 

We have recently identified a new set of HLA-A2restricted 

glioma antigens isolated by direct elution 

from tumor cells for use in glioma immunotherapy. 

These antigens were selected from a large panel 

of eluted peptides according to overexpression in 

glioma compared to normal brain and other tissues, 

and relevance in tumorigenesis. In vitro peptide 

immunogenicity was investigated in healthy individuals, 

guiding the final vaccine peptide composition. 

Here, in order to validate the use of the novel peptide 

set for multipeptide vaccination, T cell reactivity 

and absence of tolerance was investigated in patients 

with malignant glioma. In addition, peptidespecific 

T cell function was assessed in vitro. All 

patients responded to several glioma peptides, suggesting 

that this multipeptide vaccine may benefit 

the majority of HLA-A2+ patients. Moreover, 

glioma peptide-specific CD8+ T cell clones generated 

from the blood of glioma patients were able to 

efficiently kill antigen-expressing tumor cells, but 

not K562 cells. Finally, CD8+ T cells specific for a 

brevican epitope were detected at the tumor site in 

one patient, showing that, for this antigen, spontaneously 

elicited specific T cells are retained at the 

tumor site. Altogether, our results validate the use 

of the ten novel glioma antigens in multipeptide 

vaccination and adoptive cell therapies for patients 

with glioma. 

57

L120 Hilf | Therapeutic vaccination 

IMA950 - a novel multi-peptide cancer vaccine for treatment of 

glioblastoma 

Norbert Hilf 1 , Oliver Schoor 1 , Valérie Dutoit 2 , Toni Weinschenk 1 , Steffen Walter 1 , Peter Lewandrowski 

1 , Sylvia Flohr 1 , Claudia Trautwein 1 , Cécile Gouttefangeas 3 , Stefan Stevanovic 3 , 

Hans-Georg Rammensee 3 , Philipp Beckhove 4 , Christel Herold-Mende 5 , Pierre-Yves Dietrich 2 , 

Harpreet Singh 1 

1 

immatics biotechnologies GmbH, Tuebingen, Germany 

2 

Laboratory of Tumor Immunology, Oncology, Geneva University Hospital, Geneva, Switzerland immatics bio 

technologies GmbH, Tuebingen, Germany 

3 

Department of Immunology, Institute for Cell Biology, University of Tuebingen, Germany 

4 Translational Immunology Group, German Cancer Research Center, Heidelberg, Germany 

5 Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Germany 

58 

Despite promising results of various therapeutic 

approaches currently under investigation, including 

active immunotherapy, glioblastoma (GBM) 

remains one of the most fatal tumor types. IMA950 

is a novel peptide vaccine aiming at the induction 

of a broad T-cell response against a variety of 

tumor-relevant antigens expressed by GBM cells. 

IMA950 consists of 11 synthetic tumor-associated 

peptides (TUMAPs). Nine of the TUMAPs bind to 

the human leukocyte antigen (HLA) class I allele 

A*02 expressed by ~ 45% of the Caucasian population 

and two TUMAPs bind to various HLA class 

II (DR) alleles expressed by the majority of patients. 

Therefore, cytotoxic T-lymphocyte (CTL) as well as 

T helper responses are expected to arise from vaccination 

with IMA950. 

IMA950 was rationally designed employing the 

XPRESIDENT approach: More than 3,000 different 

HLA-A*02 peptides were identified from more than 

30 primary GBM specimens by mass spectrometry. 

TUMAPs were ranked according to a variety of 

criteria, including degree and frequency of mRNA 

over-expression of the source antigens in GBM 

compared with normal tissues, known relevance of 

the source antigens in oncogenic processes, in vitro 

immunogenicity in healthy donors, and feasibility 

of pharmaceutical development. 

Analysis of the „over-presentation“ on GBM samples 

directly at the peptide level confirmed the relevance 

of the finally selected TUMAPs using a novel 

approach allowing label-free HLA-peptide quanti- 

tation directly by mass spectrometry. 

In conclusion, the strong GBM association of IMA950 

TUMAPs indicated by overpresentation together 

with pre-clinical immunogenicity results suggest 

that this multipeptide vaccine may be a benefit to 

the majority of HLA-A*02-positive patients. Clinical 

development is planned to start within 2010 with 

several phase I trials investigating the safety and 

immunogenicity of the vaccine in different patient 

populations and using various immunomodulatory 

regimens in order to explore the most promising 

strategy for further development.

L121 Pawlowski | Therapeutic vaccination 

Synergy of the immunomodulators GM-CSF and Imiquimod for 

peptide-based therapeutic vaccines 

Nina Pawlowski, Norbert Hilf, Sylvia Flohr, Toni Weinschenk, Oliver Schoor, Harpreet Singh 

immatics biotechnologies GmbH, Tuebingen, Germany 

All FDA- or EMA-approved drugs currently used 

as immunomodulators with therapeutic peptidebased 

cancer vaccines are of only moderate efficacy 

in terms of direct activation of antigen-presenting 

cells which is a key prerequisite for efficient priming 

of cytotoxic T cells (CTLs). Because the reliable 

induction of tumor-directed immune responses is 

crucial for the efficacy of therapeutic cancer vaccines, 

novel immunomodulator regimens have to be 

introduced into clinical testing to achieve a better 

clinical outcome. Therefore, we analyzed safety 

and efficacy of several approved immunomodulatory 

substances and combinations thereof in a dedicated 

pre-clinical screening program. 

The combination of subcutaneously (s.c.) applied 

granulocyte-macrophage colonystimulating factor 

(GM-CSF) with topical application of the TLR7 

ligand imiquimod resulted in synergistic enhancement 

of peptide-vaccine induced CTL responses 

without any signs of toxicity or cumulated adverse 

effects. 

GM-CSF injected s.c. combined with imiquimod 

cream applied topically to the skin of C57BL/6 

mice at the vaccination site significantly increased 

the number of vaccinespecific CTLs induced by 

s.c. vaccinations with model MHC class I-binding 

peptides compared with either substance alone. 

The induced immune response was comparable to 

the effect of CpG deoxyoligonucleotides or poly-IC, 

two potent immunomodulators currently not FDA-/ 

EMA-approved and therefore not widely available 

for clinical trials in humans. However, the timing 

of imiquimod application in relation to the vaccination 

schedule is crucial, as the synergistic effect 

of imiquimod and GM-CSF was only observed, if 

imiquimod was applied at the day of vaccination 

and/or 24 h later, but not if imiquimod was applied 

the day before peptide vaccination. 

In total, 45 mice were immunized with the combination 

of GM-CSF and imiquimod without any 

signs of local or systemic toxicities. Further results 

from in vitro activation assays with human PBMCs 

and isolated antigen-presenting cells and the distinct 

molecular mechanisms of immunostimulation 

employed by both substances provide further 

support for the anticipated safety of this regimen 

in humans. 

GM-CSF and imiquimod is therefore considered as 

a promising and safe alternative immunomodulator 

regimen for upcoming immunotherapeutic approaches 

and is currently explored by the authors 

in a clinical trial in colorectal cancer patients. 

59

L122 van de Ven | Therapeutic vaccination 

Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives 

induces rapid dendritic cell differentiation 

Rieneke van de Ven 1,2 , Anneke Reurs 1,3 , Pepijn Wijnands 1,3 , Sandra van Wetering 3 , Ada 

Kruisbeek 3 , Erik Hooijberg 1 , George Scheffer 1 , Rik Scheper 1,3 and Tanja de Gruijl 2 

1 

Department of Pathology, VU University medical center, Amsterdam, The Netherlands 

2 

Medical Oncology Laboratory, VU University medical center, Amsterdam, The Netherlands 

3 DC Prime B.V., Amsterdam, The Netherlands 

60 

Appropriate activation of Dendritic Cells (DC) is 

essential for successful active vaccination and induction 

of cell-mediated immunity. The scarcity of 

precursor cells, as well as long culture methods, 

has hampered wide-scale application of DC vaccines 

derived from CD34+ precursors, despite their 

suggested superior efficacy over the more commonly 

applied monocyte-derived DC (MoDC). Here, employing 

the CD34+/CD14+ Acute Myeloid Leukemia-derived 

human DC progenitor cell line MUTZ3, 

we show that cytostatic anthraquinone-derivatives 

(i.e. the anthracenedione mitoxantrone and the 

related anthracyclin doxorubicin) induce rapid differentiation 

of CD34+ DC precursors into functional 

antigen presenting cells (APC) in a three-day 

protocol. The drugs were found to act specifically 

on CD34+, and not on CD14+ DC precursors. Importantly, 

these observations were confirmed for 

primary CD34+ and CD14+ DC precursors from 

peripheral blood. Mitoxantrone-generated DC were 

fully differentiated within three days and after an 

additional twenty-four hours of maturation, were 

as capable as control 9-day differentiated and 

matured DC to migrate towards the lymph nodehoming 

chemokines CCL19 and CCL21, to induce 

primary allogeneic T cell proliferation, and to prime 

functional MART1-specific CD8+ T lymphocytes. 

Anthraquinone-derivatives like mitoxantrone may 

thus be employed either as a differentiation-inducing 

agent for rapid in vitro generation of DC, or 

might even be exploited to mature DC precursors 

in vivo in support of DC-based therapies.

Immune monitoring 

61

021 Santegoets | Immune monitoring 

Immune and clinical response monitoring in patients with metastatic 

hormone-refractory prostate cancer receiving combined 

Prostate GVAX and anti-CTLA4 immunotherapy 

Saskia J.A.M. Santegoets 1 , A.J.M. van den Eertwegh 1 , A.G.M. Stam 2 , R.J. Scheper 2 , S.M. 

Lougheed 1 , P.E.T. Scholten 2 , B.M.E. von Blomberg 2 , H. Gall 1 , K. Jooss 3 , N. Sacks 3 , T. Harding 

3 , K. Hege 3 , I. Lowy 4 , W.R. Gerritsen 1 , and T.D. de Gruijl 1 

1 Department of Medical Oncology, VU University medical center, Amsterdam, The Netherlands 

2 Department of Pathology, VU University medical center, Amsterdam, The Netherlands 

3 Cell Genesys Inc., South San Francisco, CA, USA 

4 Medarex In.c, Bloomsbury, NJ, USA 

62 

The effects of a GM-CSF-secreting allogeneic pro- 

state cancer vaccine (Prostate GVAX) and the anti- 

CTLA4 antibody Ipilimumab were investigated in 

a Phase I dose escalation/expansion trial of patients 

with metastatic, hormone-refractory prostate 

cancer (mHRPC). Patients received a 500 million 

cell GVAX priming dose on day 1 followed by 12 biweekly 

intradermal administrations of 300 million 

cells, while Ipilimumab was administered every 4 

wks from day 1 for a total of 6 times. Initially, 12 patients 

were enrolled in cohorts of 3 and each cohort 

was assigned an escalating dose of Ipilimumab at 

0.3, 1, 3 or 5 mg/kg. 16 additional patients were 

enrolled in the expansion cohort of 3 mg/kg Ipilimumab. 

PSA declines of more than 50% (Partial 

Response, PR) were observed in 5 of 22 patients 

in the 3-5 mg/kg Ipilimumab dose cohorts, and 

were associated with Autoimmune Breakthrough 

Events (ABE), including Grade 2 or 3 hypophysitis 

and Grade 3 alveolitis. PSA stabilizations (Stable 

Disease, SD) were observed in 1/3 patients in lower 

(0.3 and 1 mg/kg), and in 7 of 22 in the higher (3-5 

mg/kg) dose levels. Moreover, regressing bone and 

lymph node metastases were observed in 2/5 PR 

patients. 

Immune response monitoring was performed to 

identify changes that might predict or correlate 

with clinical efficacy. Significant increases in frequencies 

of activated and effector CD4+ and CD8+ 

T cells were observed by HLA-DR and ICOS expression 

upon administration of high (3 and 5 mg/kg) 

but not of low (0.3 and 1 mg/kg) Ipilimumab doses; 

a relation to clinical behaviour was only observed 

for HLA-DR with earlier and more pronounced increases 

in patients with PR or SD. As an indication 

of tumor-specific responsiveness HLA-Tetramer 

(Tm) and seroreactivity to NY-ESO and PSMA were 

tested. For NY-ESO, therapy-induced increased seroreactivity 

was observed in 6/28 patients, which in 

2 patients was confirmed to coincide with increased 

Tm reactivity. PSMA seroconversions were observed 

in a total of 12/28 patients (and, of note, in 

4/5 PR), but no Tm positivity at any time was found 

in a total of 15 patients tested. PSMA seroconversion 

in the higher dose levels was associated with 

increased overall survival (P=0.062). In addition, 

significantly increased ex vivo levels of IL-4 in both 

CD4+ and CD8+ T cells were observed in patients 

with PR or SD (P

022 Pfirschke | Immune monitoring 

The repertoire of melanoma antigens recognized by T cell responses 

in malignant melanoma patients 

Christina Pfirschke 1 , Christoffer Gebhardt 2 , Alexander Enk 2 , Philipp Beckhove 1 

1 Translational Immunology Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany 

2 Department of Dermatology, University of Heidelberg, Heidelberg, Germany 

Malignant melanoma, the leading cause of skin cancer 

related death, is worldwide constantly increasing. 

During the recent years the development of novel, improved 

immunotherapeutic strategies have therefore 

become highly important. Pre-existing memory CD4 

and CD8 T cell responses are present in patients with 

many tumor entities and provide a repertoire of functional 

and potentially productive immune cells. Boosting 

patients tailored of such T cells with selected 

long peptides resembles a new strategy in melanoma 

immunotherapy. 

We here evaluated the presence and specificity of 

the repertoire of preexisting tumor-antigen specific 

T cells in 38 melanoma patients analyzed 1-4 month 

after primary tumor rejection. We have determined by 

an ex vivo short-term IFNγ ELISPOT assay the reactivity 

of spontaneously induced T cells in the blood of 

malignant melanoma patients against a broad variety 

of melanoma tumor-associated antigens (TAAs). To 

this end, long peptides (50aa) that cover several MHC 

class I- and II-restricted epitopes were used as test antigens 

while human IgG was investigated as negative 

control antigen. As antigen presenting cells, we used 

dendritic cells generated in vitro from monocytes 

and pulsed with different synthetic 50aa peptides 

derived from immunodominant regions of described 

melanoma TAAs like MelanA/MART-1, tyrosinase, 

gp100/pmel17, NY-Eso-1, p53, MDM2, NA17-A, TRP2, 

MAGEA1, MAGE-C2, GAGE-1, MIF and RAB38/NY- 

Mel-1. In order to analyze the role of regulatory T cells 

(Tregs) in suppressing TAA-specific T cell responses, 

Tregs were depleted from a part of the purified CD3+ 

T cell fractions using αCD25-magnetic beads. 

More than 80% of the analyzed T cell samples of melanoma 

patients reacted against at least one tested 

peptide. T cells of HLA-A2 positive and HLA-A2 negative 

patients recognized the polypeptides to a similar 

extent. Additionally, more than 70% of the responding 

T cell samples reacted in a polyvalent manner 

to 4 or more tested peptides at the same time. Some 

peptides, such as MelanA/MART-1 (56%), tyrosinase 

(55%) and NA17-A (50%), as well as MAGE-A1 

(44%), MDM2 (42%), GAGE-1 (41%), RAB38/NY-Mel 

(40%), TRP2 (35%) and p53 (35%) were preferentially 

recognized whereas other melanoma TAAs, such 

as MIF (30%), MAGE-C2 (29%), gp100/pmel17 (24%) 

and NY-Eso-1 (18%) elicited lower spontaneous T cell 

responses. Interestingly, the depletion of Tregs from 

the investigated T cell fractions did not result in an 

increase of T cell response compared with the non 

Treg depleted T cell samples. 

Summarized, our data demonstrate that in a majority 

of malignant melanoma patients in the peripheral 

blood a polyvalent repertoire of tumor-reactive 

memory T cells exists. In contrast to other kinds of 

tumors investigated, including colon cancer, for malignant 

melanoma we did not detect a major role of 

Tregs for control of tumor reactive memory T cells. 

Moreover, some analyzed antigens appear to be dominant 

targets of spontaneous effector T cell responses 

providing a potential new criterion for selection of 

vaccine antigens for melanoma immunotherapy. 

63

023 Veron | Immune monitoring 

Selection, validation and evaluation of a 30-Plex Luminex kit 

for immunomonitoring of a phase I clinical trial 

Laure Veron 1 , Delphine Olivier 1 , Bérangère Marie-Bastien 2 , Benoît Grellier 2 , Edwige Bonfils 2 , 

Bastien Calmels 2 , Philippe Ancian 2 , Jean Yves Bonnefoy 2 , Geneviève Inchauspé 1 , Christine Bain 1 

1 

Transgene SA, Centre of Infectiology, LYON, FRANCE 

2 

Transgene SA, Parc d‘innovation, ILLKIRCH GRAFFENSTADEN, FRANCE 

64 

Multiparametric technologies, such as the Luminex 

Bead Array technology, deserve to be evaluated 

for their potency to monitor immune responses 

induced by immunotherapeutic products. 

In a preliminary step, the Linco human 30-Plex kit 

was selected among three commercial kits based on 

technical (sample volume, time consumption and 

internal quality controls) and performance criteria 

(sensitivity) for further validation. The validation 

study was designed to evaluate the performance of 

the Linco 30-Plex kit for the measurement of analyte 

concentrations in antigen-stimulated PBMC culture 

supernatants. We also evaluated the impact of the 

in vitro stimulation step on the precision of this 

read-out for the determination of antigen-specific 

immune responses. Our data revealed that 1/ Accuracy 

and precision of the Linco 30-Plex assay were 

within acceptable limits for immunoassays, except 

for MCP-1 (Monocyte Chemotactic Protein-1) ; 2/ 

Depending on the analyte, a matrix effect of the 

complete medium was observed, especially at the 

1/10 dilution ; 3/ Linearity of dilutions was observed, 

especially for analytes that displayed a good 

recovery at low concentrations and 4/ The in vitro 

stimulation step was the main source of variability 

and should be taken into account for the analysis 

of samples from clinical trials. 

This Linco 30-plex kit was then evaluated within 

the framework of a phase I clinical trial in 15 HCV 

(Hepatitis C virus)-chronically infected patients 

who received escalating doses of TG4040, a MVA 

(Modified vaccine Ankara) -based therapeutic 

vaccine encoding 3 HCV non structural proteins. 

Different cytokine profiles were observed in the 15 

patients, especially a contrasting profile between 

the best TG4040 responder, showing a decrease 

of more than 0.5 log of the viral load (VL), and a 

TG4040 non responder patient (ie who displayed 

less than 0.5 log VL decrease), with respectively a 

broad panel of Th1/pro-inflammatory biased cytokines 

for the former and a limited Th2 biased cytokines 

for the latter. 

Overall, Luminex technology can be used to follow 

immune responses after therapeutic vaccination. 

Different parameters however need to be carefully 

taken into account for a correct interpretation of 

the data, such as matrix effect, and particularly the 

variabilitity of the in vitro stimulation step

024 Zupke | Immune monitoring 

Nanoparticles for efficient labeling of dendritic cells to be used 

in cellular immunotherapy 

Oliver Zupke 1 , Eva Distler 1 , Daniela Baumann 2 , Dennis Strand 3 , Ralf Georg Meyer 1 , Katharina 

Landfester 2 , Wolfgang Herr 1 , Volker Mailänder 1 , 2 * 

1 Dept. of Medicine III, Hematology & Oncology, University Medical Center, Mainz, Germany 

2 Max Planck Institute (MPI) for Polymer Research, Mainz, Germany 

3 Dept. of Medicine I, University Medical Center, Mainz, Germany 

The goal of this project is to provide a platform for 

the evaluation of cellular immune therapies by monitoring 

the fate of administered cells in vivo over 

time. Therefore, the effective labeling of immune 

cells is essential. Here, immunostimulatory cells 

like dendritic cells (DCs) and immune effector cells 

like CD8 and CD4 T lymphocytes are of high interest. 

In the first step, a broad panel of polymeric 

nanoparticles (NPs) with different surface functionalities 

(synthesized at the MPI for Polymer Research, 

Mainz) was screened for their suitability as 

labeling agents for human monocyte-derived DCs. 

These NPs contain a fluorescent dye (PMI, perylenmonoimide), 

allowing the quantitation of cellular 

uptake by flow cytometry. We observed that aminofunctionalized 

NPs are most efficient for DC labeling. 

In subsequent experiments, the concentration 

as well as the incubation time of NPs was optimized, 

regarding a maximal cellular uptake and on 

the same time minimal toxic effects, as determined 

by 7-AAD stainings. To confirm intracellular localization 

and to exclude mere attachment of NPs at the 

cell surface, NP-loaded immature and mature DCs 

were also analyzed by confocal laser scanning microscopy. 

Kinetic studies demonstrated that labeled 

mature DCs still contained high amounts of NPs 

for a culture period of up to 8 days, what will be of 

importance for later in vivo trafficking studies. In 

further experiments, the influence of NP loading 

on phenotype and cellular function of DCs was 

investigated. Flow cytometry analysis of different 

maturation, homing, and costimulatory molecules 

demonstrated that NP labeling by this protocol 

had no significant influence on DC phenotype. A 

possibly negative influence of NP loading on the 

immunostimulatory functions of DCs could be excluded 

on the basis of allo-MLR (mixed lymphocyte 

reaction) and cytokine ELISPOT assays. CD4 and 

CD8 T cells stimulated in MLRs with NP-labeled, 

allogeneic DCs demonstrated comparably strong 

proliferation in 3H-Thymidin assays compared to 

stimulation with unlabeled DCs. In addition, IFN-γ 

spot production of alloreactive T cells (stimulated 

with HLA-mismatched DCs) as well as virus-specific 

memory T cells (stimulated with CMV peptideloaded 

DCs) was not impaired by NP labeling of the 

antigen-presenting cells. Furthermore the ability 

for processing viral protein antigens (Influenza A) 

and their presentation via MHC II molecules to CD4 

T cells was not affected by NPs. 

In summary, we developed a protocol using amino-functionalized, 

fluorochrome-containing polymeric 

nanoparticles that allows the efficient labeling 

of human dendritic cells, without significant 

negative impact on cellular phenotype or function 

of DCs as antigen-presenting cells. Currently, the 

protocol is adapted to human leukemia-, virus-, and 

alloreactive T lymphocytes. Furthermore, experiments 

with superparamagnetic iron oxide NPs are 

ongoing, with respect to intended in vivo trafficking 

studies of transferred immune cells in humanized 

mice by magnetic resonance imaging. 

65

025 Savelyeva | Immune monitoring 

Vaccination and immune memory: modelling the bystander 

activation of CD4+ memory T cells in mice 

Natalia Savelyeva, Amy Suchacki, Stephen Thirdborough, Freda K. Stevenson & Gianfranco Di Genova 

Cancer Sciences Division, Southampton University Hospitals, Tremona RD, Southampton, UK 

66 

CD4+ T-helper (Th) cells have a key role in pro- 

moting both B and T cell-mediated immunity. 

Knowledge of the mechanisms involved in the 

maintenance of vaccine-induced or natural CD4 

T cell immunity is crucial for the development of 

successful vaccination strategies against infectious 

diseases and cancer. We previously demonstrated 

that in healthy volunteers vaccination with tetanus 

toxoid (TT) induced bystander activation of Th 

memory cells of unrelated specificities; however 

antibody responses by B cells remained vaccinespecific 

(Di Genova G et al. Blood 2006). We hypothesized 

that this bystander activation could 

represent a mechanism which contributes to their 

long-term maintenance. We have now modelled 

this phenomenon in mice, confirming the results 

observed in humans and paving the way to a better 

understanding of the immunological mechanisms 

responsible for it. Briefly, in a first model two populations 

of CD4+ memory T cells specific for two 

distinct antigens were generated simultaneously in 

C57BL/6 mice. When mice were re-challenged with 

one of the two antigens, this caused not only the 

expected recall immune response but also expansion 

in the number of cytokine-producing cells specific 

for the unrelated antigen. In a second model, 

CD4+ TCR transgenic T cells (OT-II), either naïve or 

previously activated in vitro with cognate antigen, 

were CFSE-labelled and transferred into wild type 

recipient mice which were immune to TT. Recipient 

mice were then challenged with TT antigen and 

susceptibility of OT-II cells to bystander activation 

and proliferation was tested. Antigen-activated but 

not naïve cells were responsive and underwent bystander 

proliferation. This was proportional to the 

strength of the TT-specific memory T-cell response 

and appeared to be mediated by the common 

gamma chain cytokines IL-2 and IL-7. These data 

support the principle that CD4+ T cells at various 

stages of differentiation, from activated to memory 

cells, can respond to cytokines generated during 

parallel immune responses, such as those induced 

in human subjects by vaccines or natural exposure 

to environmental antigens. This might represent a 

mechanism which could contribute to their antigenindependent 

maintenance. The findings have high 

relevance for the measurement of T-cell responses 

in patients who are being vaccinated.

026 Tanriverdi-Akhisaroglu | Immune monitoring 

Spontaneous tumour antigen specific T cell responses occur 

frequently in patients with Carcinoma of Unknown 

Primary and are predominantly directed against EGFR, 

telomerase, MAGE-3 and P53 

Serpil Tanriverdi-Akhisaroglu 1 , Harald Löffler 2 , Yingzi Ge 1 , Kim Pietsch 1 , Andreas Bonertz 1 , 

Alwin Krämer 2 , Philipp Beckhove 1 

1 Division of Translational Immunology 

2 Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, Heidelberg 

Introduction: Carcinoma of unknown primary 

origin (CUP) represents a so far largely uncharacterized 

tumour entity. Since treatment options 

and conventional therapy regimens only provide 

very limited success (median overall survival of 

3-11 months), alternative treatment options need 

to be assessed. Tumour immunotherapy has recently 

proven clinical efficiency and might provide an 

option for the treatment of CUP. 

Purpose: As a first basis for the development of 

tumour immunotherapy for CUP, we here evaluated, 

to what extent CUP patients develop spontaneous 

tumour antigen specific T cell responses. In 

addition, we characterized the antigen specificities 

of tumour-reactive T cells of CUP patients in comparison 

to patients with colorectal and pancreatic 

carcinoma, as well as breast cancer. 

Patients and Methods: We analyzed T cell responses 

from peripheral blood of 18 CUP patients. The presence 

of tumour antigen specific T cells was assessed 

by short term ex vivo IFN-γ ELISPOT assay. 

As test antigens, we used a broad panel of long 

synthetic peptides derived from immunogenic sequences 

of the following defined tumour antigens: 

MUC-11-100, MUC-1(1137-157)5, EGFR(479-528), 

p53(118-167), Her-2/neu(351-384), CEA(569-618), 

Mage3(271-314-157), NYESO1, survivin(93-142), 

telomerase(958-1007), heparanase(1-50, 163-212). 

As negative control antigen, we used human IgG. 

Test or control antigens were loaded on autologous 

DCs at a concentration of 200µg/ml and pulsed DCs 

were incubated on coated IFN-γ ELISPOT plates for 

40 hours. Spots were measured automatically by an 

ELISPOT reader and T cell reactivity was assumed 

if spot numbers in triplicate test wells were significantly 

increased compared to triplicate control 

wells. Test results were compared to results of peripheral 

blood-derived T cell samples from patients 

with pancreatic- , colorectal- or breast cancer or 

from healthy volunteers. 

Results: Interestingly, we observed in all (100%) 

patients pre-existing, tumour antigenspecific T 

cells with an average number of 3.4 antigens simultaneously 

recognized. This compares favourably to 

spontaneous T cell responses detectable in patients 

with pancreatic cancer (85%), breast cancer (61%), 

colorectal cancer (60%) and strongly exceeded 

the minor rates of TA-specific T cell responses in 

healthy individuals. CUP patients developed predominant 

T cell responses against EGFR (50%), 

telomerase (47%), Mage3 (40%) and p53 (39%), 

whereas responses against the same peptides were 

less frequent in CRC patients (12%,0%,20% and 

21%, respectively), pancreatic carcinoma patients 

(23%,20%,23% and 26%) and also in breast cancer 

patients (47 %,21%, 29% and 41%). Particularly, T 

cell responses against telomerase were significantly 

increased in CUP patients as compared to the 

other defined tumour entities (p=0.03). 

Conclusion: CUP patients develop polyvalent T cell 

responses against a distinct repertoire of tumour 

antigens, particularly against EGFR, telomerase, 

MAGE-3 and P53, suggesting that these antigens 

may be candidates for antigen specific immunotherapy 

against CUP. 

67

027 Low | Immune monitoring 

Culturing PBMC in the presence of antigen results in a measurable 

expansion of memory T cells 

Lindsey Low, Ann Mander, Kathy Tier, Christian Ottensmeier 

Cancer Research UK Clinical Centre, Cancer Sciences Division, University of Southampton 

68 

Following an in vivo encounter with an antigen, 

there is a primary expansion of effector CD8 T cells, 

followed by a contraction in which 90 – 95% of 

effector cells die. A small population of memory 

cells remain which can persist long term, and will 

expand to produce effector cells if the antigen is encountered 

again. Culturing of PBMC in the presence 

of antigen is believed to stimulate this expansion, 

producing antigen-specific effector cells which can 

be detected using standard immunological assays. 

The protocol was developed using cryporeserved 

PBMC from HLA A2 positive heathy donors, cultured 

in the presence of IL-2 with the viral peptide 

antigens, cytomegalovirus (CMV), CMV pp65 493- 

499, (NLVPMVAVT); influenza A, Matrix 1 58-66, 

(GILCFVFTL); Epstein Barr virus (EBV), BMLFI 

259-217 (GLCTLVAML), and measles, non-structural 

C protein 84-92 (KLWESPQEI). Variables optimized 

included cell concentration, peptide concentration, 

well size, time spent in culture, feeding 

regimen, medium and use of serum. The resulting 

protocol was shown to give an optimum yield of antigen-specific 

effector cells at the end of the culture 

process. Cultured cells were used in immunological 

assays to determine phenotype and fuctionality. 

FACS analysis of pre- and post-culture cells has been 

used to show a measureable post-culture increase 

in the population of effector cells. Use of cultured 

cells in standard IFNγ ELISpots shows a noticeable 

amplification of antigen-specific response rates 

when compared to non-cultured cells for both the 

test viral antigens and for low frequency tumour 

associated antigens in clinical trial usage. 

This indirect measurement of the memory cell population 

provides a sensitive and reliable method to 

monitor the long term effectiveness of immunotherapeutic 

vaccination.

028 Laske | Immune monitoring 

T-cell immunomonitoring in patients with renal cell carcinoma 

after multi-peptide vaccination 

Karoline Laske 1 , Annemarie Dröge 1 , Susan Feyerabend 2 , Jörg Hennenlotter 2 , Jens Bedke 2 , Patricia 

Hrstic 1 , Stefan Stevanovic 1 , Arnulf Stenzl 2 , Cécile Gouttefangeas 1 , Hans-Georg Rammensee 1 

1 Institute for Cell Biology, Department of Immunology, Eberhard Karls University Tuebingen, 

Auf der Morgenstelle 15, 72076 Tuebingen, Germany 

2 Department of Urology, Eberhard Karls University Tuebingen, Hoppe-Seyler-Str. 3, 72076 Tuebingen, 

Germany 

In this clinical trial, patients with advanced or 

metastatic renal cell carcinoma after complete resection 

receive a multi-peptide cocktail containing 

HLA-class I and II binding peptides. The vaccine is 

applied either intradermally with GM-CSF as adjuvant 

or subcutaneously in Montanide ISA52 at 

18 vaccination time points over 12 months or until 

progression. Computer tomography imaging is performed 

to evaluate progression-free survival every 

3 months. HLA-A*02 positive or negative patients 

receive an individual peptide cocktail containing 

between 3 and 12 HLA-class I binding peptides. 

These peptides are known epitopes or ligands 

derived from selected tumor-associated antigens. 

Furthermore, the peptide cocktail for HLA-A*02 positive 

patients contains one peptide (from the influenza 

virus) as recall control and one peptide (from 

HBV) as priming control for the vaccination. 

Additionally, 3 to 4 HLA-class II binding peptides 

are included for CD4+ T-cell stimulation. Until 

now, 17/40 patients have been recruited and T-cell 

monitoring was performed for 9 patients. Blood 

was collected at each vaccination time point, serum 

and peripheral blood mononuclear cells (PBMCs) 

were isolated and frozen. For T-cell monitoring, 

PBMCs from different time points were thawed and 

stimulated with the individual peptide cocktail for 

12 days. Detection of specific T-cells was performed 

by IFN-gamma Elispot, tetramer staining (for 

HLA-A*02 positive patients) and intracellular cytokine 

staining. So far, all 7 monitored HLA-A*02 

positive patients developed a strong CD4+ T-cell 

response against 2 of the 4 HLAclass II binding 

epitopes (derived from carbonic anhydrase 9 and 

cyclin-D1 protein) which were detected by Elispot 

and further characterized by intracellular cytokine 

staining. We could detect a CD8+ T-cell response 

in 4 of 7 HLA-A*02 positive patients. The most frequently 

recognized peptides are from the proteins 

cyclin-D1 and guanylate cyclase 1. 

69

029 Brix | Immune monitoring 

Culturing PBMC in the presence of antigen results in a measurable 

expansion of memory T cells 

Liselotte Brix, Tina Jakobsen, and Henrik Pedersen 

Immudex, Copenhagen, Denmark 

70 

Sensitive and reliable monitoring of cellular immune 

responses is becoming increasingly important in 

cancer vaccine and immunotherapeutic development. 

Flow analysis using conventional fluorescent 

MHC multimers like Tetramers and Pentamers has 

made a great impact in this field enabling visualization, 

enumeration and phenotypic characterization 

of antigen-specific T-cells. However, shortcomings 

such as difficulties in detecting low-affinity interactions, 

e.g. tumor-specific T-cell responses, low 

reproducibility and reagent stability are still prominent 

problems. 

MHC Dextramer reagents are a new generation of 

fluorescent MHC multimers that solve some of those 

problems. Thus, we have used MHC Dextramers to 

detect low-affinity T cells (i.e. T cells carrying TCR 

with low affinity for the MHC-peptide complex). 

In contrast to Tetramers, the Dextramers allowed 

enumeration of T cells carrying TCRs with a Kd of 

> 250 µM. To our knowledge this is the first time 

that antigen-specific T cells with such low-affinity 

TCR have been detected with a fluorescent MHC 

multimer reagent. 

When staining T cells of higher affinity-TCR’s (5 

µM – 100 µM), Dextramers had much higher resolution 

than the corresponding Tetramers. Dextramers 

thus provide a more reliable means for identification 

of true positive antigen-specific T cells. 

The increased sensitivity and resolution of the 

Dextramers probably reflects the higher number of 

MHC-peptide complexes and fluorochromes, since 

the high number of MHC-peptide complexes increases 

their avidity for the specific T-cells, and the 

higher number of fluorochromes enhances staining 

intensity. As a result, Dextramers have higher sensitivity, 

higher staining intensity and higher signalto-noise 

ratio. 

The dextran backbone of the MHC Dextramer 

stabilizes the attached proteins. When analyzing 

the stability of the MHC Dextramers, a given 

blood sample could be stained reproducibly well 

using the same batch of a MHC Dextramer for 

more than 4 month. Likewise, different batches 

of MHC Dextramers provided similar frequencies 

of antigen-specific T cells with comparable staining 

intensities when staining the same sample. 

We also investigated the impact of freezing and 

thawing MHC Dextramers. Surprisingly, the MHC 

Dextramer reagent could be subjected to repeated 

freeze-thaw cycles without affecting the staining 

intensity, resolution or the number of positive cells 

detected. 

In summary, MHC Dextramers are sensitive, highly 

stable reagents that are suitable to minimize assayto-assay 

variation, and that are particularly useful 

reagents in the immune monitoring of cancer immunotherapy.

030 Brix | Immune monitoring 

MHC Dextramers: improved detection of antigen-specific T-cells 

in fluid samples and in situ 

Liselotte Brix, Tina Jakobsen, and Henrik Pedersen 

Immudex, Copenhagen, Denmark 

Sensitive and reliable monitoring of cellular immune 

responses is becoming increasingly important in 

vaccine and immunotherapeutic development. 

Flow analysis using fluorescent MHC multimers 

has made a great impact in this field enabling visualization, 

enumeration and phenotypic characterization 

of antigen specific T-cells. Here we discuss 

how MHC Dextramers, a new generation of MHC 

multimer reagents, can be used for advanced detection 

of antigen specific T-cells in comprehensive 

applications. 

MHC Dextramer reagents carry a higher number 

of MHC-molecules and more fluorochromes than 

conventional MHC multimers. This increases their 

avidity for the specific T-cell and enhances their 

staining intensity, thereby increasing resolution and 

the signal-to-noise ratio. Furthermore the dextran 

backbone of the MHC Dextramer stabilizes the attached 

MHC-molecules and fluorochrome proteins. 

This allows specialized applications such as in situ 

staining (e.g. detection of antigen-specific T-cells 

in frozen tissue sections), detection of very rare 

events, combined staining with high numbers of 

MHC Dextramer reagents in same tube and MHC 

Dextramer staining combined with extracellular 

and intracellular staining for e.g. surface proteins 

and cytokines. 

Such specialized applications are useful in improving 

immune monitoring of cancer immunotherapeutics. 

71

L124 Zhang | Immune monitoring 

Goal of ELISPOT proficiency accomplished: ELISPOT assays provide 

reproducible results among different laboratories for T-cell 

immune monitoring — even in hands of ELISPOT novices 

W. Zhang 1 , R. Caspell 1 , A. Y. Karulin 1 , M. Ahmad 2 , N. Haicheur 3 , A. Abdelsalam 4 , K. 

Johannesen 5 , V. Vignard 6 , P. Dudzik 7 , K. Georgakopoulou 8 , A. Mihaylova 9 , K. Silina 10 , N 

Aptsiauri 11 , V. Adams 12 , P. V. Lehmann 1,13 , and S. McArdle 2 

1 Cellular Technology Ltd., Shaker Hts. Ohio, USA 

2 Nottingham Trent University, Nottingham, UK 

3 Hôpital Européen Georges Pompidou, Paris, France 

4 University of Pittsburgh, Pittsburgh, PA, USA 

5 

Norwegian Radium Institute, Oslo,Norway 

6 

INSERM, Nantes, France 

7 

Jagiellonian University Medical College, Krakow, Poland 

8 Immunology Center, St. Savas Cancer Hospital, Athens, Greece 

9 

UMBAL “Alexandrovska”, Sofia, Bulgaria 

10 

Biomedical Research Study Centre, Riga, Latvia 

11 

Hospital Universitario Virgen De la Nieves, Granada, Spain 

12 

Onyvax Ltd, London, UK 

13 

Department of Pathology, Case Western Reserve University, Cleveland OH, USA 

72 

T cell monitoring remains challenging in tumor 

vaccine trials due to the necessity of testing live 

cells in functional assays, and the low frequencies 

of tumor antigen-specific T cells. Recent multi-center 

initiatives that aimed at harmonizing T 

cell assays have drawn attention to alarming- 30-, 

20- and 150- fold inter-laboratory variations in test 

results for ELISPOT, ICS and tetramers, respectively 

(Immunity, 2009, 31: 527-528). The authors concluded: 

“The high degree in variability makes the 

comparison between any two labs become a game 

of chance”. Puzzled and alarmed by this message, 

we undertook a similar effort for ELISPOT together 

with a European consortium, NEUCAPs. For 

our study, we required that all participants from 

the eleven reporting labs follow the same detailed 

protocol using one uniform platform, and that the 

study participants had never previously conducted 

an ELISPOT assay (the results of their first attempt 

were recorded for the study). While three of the 

labs failed with the basic logistics of the trial, eight 

detected the peptide-specific CD8+ T-cells in frequencies 

approximating the values established by 

the Reference Laboratory. These results show that 

ELISPOT can produce comparable, reliable data 

(even from untrained personnel) if a standardized 

platform for the assay and data analysis is followed. 

Since ELISPOT assays have been qualified 

and validated for regulated studies, they are ideal 

candidates for robust and reproducible monitoring 

of Tcell immunity in tumor vaccine trials.

031 Gross | Immune monitoring 

Monitoring of vaccine-specific regulatory and helper CD4+ 

and cytolytic CD8+ T cells after vaccination with autologous 

antigen-loaded dendritic cells 

Stefanie Gross, Annett Hamann, Gerold Schuler, Eckhart Kämpgen, Beatrice Schuler-Thurner 

Department of Dermatology, University of Erlangen, Germany 

74 

Cancer vaccines have been shown to induce tumorspecific 

CD8+ cytolytic T cells (CTL) in the blood 

of cancer patients. However, clinical outcome often 

remains poor. This ‚‘cancer vaccine paradox‘‘ can 

nowadays be readily explained by tumor-escape 

and immune-regulatory mechanisms. It has been 

suspected, that cancer vaccines of different types 

might not only induce the desired CTL response 

but simultaneously also expand antigen-specific 

regulatory T cells (Treg), thus counteracting productive 

immune responses and preventing clinical 

efficacy of cancer vaccines. Of note is that it has 

been claimed that mature dendritic cells (DC) are 

not only expanding antigen-specific helper T cells 

but also the disadvantageous Tregs. The induction 

of antigen-specific Tregs by DC and other vaccines 

has, however, not yet been studied systematically 

by advanced immunomonitoring techniques. 

Frequencies and nature of vaccine-induced CD8+ 

cytolytic and CD4+ helper or regulatory T cells in 

vaccinated cancer patients were analyzed ex vivo 

by 9 color flow cytometry. MHC Class I and MHC- 

Class II multimer-staining for different tyrosinase, 

MelanA, gp100, NY-ESO1 or MAGE3 epitopes was 

combined with intracellular staining for different 

signature-transcription factors such as FoxP3 

(Treg), GATA-3 (Th2) and RoR t (Th17) for CD4+ 

T cells or function-associated molecules like perforine 

and granzyme B for CD8+ T cells. 

In contrast to common belief our data so far do 

not support the notion that tumor vaccination with 

autologous mature dendritic cells bears the risk 

to induce immuno-suppressive regulatory T cells. 

Remarkably, the same vaccine induced different 

immune responses in patients (Th1, Th2, granzyme 

B), pinpointing the likely benefit of patientselection 

and vaccination at early disease stages.

032 Zelba | Immune monitoring 

NY-ESO-1-specific T cells in long-term melanoma survivors 

Henning Zelba 1 , Benjamin Weide 2 , Claus Garbe 3 , Graham Pawelec 1 , Evelyna Derhovanessian 1 

1 Department of Internal Medicine II, Section for Transplantation Immunology and Immunohaematology, 

University Hospital, Tuebingen, Germany 

2 Department of Dermatology, Division of Dermatooncology, University Hospital, Tuebingen, Germany 

Immunotherapy has now become a potentially 

effective cancer treatment modality for certain 

patients. Theoretically any protein expressed abnormally 

by the tumour can serve as a target for 

antibodies or T cells, and there is an ever-increasing 

list of possible antigen targets. However, it 

is not clear whether targeting particular antigens 

can result in a better clinical outcome than others. 

Moreover, non-immunotherapeutic treatments may 

also result in the generation of immune responses 

against certain of these target antigens, which may 

be associated with improved patient survival. 

To test this, we are analysing exceptionally longterm 

melanoma survivors (LTS) in comparison to 

patients with bad prognosis who progressed during 

treatment and died within the usual time period. 

Patients included those receiving chemotherapy ( e. 

g. Dacarbazin, Temodal,…) and Immunotherapy ( 

e. g. IL-2 injection, ...). 

We analysed CD4+ and CD8+ T-cell responses 

against the four common melanomaassociated antigens 

Melan-A, MAGE-A3, Survivin and NY-ESO- 

1. Antigen-specific T-cell responses were detected 

using peptide mixtures spanning the whole sequence 

of the molecule after one round of in vitro 

sensitization for 12 days. Intracellular production 

of six different cytokines (IFN-γ, TNF, IL-2, IL-4, 

IL-10 and IL-17) was measured simultaneously in 

both CD4+ and CD8+ T-cells by 14 colour multiparameter 

flow cytometry, allowing analysis of 

phenotype and function (TH1, TH2, TH17) at the 

single-cell level. 

In this way, we have identified NY-SO-1-specific T 

cells in 7 of 10 LTS but in only 1 of 6 short-term survivors. 

The prevalence of T-cells specific for Melan- 

A, MAGE-A3 or Survivin did not differ between LTS 

and other patients. Similar results were obtained 

in an independent vaccination trial targeting the 

above antigens. NY-ESO-1-specific immune responses 

were found in 3 of 5 patients with a good 

clinical outcome after vaccination, but in only 1 of 

7 patients with a poor clinical outcome. MAGE-A3specific 

responses were induced in the majority of 

patients after vaccination, but there was no difference 

between LTS and poor responders. 

In all LTS patients, as well as in the patients with 

good clinical outcome in the vaccination trial, 

CD4+ and CD8+ NY-ESO-1-specific T cells produced 

high levels of IFN-γ and TNF. In contrast, 

NY-ESO-1-specific T-cells in the two patients with 

bad prognosis did not produce any or only small 

amounts of these cytokines. Taken together, our 

data suggest that NY-ESO-1 is likely to be a promising 

target antigen for melanoma patients and 

might be a good marker to predict the clinical 

outcome of individual patients. 

75

033 Singh | Immune monitoring 

A stable standard sample for harmonisation and validation 

of T cell assay 

Satwinder Kaur Singh 1 , Bart Tummers 1 , Ton Schumacher 2 , Kees Franken 3 , Marij Welters 3 , 

Cedrik M. Britten 4 , Sjoerd H. van der Burg 1 

1 Deparment of Clinical Oncology, Leiden University Medical Center, Leiden, the Netherlands 

2 Department of Immunology, Dutch Cancer Institute, Amsterdam, the Netherlands 

3 Immunohematology and Blood transfusion, Leiden University Medical Center, Leiden, the Netherlands 

4 Third Medical Department, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany 

76 

Immune monitoring of clinical trials is a challenging 

task as different methods and protocols are applied 

to quantify vaccine-induced T cell responses. Heterogeneity 

in protocols and lack of standards make it 

difficult to compare results obtained from different 

clinical trials. Harmonisation of quantitative assays 

critically depends on samples containing defined 

numbers of antigen-specific T cells, however, the 

availability of such samples is limiting at best. 

We have developed a standard sample using the 

HLA-A2-restricted NY-ESO-1-specific T cell receptor 

(TCR)-transduced T cells spiked into autologous 

PBMC population. The NY-ESO-1- specific TCR 

was retrovirally transduced into purified CD8+ 

T cells and NY-ESO-1-specific T cells were sorted 

using HLA-peptide tetramer to obtain >99% TCRtransduced 

pure CD8+ T-cell population. These 

purified cells were clonally expanded and then 

spiked either at a concentration of 0.25% or 0.5% 

into autologous PBMCs. Multiple batches were extensively 

tested for stability up to 6 months in time. 

The percentage of detected tetramer-positive T cells 

in subsequently tested frozen vials varied little to 

the calculated number of spiked cells (CV

034 Guidoboni | Immune monitoring 

Changes of immune cell infiltrates induced by dendritic cell 

vaccination in melanoma patients‘ tumor tissues: 

an immunohistochemical study 

Massimo Guidoboni 1 , Laura Ridolfi 1 , Annamaria Granato 1 , Massimiliano Petrini 1 , Laura 

Fiammenghi 1 , Valentina Ancarani 1 , Elena Pancisi 1 , Linda Valmorri 1 , Angela Riccobon 1 , 

Roberta Gafà 2 , Giovanni Lanza 2 , Dino Amadori 3 , Ruggero Ridolfi 1 

1 Immunotherapy and Somatic Cell Therapy Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei 

Tumori, Meldola (FC), Italy 

2 Department of Pathology, University of Ferrara, Italy 

3 Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola (FC), Italy 

We have been treated more than 40 patients in a 

phase I/II study of therapeutic vaccination using 

DC pulsed with autologous tumor lysate or homogenate 

in advanced melanoma. Objective responses 

are observed in a limited number of patients, clinical 

responses rates as evaluated by classical responses 

criteria are still disappointing, and we are not able 

to identify patients who will benefit from DC vaccination. 

Spontaneous and vaccine-induced antitumor 

immune responses have been extensively characterized 

in peripheral circulating compartment, 

and in some cases this approach predicted clinical 

response to immunotherapy; however, we do not 

know whether this translate into efficient immune 

cell recruiting in all tumor sites. To shed light on 

changes induced by DC vaccine on the composition 

of immune effectors in tumor sites, we evaluated 

tumor infiltrating lymphocytes (TILs) in 12 

tumor biopsies taken from 4 patients before and at 

different times after DC vaccination for metastatic 

melanoma. Two patients showed complete clinical 

responses (CR) after at least 6 courses of vaccine. 

In one of these patients DC vaccination induced 

huge amount of CD8+ activated cytotoxic lymphocytes 

(24,89 CD8+ TILs/100 melanoma cells) 

in one tumor site compared to prevaccine biopsy; 

however, negligible levels of CD8+ TILs together 

with considerable amount of CD4+ cells were found 

in one other relapsed site, suggesting that immune 

escape had occurred. The other patient showing 

CR underwent relapse and progressive surgical de- 

bulking of metachronous localizations led to surgical 

CR still lasting. Tumor biopsies taken after 

vaccination showed lower amount of CD8+ TILs 

than those collected before (2,13 and 1,38 vs 8,44 

CD8+ TILs/100 tumor cells), showing that complete 

surgical removal of „immune escaped“ lesions 

may lead to persistent CR in patients with good 

clinical status. One patient undergone progression 

after Ipilimumab failure showed considerable 

levels of CD8+ TILs. After 9 courses of DC vaccine 

with stable disease (SD), she relapsed and progressing 

lesion was removed, with decreased amount of 

tumor infiltrating CD8+ lymphocytes in this tumor 

site. The last patient has been vaccinated for 4 years 

with still lasting SD, and further tumor biopsies 

were taken to prepare additional doses of vaccine. 

Treatment induced lower increase of CD8+ TILs 

in postvaccine biopsies than those observed in the 

other patients (3,28 and 1,93 vs 1,12 CD8+ TILs/100 

tumor cells), suggesting that lower immune pressure 

might allow to reach an equilibrium between 

immune system and tumor which slow occurrence 

of immune escape. Although performed on a 

limited number of cases, this study firstly explored 

the possible role for „tissue immune monitoring“ in 

the clinical setting and strongly indicate its use to 

direct clinical decision in multimodal therapeutic 

approach. 

77

035 Voland | Immune monitoring 

Characterisation and functional analysis of T-cell responses in 

melanoma patients vaccinated with peptide-loaded 

dentritic cells 

Steve Voland 1 , Stefanie Gross 1 , Beatrice Schuler-Thurner 1 , Pierre Coulie 2 , and Gerold Schuler 1 

1 Dept. Dermatology, University Hospital Erlangen, Erlangen, Germany 

2 de Duve Institute and Université catholique de Louvain, Brussels 

78 

T cell responses in melanoma patients vaccinated 

with autologous monocyte-derived dendritic cells 

(DC) loaded with peptides from different tumorassociated 

antigens (TAA) were characterized for 

their functional capacity. 

To assign a certain functional capacity (cytolytic 

activity, cytokine production and degranulation 

upon restimulation) to specific T cell clones we 

chose a limiting-dilution based approach. Frozen 

aliquots of PBMC from 5 melanoma patients who 

had received four vaccinations were thawed, 

loaded with peptide and seeded in a 96well plate 

followed by a 14-day culture. The cells were restimulated 

with autologous peptide-loaded PBMC 

at day 7. By splitting the cells two times during 

the 14-day culture four plates with identical clonal 

composition were obtained. Comparative analyses 

of each corresponding well were performed with 

the following assays: 

1. percentage of peptide-specific T cells, determined 

by MHC tetramer binding 

2. intracellular cytokine production (interferon-γ, 

interleukin 2, TNF-α) and degranulation (by CD107a 

mobilization) after antigenic stimulation 

3. cytolytic activity determined by a standard 51Crrelease 

assay 

In all 5 patients vaccine-specific CD8+ T cells were 

detected after in vitro presensitation with peptide. 

Detected responses differed in magnitudes and 

overall functional capacity. In most cases a positive 

correlation between lytic activity and antigenspecificity 

(MHC tetramer positivity) was found. 

Furthermore the lytic activity correlated positively 

with certain cytokine profiles with a pronounced 

IFN-γ and TNF-α proportion and to a lower extend 

also with IL-2 and CD107a. 

From our data can be concluded that vaccination 

with autologous monocyte-derived DCs loaded 

with TAA-derived peptides is capapble to induce 

antigen-specific CD8+ T cells, with the potential 

to produce different immune-stimulatory cytokines 

and which show cell mediated cytotoxitcity. 

Further investigations of the induced T cells will be 

conducted to determine the breadth of the induced 

immune response by analysis of the different T cell 

clones and their affinities.

036 Wagner | Immune monitoring 

GPI-anchor negative T cells with impaired effector function persist 

after Alemtuzumab mediated T-cell depletion 

Eva Maria Wagner 1 , Aline Naomi Lay 1 , Caroline Goetze 1 , Diana Wolff 1 , Julia Hemmerling 1 , 

Matthias Theobald 1 , Wolfgang Herr 1 , Ralf Georg Meyer 1 

1 Department of Hematology/Oncology at the University Medical Center of the Johannes Gutenberg-University 


The monoclonal anti-CD52 antibody Alemtuzumab 

is frequently used for T-cell depletion (TCD) in the 

context of allogeneic hematopoietic stem cell transplantation 

(HSCT). We have previously demonstrated 

that substantial proportions of reconstituting T 

cells remained CD52 negative for years, especially 

in patients who did not receive donor lymphocyte 

infusions. CD52 is a glycosylphosphatidylinositol 

(GPI)-anchored molecule of unknown function. By 

flow-cytometry, we demonstrated that the lack of 

CD52-expression was associated with decreased 

expression of further GPI-anchored molecules like 

CD55 and CD59. We therefore analyzed CD52 negative 

T cells of 20 patients after HSCT with a fluorescent 

aerolysin staining (FLAER) that directly labels 

the GPI-anchor and found that the loss of CD52 and 

further GPI-anchored proteins in T cells was due to 

the loss of the GPI-anchors themselves. In contrast, 

CD52 positive T cells of the same patients showed 

no reduced FLAIR-signal and normal expression 

of CD55 and CD59. Patients who underwent Alemtuzumab-mediated 

TCD prior to allogeneic HSCT 

suffer from long-lasting immunologic dysfunction 

even when peripheral T cell counts have reconstituted 

to normal range. Hence, we compared GPI-anchor 

positive and negative T cells for their response 

to viral and allogeneic stimuli. When FACS-sorted 

CD52 negative and positive T cell populations were 

expanded in vitro (IL2, OKT3, feeder-cells), we observed 

that GPI-anchor expression remained absent 

in the CD52 negative T-cell cultures. In contrast, the 

CD52 positive T-cell cultures showed constant expression 

of GPI-anchors for more than 16 weeks. In 

addition, the CD52 expression had no influence on 

the growth-kinetics following antigen-independent 

stimulation with IL2 and OKT3. However, CD52 positive 

T cells showed enhanced proliferation after specific 

stimulation with CMV-pp65 peptides. In IFN-γ 

ELISPOT assays using CMV-peptide (pp65 and IE-1) 

loaded autologous dendritic cells as stimulators, 

specific spot production was only detected among 

the GPI-anchor positive T cells. To investigate on 

T-cell function ex vivo, we performed IFN-γ Secretion-assays 

with T cells directly isolated from patients. 

CD52 negative T cells again showed a reduced 

IFN-γ-secretion after stimulation with either autologous 

DCs loaded with the CMV-peptides or with 

allogeneic DCs. Nonetheless, T cells with CMV-pp65 

specific T cell receptors were present in the CD52 

positive as well as the CD52 negative subpopulations, 

as proven by tetramer-staining. Despite the 

functional differences, the percentages of naïve and 

memory T cells did not differ between both populations. 

In summary, we demonstrated that after Alemtuzumab-based 

TCD, substantial proportions of 

reconstituting donor T cells lost the expression of 

GPI-anchors. GPI-anchor negative T cells showed 

reduced proliferative capacity as well as reduced 

IFN-γ secretion in response to CMV or allogeneic 

stimulation. Our data suggest that a loss of GPI-anchored 

molecules might contribute to an impaired 

function of reconstituting donor T cells after Alemtuzumab-mediated 

TCD. We will further investigate 

on the impact of different GPI-anchored molecules 

on effector-functions of different T-cell subsets. 

79

037 Aarntzen | Immune monitoring 

Skin-derived functional specific Tcells predict clinical outcome 

upon DC vaccination in stage III and IV melanoma patients 

Erik Aarntzen 1,2 , Annemiek de Boer 1 , Nicole Meeusen-Scharenborg 1 , Danita Schuurhuis 1 , 

Gerty Schreibelt 1 , Joost Lesterhuis 1,2 , Hans Jacobs 1 , Michelle van Rossum 3 , Carl Figdor 1 , 

Gosse Adema 1 , Kees Punt 2 , Jolanda de Vries 1 

1 

Centro de investigación médica aplicada (CIMA) 

2 

Bristol Myers-Squibb Pharmaceutical research institute, Princeton, NJ 

3 Sidney Kimmel Cancer Center. Johns Hopkins Medical School. Baltimore, BA 

80 

Purpose: To improve immunotherapy against 

cancer, tumor-specific immunomonitoring is essen- 

tial. In this study, we analyze the predictive value of 

T cell cultures from Delayed Type Hypersensitivity 

(DTH) skin sites as monitoring tool after dendritic 

cell (DC) vaccination in melanoma patients. 

Patients and methods: In our ongoing trials, HLA- 

A2.1 positive stage III and IV melanoma patients 

are vaccinated with mature DC loaded with the 

tumor associated antigens gp100 and tyrosinase. 

After vaccination, tumor-specific response is routinely 

evaluated in blood and cultures from DTH 

infiltrating lymphocytes (DIL). 

Results: Almost all stage III and IV patients show 

both proliferative and humoral responses to the 

control antigen KLH. Tumor-specific CD8+ Tcells 

are rarely detected in peripheral blood (6 out of 73 

patients tested). In the vast majority of patients, 

sufficient cell numbers are obtained from DIL cultures 

for tetramerstaining and functional analysis. 

We show a strong predictive value of the presence 

of functional specific CD8+ Tcells in DIL cultures 

for favorable clinical outcome in both stage III 

(n=75) and IV (n=85) melanoma patients. 

Conclusion: Our findings show that the presence 

of functional tumor-specific Tcells from DTH skin 

biopsies highly predict clinical outcome.

038 Moodie | Immune monitoring 

Response definition criteria for ELISPOT assays revisited 

Zoe Moodie 1 , Leah Price 2 , Cécile Gouttefangeas 3 , Ann Mander 4 , Syliva Janetzki 5 , Marij J.P. 

Welters 6 , Christian Ottensmeier 4 , Sjoerd H. van der Burg 7 and Cedrik M. Britten 8 

1 Statistical Center for HIV/AIDS Research & Prevention (SCHARP) Fred Hutchinson Cancer Research 

2 Department of Biostatistics, New York University, New York, NY USA, Center, Seattle, WA USA 

3 Department of Immunology, University of Tuebingen, Tuebingen, Germany 

4 Experimental Cencer Medicine Centre and Cancer Sciences Division, Southampton University Hospitals, Southampton, UK 

5 ZellNet Consulting, Inc., Fort Lee, NJ, USA 

6 Department of Immunhematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands 

7 Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands 

8 8 III. Medical Department, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany 

No consensus exists on to how to determine if an 

immune response occurred based on raw data from 

an ELISPOT assay. The goal of this study was to 

enable investigators to understand currently available 

methods for response determination and to rationally 

select and apply a scientifically sound method appropriate 

to their specific laboratory setting. Five representative 

approaches were applied to data sets from 

the CIMT Immunoguiding Program and the response 

detection and false positive rates of each approach 

were compared. Simulation studies were also performed 

to compare empirical and statistical approaches. 

We conclude that both empirical and statistical approaches 

may serve as appropriate response definition 

criteria. However, we recommend the use of a nonparametric 

statistical test. To enable use of the recommended 

statistical tests, a web-based user interface 

was created: http://www.scharp.org/zoe/runDFR/. 

This interface allows the user to upload data from an 

ELISPOT assay and obtain the response determination 

based on the recommended statistical tests. 

Furthermore, we recommend the use of six medium 

control wells or four wells each for both medium 

control and experimental conditions be performed in 

order to increase the sensitivity of the assay. The study 

provides an estimate for replicate variation and limit 

of detection that can be expected from an average 

lab. The authors recommend that replicates with high 

variation and below the estimated limit of detection 

should be filtered out or interpreted with caution. 

81

039 Attig | Immune monitoring 

Recommendations for HLA-peptide multimer staining experiments 

- Results from the second HLA-peptide multimer proficiency 

phase organized by the Cancer Immunotherapy Consortium 

Sebastian Attig 1 *, Leah Price 2 *, Sylvia Janetzki 3 , Michael Kalos 4 , Michael Pride 5 , Lisa Mc 

Neil 5 , Tim Clay 6 , Jianda Yuan 7 , Kunle Odunsi 8 , Axel Hoos 9 , Pedro Romero 10 , Cedrik M. Britten 

1,11 for the CRI-CIC Assay Working Group 

1 

Division of Translational and Experimental Oncology, Department of Internal Medicine III, University Medical 

Center of the Johannes-Gutenberg University, Mainz, Germany 

2 

Department of Biostatistics, New York University, New York, NY USA 

3 

ZellNet Consulting, Inc., Fort Lee, NJ USA 

4 

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Abramson 

Family Cancer Research Institute, Philadelphia, PA USA 

5 

ZellNet Consulting, Inc., Fort Lee, NJ, USA 

6 

Surgery and Immunology, Duke University Medical Center, Durham, NC, USA 

7 

Ludwig Center for Cancer Immunotherapy, Immunology Program, Sloan-Kettering Institute, New York, 

NY USA 

8 

Departments of Gynecologic Oncology and Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA 

9 

Bristol-Myers Squibb, Wallingford, CT USA 

10 

Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University 

Hospital (CHUV), Lausanne, Switzerland 

11 

Clinical Development, BioNTech AG, Mainz, Germany 

82 

The Cancer research Institute‘s Cancer Immuno- 

therapy Consortium (CRI-CIC) has conducted a 

HLA-peptide multimer (MULTIMER) proficiency 

panel focusing on the impact of DUMP channel use. 

The study showed that the introduction of a dump 

channel did not impact on the detection rate of participating 

labs but did reduce the non-specific MUL- 

TIMER binding within the group. Both the use of 

DUMP channel and dead cell marker may decrease 

the amount of false positive events in CD8-positive 

population thus leading to a lower limit of detection 

of the test and provide a more accurate measure of 

the true MULTIMER specific binding. In 88% of 

all the CMV-MULTIMER replicates a response was 

detected, demonstrating that low frequency CMV 

responses were successfully detected by most of 

the labs. Variation of duplicate staining was below 

30% in 70% of participating labs indicating a very 

good reproducibility of results even for low frequency 

T cell responses. Introduction of a dump 

channel also decreased the variability in reported 

frequencies of pp65-specific CD8 T cells across the 

group. However, the response detection rates for 

the Melan-A responses were much lower which was 

due to the particular staining characteristics of the 

Melan-A-specific MULTIMER. Results from this 

proficiency panel phase clearly confirm the value 

of previously published harmonization guidelines, 

and have led to additional recommendations on 

assay performance and how to best present data 

from MULTIMER experiments.

Cellular therapy 

83

040 Kotsiou | Cellular therapy 

Application of MHC class I single chain trimers for leukaemia 

immunotherapy 

Eleni Kotsiou 1,2 , Julian Dyson 2 , Keith G Gould 1 

1 

Department of Immunology, Wright-Fleming Institute, St Mary’s campus, Imperial College, 

London W2 1PG, UK 

2 

Immunobiology Section, Commonwealth Building, Hammersmith Hospital, Imperial College, 

London W12 ONN, UK 

84 

Major Histocompatibility Complex (MHC) class I 

single chain trimers (SCTs) consist of antigenic 

peptide, β2-microglobulin and MHC heavy chain 

all joined together in a single polypeptide molecule 

via flexible linkers. SCTs are extremely valuable 

tools for the stimulation of CD8+ T cell responses 

because of their enhanced stability compared to 

conventional MHC molecules and can therefore be 

used for the expansion of peptide specific CD8+ T 

cells either in vitro or in vivo. 

We have expressed the human minor histocompatibility 

(H) antigens, HA-1 and HA-2 (expressed only 

on haematopoietic cells and restricted by human 

leukocyte antigen (HLA) A2) and the mouse HY 

minor transplantation antigens originating from 

the Smcy and Uty genes (restricted by H2-Db) in the 

SCT format. For the HLA-A2 SCTs, different forms 

were constructed by modifying the CD8 binding 

region (CD8 enhanced and non-binding variants) 

whereas in both mouse and human SCTs we introduced 

cysteine residues in the heavy chain and 

first linker that have been shown to form a disulfide 

bond, anchoring the peptide more efficiently 

(disulfide-trap (dt) variants). 

All human and mouse SCTs had good cell surface 

expression as determined by specific antibody 

staining of TAP2- deficient CHO cell transfectants. 

Soluble forms of HA-1 and HA-2 SCTs were obtained 

by retroviral transduction of human embryonic 

kidney (HEK) 293 T cells, and subsequent purification 

of the soluble protein from culture superna- 

tant. As the HA-1 and HA-2 minor H antigens are 

only expressed on normal and malignant haematopoietic 

cells, they are good candidates for therapeutic 

targeting. For this reason soluble SCTs were 

used for the in vitro expansion of allo-restricted 

CD8+ T cells specific for HA-1 and HA-2 minor H 

antigens from HLA-A2 negative individuals. Furthermore 

the minor male specific transplantation 

antigens DbUty and DbSmcy were expressed as 

SCTs and dtSCTs and will be introduced into bone 

marrow dendritic cells by retroviral transduction. 

Their potential to expand antigen specific CD8+ 

T cells in vitro and in vivo as a vaccination model 

will be assessed. Further results will be presented 

illustrating the applications and functions of MHC 

class I SCTs.

041 Wittmann | Cellular therapy 

Comparing genetically engineered T cells for chimeric TCR 

versus CD16 + Trastuzumab for adoptive immunotherapy 

against HER2 positive breast carcinomas 

Sandrine Valsesia-Wittmann 1 , Béatrice Clémenceau 2 , Anne-Catherine Jallas 1 , Anne-Claire 

Doffin 3 , Jenny Valladeau 3 , Raphaël Rousseauy, Christophe Caux 1,3 and Henri Vié 2 

1 Centre Léon Bérard -plateforme d’Innovation en Immunomonitoring et Immunothérapie- 

28 rue Laennec-69008 LYON, France 

2 INSERM CRCNA U892 - Institut de Recherche Thérapeutique de l‘Université de Nantes- 

44007 NANTES, France 

3 CLB-INSERM U590, Cytokines et cancers, Lyon, France 

4 Roche, Bale, Switzerland 

The HER2 receptor is overexpressed in 25% of 

breast cancers and is associated with poor prognosis. 

Trastuzumab, a monoclonal antibody targeting 

HER2 has been demonstrated to improve survival 

of HER2 overexpressing metastatic breast cancer. 

However, majority of patients who initially respond 

to trastuzumab develop resistance within one year 

of treatment initiation, and in the adjuvant setting 

15% of patients still relapse despite trastuzumabbased 

therapy. 

The goal of this project is to develop and compare 

efficiency of two adoptive immunotherapy approaches 

by genetically engineered T cells to overcome 

this resistance. For the first approach, we developed 

T lymphocytes armed with an anti-HER2 

chimaeric TCR (scFvanti-Her2(FRP5)-CD28TM- 

CD3zeta) to directly kill HER2 positive cells. For 

the second approach, we developed T lymphocytes 

armed with a high affinity IgG FcR (FcgRIIIa, CD16) 

linked to its transduction chain FceRIg (CD16/g). 

In this latter case, the HER2 antigen is pre-targeted 

by trastuzumab to induce a “two step killing” 

through Antibodies Dependant Cellular Cytotoxicity 

(ADCC). 

Results obtained in vitro demonstrated the high 

direct killing efficiency of anti-HER2 chimaeric 

TCR CTL against HER2 amplified breast carcinoma 

cells BT474 or SKBR3 (>75%). However, abnormal 

reactivity of these CTL against undetectable HER2 

expression was observed (15 to 30% of lysis). This 

constitutive non specific activation of chimaeric 

TCR might represent an important limitation point 

for clinical use. 

On the other hand, “two step killing” with CD16/g- 

CTL demonstrated a low but very specific cytotoxicity 

against HER2 positive cells through ADCC 

only in the presence of Trastumumab (20% to 

40% of lysis at 30:1 ET). The moderate efficiency 

of specific lysis is linked to low levels of CD16/g 

expression and uncorrelated to level of HER2 targeted 

antigen expression. In order to improve our 

strategy and before transfert in animal model, we 

are developing new constructs with: (i) modification 

of the transduction domain of chimaeric TCR 

to block constitutive activation and limit non specific 

lysis and (ii) increase the efficiency of CD16/g 

expression at the surface of effectors to enhance 

ADCC mechanism. 

Furthermore, we are comparing in vivo efficacy 

of both approaches (“one step” TCR vs “two 

step”ADCC) to induce regression of HER2 + or - 

breast carcinoma xenograft in immunodeficient 

mice model NOD-SCID b2-/-. 

Our last results will be presented. Because the 

alteration of ADCC mechanisms during trastuzumab 

treatment is one rational explanation for 

the acquired resistance, improving effectors might 

represent a safe adjuvant treatment to prevent resistance. 

85

042 Weigand | Cellular therapy 

Generation of CD4+ T cells with specificity for FMNL1 

Luise Weigand 1 , Xiaoling Liang 1 , Ingrid Schuster 2 , Elfriede Eppinger 1 , Yanyan Han 1 , 

Matthias Schiemann 3 , Elisabeth Kremmer 2 , Andreas Moosmann 4 , Josef Mautner 5 , Christian 

Peschel 1 , Angela Krackhardt 1 

1 

Department of Hematology/Oncology, Klinikum Rechts der Isar, Technische Universität München, 

Munich, Germany 

2 

Division Helmholtz Zentrum München – German Research Center for Environmental Health, 

Institute of Molecular Immunology, Munich, Germany 

3 

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, 

Munich, Germany 

4 

Helmholtz Zentrum München – German Research Center for Environmental Health, Clinical Cooperation 

Group Molecular Oncology and Ludwig-Maximilians-Universität München, Klinik für Hals-Nasen- und 

Ohrenkrankheiten, Munich, Germany 

5 

Helmholtz Zentrum München – German Research Center for Environmental Health, Institute for Clinical 

and Molecular Biology, Munich, Germany 

86 

Adoptive T cell therapies in the context of allogeneic 

stem cell transplantation have curative potential, 

especially in hematological malignancies. Patients 

with leukemic relapse after stem cell transplantation 

are treated with donor lymphocyte infusions 

which may induce a beneficial Graft-versus-leukemia 

effect but also detrimental Graft-versus-host 

disease. This approach may be improved by transferring 

T cells with selected specificities. We have 

previously generated allorestricted CD8+ T cells 

with specificity for the SEREX-derived tumor-associated 

antigen formin-like protein 1 (FMNL1). As 

it has been repeatedly shown, that CD4+ T cells 

play a critical role in tumor eradication and maintenance 

of immune memory, the aim of this project 

was to generate CD4+ T cells directed towards a 

peptide derived from FMNL1. 

We used two different methods to isolate FMNL1specific 

CD4+ T cells: Dendritic cells were pulsed 

with either one out of nine peptide pools from an 

FMNL1 peptide-library or recombinant protein to 

prime autologous PBMC or CD45RO- T cells. Using 

dendritic cells pulsed with overlapping peptides 

derived from the FMNL1 peptide library as stimu- 

lator cells, several T cell clones (e. g. Ga3.7) highly 

specific for the same FMNL1 peptide could be 

isolated. However, these clones did not recognize 

natural targets. In contrast, using protein-pulsed 

dendritic cells as stimulator cells, we were able to 

generate CD4+ T cell clones reactive against cells 

naturally expressing FMNL1. Intensive testing of 

one specific clone (Aa2.2) showed that recognition 

of target cells could be enhanced by transduction 

with FMNL1 and blocked by an HLA-DR specific 

antibody. Moreover preliminary data show, that 

Aa2.2 recognizes HLA-DR matched chronic lymphocytic 

leukemia cells. Both, the peptide-specific 

clone Ga3.7 and Aa2.2 show a T helper cell type 2 

(TH2) cytokine profile with antigen-specific secretion 

of IL-5, IL-10 and IL-13. TCR genes of relevant 

clones have been isolated and are, actually, transduced 

in indicator cell lines in order to confirm the 

MHC restriction element and to define the recognized 

epitope.

043 Liang | Cellular therapy 

Transfer of human T-cell receptor (TCR) containing murine 

chimeric constant betagamma-chain sequences reduces the risk 

of mixed heterodimers and enhanced the proliferation of 

transduced T cells 

Xiaoling Liang 1 , Yanyan Han 1 , Ingrid G. Schuster 2 , Luise U. Weigand 1 , Elfriede Eppinger 1 , 

Dirk Busch 3 , Wolfgang Uckert 4 , Christian Peschel 1 , Angela Krackhardt 1 

1 Technical University Munich, Klinikum Rechts der Isar, Medizinische Klinik III, 

Innere Medizin mit Schwerpunkt Hämatologie und Onkologie, Munich, Germany 

2 Helmholtz Zentrum München-German Research Center for Environmental Health, 

Molecular Immunology, Munich, Germany 

3 Technical University Munich, Medical Microbiology, Immunology and Hygiene, Munich, Germany 

4 Max Delbrück Centrum, Berlin, Germany 

Adoptive transfer of tumor-associated antigen (TAA) 

or virus specific T-cell receptor (TCR)-transduced 

T cells may represent an attractive and promising 

approach to specifically treat malignant diseases 

and has been previously successfully applied in the 

clinic. However, there are different concerns which 

need to be addressed to further improve this therapeutic 

approach: First, the formation βγof heterodimers 

between endogenous and transduced TCR 

chains may abrogate specific TCR function and 

harbours a particular risk for unknown specificities. 

Second, longterm survival of TCR-transduced 

T cells has been demonstrated to be critical for the 

effectivity of this approach and needs to be further 

improved. 

We have previously identified several HLA-A2-allorestricted 

T-cell receptors with specificity for the 

breast cancer associated antigen HER2/neu and 

the hematopoietic lineage specific protein Forminlike 

protein 1 (FMNL1) which is overexpressed in 

diverse malignant cell lines and native leukemia 

cells. The FMNL1-specific TCR SK22 represents a 

weak TCR with low interchain affinity which was 

significantly improved by current optimization stra- 

tegies including murinization of constant chains 

and codon-optimization. We additionally modified 

this TCR as well as two other TCR with alternative 

specificities as HER2/neu and GP100 by introducing 

murinized constant chain betagamma (βγ) 

chimera, which results in reduced pairing of heterodimers 

in PBMC. More significantly, proliferation 

of T cells was clearly enhanced in effector cells and 

T cell clones transduced with αβγ compared to αβ 

TCR. Preliminary results suggest that also different 

cytokine patterns might be expressed. In contrast 

to a previous publication, we observed a significant 

polarization of CARMA-1 to the immunogical 

synapse in different TCR containing the chimeric 

chain combination and no decrease in Fas-Ligand 

expression in transduced T cells suggesting that 

altered CARMA-1 signaling might not be the only 

explanation for enhanced proliferation of T cells 

transduced or transgenic for the βγ-TCR. 

In conclusion, our data show that the transfer of 

TCR chain genes containing optimized murinized 

chimeric βγ-constant chains may have advantages 

and might be an alternative or synergistic choice 

for TCR optimization for adoptive T cell transfer. 

87

044 Kalos | Cellular therapy 

Clearance of established leukemia in a mouse xenograft model 

by a single injection of primary T cells gene-modified to express 

chimeric antigen receptors that target CD19 

Michael Kalos 1 , David M. Barrett 2 , Carmine Carpenito 1 , Stephan A. Grupp 2 and Carl June 1 

1 Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 

2 Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 

88 

We are evaluating the potential to use T-cells re- 

directed by genetic engineering to target human 

tumors. Chimeric antigen receptors (CAR), developed 

by combining antigen binding domains from 

antibodies or T cell receptors (TcR) to T cell signalling 

domains offer a potentially powerful platform 

to mediate tumor re-targeting. 

To re-target T cells against B cell malignancies we 

are focusing on the CD19 molecule, a membrane 

glycoprotein found on human B lymphocytes and 

expressed by the vast majority of B cell tumors. We 

have generated a CAR that consists of an extracellular 

scFv domain derived from an antibody that 

targets CD19 linked to the intracellular signalling 

domain of the TcR zeta chain. To criticaly evaluate 

the effects of including signaling domains from T cell 

costimulatory on CAR functionality and anti-CD19targeting 

CAR we introduced signalling domains 

from CD28 or CD137 (41BB) domains into the anti- 

CD19 CAR, transduced such CAR into primary 

human CD8+ and CD4+ T cells, and evaluated 

the ability of the transduced T cells to eradicate 

established aggressive CD19+ leukemia cell line 

Nalm-6 in the highly immunodeficient NOG mouse 

model. We evaluated primary lymphocytes gene 

modified to express anti-CD19-targeting CAR that 

contain TcRζ —alone, CD28-TcRζ, CD137-TcRζ—, 

and CD28-CD137-TcRζ — signaling domains. 

Mice treated with a single dose of T cells engineered 

to express each of the anti-CD19 CAR demonstrated 

statistically significant improved survival over con- 

trols animals. The CD19-CD28-CD137-ζ — CAR mediated 

the longest median overall survival and the 

most mice surviving until the end of the study at 

Day 180. In vivo bioluminescent imaging shows the 

kinetics of leukemia clearance to be rapid (within 

72 hours), and proves a sensitive tool for tracking 

disease relapse. 

These data demonstrate the potential of CAR directed 

to the CD19 molecule to effectively redirect T 

cells to eradicate established bone marrow disease 

with a single injection of CAR modified cells. Future 

efforts will focus on enhancing in vivo persistence 

and surveillance capabilities of these CAR T-cells, 

as well as investigating the ability of CAR T cells to 

clear repeated challenges of leukemia.

045 Grange | Cellular therapy 

Optimizing effector and memory properties of CD8 T cells 

for adoptive tumor immunotherapy 

Magali Grange 1 - 3 , Michel Buferne 1 - 3 , Lee Leserman 1 - 3 , Anne-Marie Schmitt-Verhulst 1 - 3 and 

Nathalie Auphan-Anezin 1 - 3 

Centre d'Immunologie de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 

13288 Marseille cedex 9, France. 

1 

INSERM U631, Parc Scientifique de Luminy, Case 906, 13288 Marseille, France 

2 

CNRS UMR6102, Parc Scientifique de Luminy, Case 906, 13288 Marseille, France 

3 

Université Aix-Marseille, Parc Scientifique de Luminy, Case 906, 13288 Marseille, France. 

Relative to normal tissues, tumor cells ectopically 

express or overexpress tumor-assoiated antigens 

(TAA). Therefore, targeted destruction of malignancies 

by enhancing CD8 T lymphocyte (TL) responses 

to TAA is an attractive therapeutic modality 

that is currently being evaluated. This protocol 

typically relies on the ex vivo expansion of TAAspecific 

CD8 effector TL in medium containing IL-2 

to support their survival and proliferation. While 

this allows the generation of large numbers of TAAspecific 

TL, it appears that these cells are terminally 

differentiated effector TL that rapidly become 

senescent. Therefore, alternatives to the use of IL-2 

need to be developed. Several groups including 

ours have shown that the IL-2R/STAT5 signaling 

reinforces the gene expression program initiated 

by TCR engagement in effector TL, including CD4 

Th2, CD4 Th1 and CD8 TL. For the latter subset 

we have shown that a constitutively active STAT5a 

transcription factor (STAT5CA) mimicked IL-2R signaling 

to enhance CD8 TL effector functions in 

vitro. Expression of the same STAT5CA mutant has 

been shown by others to promote the differentiation 

and the self renewal of hematopoietic stem 

cells, a cell population that shares a transcriptional 

signature with self renewing memory TL. 

We now report on the potential of STAT5CA expressing 

TAA-specific CD8 TL to gain both potent antitumor 

CD8 effector activity and long-term maintenance 

in vivo through the regulation of genes 

responsible for self-renewal. 

We show that expression of STAT5CA in CD8 TL 

favors their differentiation into long-lived effector 

cells which manifest the phenotypic and migratory 

properties (tissue homing) characteristic of effector 

TL and the high potential for Ag recall responses 

and absence of senescent features reminiscent of 

central memory TL. Nevertheless, the transduced 

CD8 TL return to quiescence and remain under 

control of TCR-derived signals for their survival 

and the activation of effector functions. Importantly, 

the characteristics conferred on TL by expression 

of STAT5CA were obtained with both monoclonal 

(TCR transgenic) and polyclonal CD8 TL. Therefore, 

active STAT5, by counteracting the induction of 

senescence, has distinct effects from IL-2 mediated 

signals that promote the differentiation of terminally 

differentiated effector TL. Finally, in adoptive 

therapy, STAT5CA-expressing CD8 effector TL demonstrated 

increased efficiency to cure mice with 

established tumors. 

Therefore, the novelty of our approach resides in the 

modification of a “master switch” gene as a useful 

strategy to optimize differentiation of CD8 TL into 

functional, long-lived and replication competent 

effector TL, through the concerted regulation of 

molecules involved in co-stimulation, memory fate, 

tissue infiltration, function and potential for secondary 

response. 

89

046 Singh | Cellular therapy 

Search for FLT3- ITD-reactive CD8+ T cells in the peripheral 

blood of healthy donors 

Vijay Singh 1 , Claudine Graf 1 , Volker Lennerz 1 , Catherine Wölfel 1 , Thomas Wölfel 1 

1 

III. Medizinische Klinik (Hematology & Oncology), University Medical Center of the 

Johannes Gutenberg-University, Mainz, Germany 

90 

FLT3-ITDs (fibroblast-macrophage stimulating 

factor receptor (FMS)-like tyrosine kinase receptor 

3 –internal tandem duplications) support leukemic 

transformation by constitutive phosphorylation 

resulting in uncontrolled activation and their presence 

is associated with worse prognosis. We have 

previously demonstrated that FLT3-ITD-derived neoantigens 

can be recognized by autologous AMLreactive 

CD8+ T cells (1, unpublished). We have 

chosen three immunogenic FLT3-ITDs identified in 

patients VE, IN and QQ, known to be restricted by 

HLA-B*27:05, -A*32:01 and -A*11:01, respectively. 

We set out to generate stable CD8+ T-cell populations 

and clones against these FLT3-ITDs from 

allogeneic healthy donors carrying at least one relevant 

FLT3-ITD-antigen presenting HLA I allele. 

In brief, blood-derived CD8+ T cells were stimulated 

under quasi-limiting dilution conditions with 

autologous irradiated CD8- PBMCs electroporated 

with in vitro-transcribed RNA encoding FLT3-ITD 

(2). On day 12, responder T cells were tested in 

an IFN-γ ELISPOT assay for recognition of the appropriate 

FLT3-ITD. ITD-reactive microcultures 

were restimulated twice with autologous irradiated 

CD8-PBMCs or adherent cells (monocytes) 

electroporated with FLT3-ITD mRNA. On day 26, 

to assess their HLA restriction, responder T cells 

were tested against COS-7 cells co-transfected with 

all donor HLA I alleles (predicted on the basis of 

2-digit typing) and the respective FLT3-ITD in an 

IFN-γELISPOT assay. Until now, we have analyzed 

16 healthy donors for FLT3-ITD response on d12. 

We observed ITD reactivity in seven donors. The 

frequencies of ITD-reactive T cells were estimated 

to be in the range of 4.25 x 10-7 to 2.5 x 10-6. In 

3 donors ITD-reactive T cells could be propagated 

over two further stimulation periods applying monocytes 

as presenting cells. In one donor we found 

that anti-FLT3-ITD (VE) T cells were restricted by 

HLA-Cw*07:02 whereas in the remaining donors 

the restricting HLA alleles were not identified. In 

further experiments we will try to derive stable 

T cell clones against FLT3-ITDs from allogeneic 

sources. These T cells or their receptors could be 

used in adoptive transfer therapies. 

(1) Graf C et al. Blood 109:2985-8, 2007; 

(2) Teufel R et al. Cell Mol Life Sci 62:1755-62, 2005

047 Blaudszun | Cellular therapy 

Adoptive transfer of ex vivo activated and antineoplastic drug 

loaded T lymphocytes retargeted to EpCAM expressing tumours 

André-René Blaudszun 1 , Anja Philippi 1 , Alice Borth 1 , Gerhard Moldenhauer 2 , Philipp Beckhove 

2 , Hyeck-Hee Lee 1 and Ute Steinfeld 1 

1 Cellular Immunotherapy Group, KIST Europe Forschungsgesellschaft mbH, Saarbrücken, Germany 

2 Translational Immunology Unit, German Cancer Research Center, Heidelberg, Germany 

T lymphocytes are often the first choice for adop- 

tive cell therapy (ACT) against cancers, because 

of their intrinsic cytolytic capacity. Unfortunately, 

clinical trials have shown that the effectiveness of 

ACT is limited. Among various therapeutic options, 

chemotherapy remains the mainstay of treatment 

for neoplasms. However, therapies based on antineoplastic 

drugs are often accompanied by severe 

adverse effects, because they are also harmful to 

healthy constantly dividing cells and tissues. 

Here, we describe a novel cancer therapy approach 

that combines the specificity of retargeted T cells 

with the high efficacy of chemotherapies. To enhance 

the natural cytotoxic function of human lymphocytes, 

ex vivo activated and expanded T cells were 

loaded with a liposomal preparation of the secondgeneration 

anthracycline idarubicin (LipoIda). Although 

T lymphocyte viability was impaired upon 

LipoIda treatment, mice experiments were carried 

out to test the effectiveness of the living drug carriers 

in vivo. When SKOV-3 xenograft mice were 

treated with either unloaded or idarubicin-loaded 

allogenic T cells in combination with the bispecific 

antibody EpCAM×CD3, tumour growth was more 

efficiently inhibited by the drug-containing cells. 

To analyse the observed effect in more detail, we 

compared the action of idarubicin-loaded T cells 

with the effect of a co-administration of unloaded 

T cells and free drug. Xenograft mice treated with 

drug-containing T lymphocytes showed a stronger 

inhibition of tumour growth than the control 

animals suggesting that the combined T cell-drug 

effect of LipoIda-treated T cells on malignant cell 

proliferation is rather synergistic than additive. 

Thus, redirection of antineoplastic drug-loaded 

T lymphocytes represents a promising course of 

action to treat human cancers. Since the administered 

idarubicin amount lies below conventional 

therapeutic doses, strong adverse effects typically 

caused by chemotherapy are unlikely to occur in 

our approach. 

91

048 Schendel | Cellular therapy 

High avidity T cell clones specific for tumor-associated 

antigens and how to find them 

Dolores J. Schendel 1,2 , Susanne Wilde, Daniel Sommermeyer 3 , Matthias Leisegang 3 , Stefani 

Spranger 1 , Slavoljub Milosevic 1 , Bernhard Frankenberger 1 and Wolfgang Uckert 3,4 

1 

Institute of Molecular Immunology and 

2 

Clinical Cooperation Group “Immune Monitoring”, Helmholtz Zentrum München, German Research Center for 

Environmental Health, Munich, Germany 

3 

Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 

4 Humboldt University Berlin, Institute of Biology, Berlin, Germany 

92 

Since many tumor-associated antigens (TAA) re- 

present over-expressed self-proteins, T cells with 

high avidities have been eliminated during negative 

selection in the thymus to prevent autoimmunity. 

Therefore, we compared self-restricted and allo-restricted 

T cell lines and clones with the aim to identify 

properties that discriminate high avidity from 

low avidity T cells. Autologous CD8+ T cells were 

primed using either dendritic cells (DC) of HLA-A2donors 

pulsed with in vitro transcribed (ivt) RNA 

encoding a selected TAA plus allogeneic HLA-A2 

ivt-RNA or DC of HLA-A2+ donors loaded with 

TAA encoding ivt-RNA alone. Multimer+CD8+ 

cells were sorted, cloned by limiting dilution and 

uncloned cells were expanded as bulk lines using 

antigen-independent stimulation. 

Since high-intensity multimer staining was shown 

previously to indicate strong TCR-ligand interactions, 

mean fluorescence intensities (MFI) of multimer 

binding was used as a first estimate of structural 

TCR-MHC/peptide binding affinity. A second estimate 

was made based on loss of multimer binding 

over time (i.e. multimer off-rate), since a slower 

off-rate suggests that TCR-ligand interactions are 

more stable and of higher structural affinity. Differences 

were seen directly in primed cultures and 

after sorting of bulk lines from various priming cultures. 

Multimer+CD8+ cells of HLA-A2+ donors 

had lower MFI of multimer binding compared to 

HLA-A2- donors and percentages of double-positive 

cells were also lower. A more rapid loss of 

multimer binding was measured over time. These 

characteristics of multimer binding indicated that 

self-restricted T cells were of lower TCR affinity 

compared to allo-restricted T cells. At the clonal 

level, we observed more disparities regarding the 

multimer analyses since there was a trend but no 

significant correlation between multimer analysis 

and functional avidity, as measured by responses 

to titrated amounts of TAA specific peptide pulsed 

onto T2 cells. These results revealed that multimer 

MFI and off-rates alone do not conclusively predict 

functional avidity of T cell clones. These discrepancies 

were retained when TCR from selected 

clones were expressed as transgenic proteins in 

activated lymphocytes.

049 Albrecht | Cellular therapy 

IL-21 treated naive CD45RA+ T cells represent a reliable source 

for generating AML-reactive cytotoxic T lymphocytes with high 

proliferative potential and early differentiation phenotype 

Jana Albrecht 1 , Michaela Frey 1 , Daniel Teschner 1 , Alexander Carbol 2 , Matthias Theobald 1 , 

Wolfgang Herr 1 , Eva Distler 1 


2 Center for Blood Transfusion, University Medical Center, Mainz, Germany 

Specific allogeneic T cell therapy inducing selective 

and durable graft-versus-leukemia effects requires 

in vitro strategies to generate highly proliferating 

leukemia-reactive T cells with an early differentiation 

phenotype and strong effector functions. 

Here, we introduce an approach for the reliable 

generation of such leukemia-reactive cytotoxic T 

lymphocytes (CTLs) from naive CD45RA+ CD8+ 

T cells of healthy donors. The protocol includes 

the stimulation of immunomagnetically isolated 

naive T cells with primary acute myeloid leukemia 

(AML) blasts of patient origin in HLA class I-matched 

mini-mixed lymphocyte/ leukemia cultures 

(mini-MLLCs), according to our recently published 

protocol (Distler et al., Exp. Hematol. 36:451-463, 

2008). In 8 different donor/AML-patient pairs we 

succesfully isolated AML-reactive CTLs from naive 

CD45RA+ precursors. If IL-21 was added during 

the primary stimulation phase in addition to the 

previously used cytokines IL-7, IL-12, and IL-15, the 

procedure was even more efficient. AML-reactive T 

cell cultures could be easily expanded by weekly 

antigen-specific stimulation to cell numbers exceeding 

108 within a few weeks. Most CTLs expressed 

one or up to three TCR Vβ chains, suggesting 

mono- or oligoclonality. For up to 10 weeks 

of in vitro culture the majority of CTL clones and 

lines retained strong HLA class-I restricted effector 

functions (cytolysis and IFN-γ secretion). On the 

basis of reactivity pattern with patient cells, CTLs 

could be divided into two groups: Those that exclu- 

sively reacted with primary AML blasts, suggesting 

leukemia-assosiated antigens as targets, and those 

that recognized both primary AML blasts and patient-derived 

lymphoblastoid cell lines (LCLs), indicating 

minor histocompatiliby antigens as targets. 

Donor-derived LCLs or the NK cell target K562 were 

not lysed. By the use of monoclonal anti-HLA class 

I-blocking antibodies and cross reactivity analyses 

against partially HLA class I-matched leukemia 

samples, we could identify HLA-A01, -A02, -B13, 

-B15, -B57, -Cw03, -Cw06 and -Cw07 as restriction 

elements. Most importantly, leukemia-reactive 

CTLs retained expression of the early T cell differentiation 

markers CD27, CD28 (costimulation), 

CD62L, CXCR4 (homing) and CD127 (IL7Rα) for 

several weeks during in vitro expansion. For some 

of these markers, the effect was even enhanced by 

IL-21. This expression pattern may be favorable for 

CTL survival and homing in vivo after adoptive 

transfer. 

In summary, AML-reactive CTLs generated with 

IL-21 from the naive CD8+ CD45RA+ subset of 

healthy donors are promising candidates for adoptive 

immunotherapy because of their expression of 

early phenotype markers along with a high proliferative 

potential and strong effector functions. 

93

050 Klobuch | Cellular therapy 

T cell receptor RNA transfer into non-reactive human 

T lymphocytes turns them into potent CMV-specific effector 

cells 

Sebastian Klobuch 1 , Katrin Besold 2 , Bodo Plachter 2 , Mirjam H. M. Heemskerk 3 , Niels 

Schaft 4 , Ralf-Holger Voss 1 , Matthias Theobald 1 , Wolfgang Herr 1 , Simone Thomas 1 

1 Department of Hematology & Oncology, University Medical Center, Mainz, Germany 

2 

Institute of Virology, University Medical Center, Mainz, Germany 

3 

Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands 

4 Department of Dermatology, University Hospital of Erlangen, Erlangen, Germany 

94 

Cytomeglovirus (CMV)-associated disease is a 

life-threatening complication in patients after allogeneic 

hematopoietic stem cell transplantation 

(HSCT). Although antiviral drug therapy is successfully 

used to reduce the risk of CMV disease, 

long-term virus control requires the reestablishment 

of protective antiviral T cell immunity in 

the host. The latter is challenging, particularly if 

the donor is CMV seronegative or possesses only 

a weak CMV-specific memory T cell response and 

thus, no CMV-reactive T cells are being transferred 

from donor to recipient during HSCT. Grafting 

nonreactive T cells of CMV seronegative donors by 

virusantigen specific T cell receptors (TCR) may be 

an efficient means to transfer CMV specific T cell 

function into allogeneic HSCT recipients. In this 

study, we have reprogrammed T cells of CMV-seronegative 

donors with human TCR recognizing the 

immunodominant HLA-A*0201- binding epitope 

495-503 derived from the CMV pp65 protein. To 

overcome the limitations of retroviral TCR gene 

transduction that hamper clinical translation, we 

used in vitro transcribed RNA encoding CMV-specific 

TCR for electroporation of non-reactive human 

T cells. After RNA transfection of anti-CD3 stimulated 

peripheral blood mononuclear cells (PBMCs), 

TCR expression levels were sufficient to trigger 

IFN-g secretion and cytolytic activity against pp65 

peptide-pulsed target cells and moreover against 

human fibroblast upon CMV infection. Due to the 

instability of the introduced RNA molecules, TCR 

expression and effector function was measureable 

for a time period of at least 3 days. We also observed 

that TCR RNA transfection of CD4+ T cells 

turned them into potent CMVpp65/HLA-A*0201specific 

T helper cells. This was demonstrated by 

co-incubating them with immature dendritic cells 

(DC), which resulted in maturation of DC only in 

the presence of the CMV pp65 epitope. 

Next, we transfected pure naïve and memory CD8+ 

T cell subsets isolated from peripheral blood of 

CMV-seronegative donors. Although 90% of naïve 

CD8+ T cells were CMVpp65/HLA-A*0201 tetramer 

positive after electroporation, they mediated only 

marginal lysis toward CMV-infected fibroblasts. 

In contrast, memory CD8+ T cells showed strong 

TCR expression and cytotoxicity upon antigen recognition 

up to one week. In summary, our data demonstrate 

that non-reactive human T cells can be 

easily redirected with CMVpp65 TCR RNA, thereby 

gaining CMV-specific T cell effector function for a 

considerable time period. 

We therefore believe that CMVpp65 TCR RNA has 

the potential to be further developed as a therapeutic 

‘off-the-shelf’ reagent for CMV-seropositive 

patients who undergo allogeneic HSCT from CMVseronegative 

donors.

051 Distler | Cellular therapy 

Alloreactivity to HLA class I mismatch alleles mainly resides in 

CCR7+ naive and central memory CD8 T cells 

Eva Distler 1 , Saliha Asdufan 1 , Michaela Frey 1 , Udo F. Hartwig 1 , Matthias Theobald 1 , Wolfgang 

Herr 1 


Reducing the incidence of severe graft-versus-host 

disease (GVHD) remains the major challenge in 

patients who receive stem cell transplants from 

HLA-mismatch donors. Selective depletion of alloreactive 

donor T cells can prevent GVHD, but is 

still a complex technical procedure that requires 

the in vitro culture of T cells for several days. Here, 

we studied CD8 T cell subsets of 5 healthy donors, 

sorted by flow cytometry according to expression 

of the differentiation markers CD45RA, CD45RO, 

CCR7 and CD62L for the ability to respond to allo- 

HLA class I alleles in vitro. To detect pure alloreactive 

T cell responses and minimize the interference 

by other specificities, we used HLA-deficient K562 

cells transfected with single HLA-A, -B or -C alleles 

for weekly stimulations. We observed the strongest 

anti-HLA mismatch proliferation in sorted CCR7pos 

and CD62Lpos fractions, comprising naive (TN) 

and central memory CD8 T cells (TCM). In contrast, 

the CCR7neg and CD62Lneg counterpart fractions 

containing effector memory (TEM) and terminal 

effector CD8 T cells showed much lower allo-HLA 

induced in vitro expansion. The CD45RApos subset 

enriched for TN cells and devoid of TCM and TEM 

cells demonstrated less strong allo-proliferation 

compared to corresponding CD45RAneg cells. Low 

allo-proliferation was observed for CD45ROpos and 

CD45ROneg populations. Interestingly, the growth 

pattern of CD8 T cell subsets was consistent, regardless 

of HLA-A, -B, or -C mismatch alleles were 

used for stimulation. Cultures derived from all CD8 

T cell subsets were analyzed for allo-HLA class I reactivity 

by IFN-γ ELISPOT assay starting on day 11. 

The strongest anti-HLA mismatch reactivity was 

detected in the CCR7pos subset. Lower level of alloreactivity 

was observed in CD62Lpos, CD45ROneg, 

and CD45RApos fractions. All counterpart fractions 

showed even less strong anti-HLA responses. 

Again, these findings were consistent for all HLA 

alleles tested. Our results thus demonstrate that distinct 

CD8 T cell subsets vary substantially in the 

ability to respond to HLA class I mismatch alleles. 

Although all subsets enriched for TN and TCM 

produced substantial alloreactivity, the CCR7pos 

fraction exceeded others in terms of proliferation 

and effector function. Thus, in vitro removal of 

CCR7pos CD8 T cells may be a straightforward culture-independent 

allodepletion procedure. Further 

experiments comparing anti-HLA mismatch and 

self-HLA-restricted anti-leukemia reactivity of T 

cell subsets are ongoing. 

95

052 Salguero | Cellular therapy 

Dramatic early expansion of CMV-reactive human T cells in 

vivo is supported by lentiviral vector-induced engineered dendritic 

cells 

Gustavo Salguero 1 , Bala Sai Sundarasetty 1 , Dirk Wedekind 2 , Sylvia Borchers 1 , Gregor 

Warnecke 3 , Ann-Kathrin Knöfel 3 , Rainer Blasczyk 4 , Britta Eis-Vesper 4 , Eva Mischak-Weissinger 

1 , Arnold Ganser 1 , Renata Stripecke 1 

1 

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, 

Hannover Medical School, Hannover, Germany 

2 

Central Animal Laboratory, Hannover Medical School, Hannover, Germany 

3 Department of Experimental Transplantation, Hannover Medical School, Hannover, Germany 

4 Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany 

96 

Dendritic cell (DC) based immunotherapy is a 

potent strategy to induce antigen-specific responses 

against cancer and infections. Strategies 

to enhance the viability, biodistribution and immunostimulatoy 

capacity of ex vivo generated DC in 

vivo, may also be critical to determine the success 

of adoptive T cell therapies. We have previously 

shown that genetically reprogramming of human 

monocytes using bicistronic lentiviral vectors (LV) 

expressing GM-CSF and IL-4 induces self-differentiated 

DCs (SMART-DC) with improved engraftment, 

viability and biodistribution in vivo (Sundarasetty 

et al., CIMT 2009). Here, we assessed the in vivo 

potential of SMART-DCs to support engraftment 

and expansion of human T cells reactive against 

cytomegalovirus (CMV). Human SMART-DCs were 

generated from CD14+ monocytes obtained from 

CMV-seropositive donors and co-transduced with 

a LV expressing the full-length CMV protein pp65 

(SMART-DC/pp65) for analyses of antigen-specific 

responses. SMART-DC/pp65 showed a stable DC 

immunophenotype in vitro (CD209+, HLA-DR+, 

CD86+) and high viability in vitro (>3 weeks) and 

in vivo (>6 weeks). NOD.Rag1-/-.IL2rγ-/- immunodeficient 

mice were preconditioned with SMART- 

DC or with SMART-DC/pp65 injected s.c. and 7 

days later infused i.v. with autologous T cell expressing 

the firefly luciferase (fLUC). The engraftment, 

expansion and biodistribution of T cells were 

evaluated by serial in vivo bioluminescence image 

analysis, flow cytometry and immunohistochemi- 

stry. Control arms with “conventional” DCs alone 

or pulsed with pp65 overlapping peptides were included. 

SMART-DC-preconditioned mice showed 

significantly enhanced engraftment of fLUC-T cells 

in spleen (day 14, 6-fold p

053 Stolle | Cellular therapy 

Generation of HCMV-/TAA-bispecific human T cells by 

either the genetic equipment of HCMV+ T cells with 

tumor-reactive TCRs or the combined retroviral transduction 

of bulk human T cells with HCMV-/TAA-specific TCRs 

Diana Stolle 1 , Beate Hauptrock 1 , Hakim Echchannaoui 1 , Edite Antunes 1 , Simone Thomas 1 , 

Sebastian Klobuch 1 , Wolfgang Herr 1 , Matthias Theobald 1 , Ralf-Holger Voss 1 


Immune suppression after allogeneic stem cell 

transplantation causes reactivation of human cytomegalovirus 

(HCMV) and comes along with increased 

mortality in HCMV positive patients. The 

aim of cellular immunotherapy is the eradication 

of tumor cells and the decrease of the HCMV viral 

load by adoptive transfer of HCMV-/tumor associated 

antigen (TAA)-bispecific T lymphocytes. 

Therefore, we performed two different strategies: 

First, we generated HCMV-reactive (HCMV+) T 

cells from seropositive human blood donors using 

pp65(NLVPMVATV(495-503)) peptide-specific stimulation. 

In this case one or two restimulations 

led to the efficient accumulation of up to 80 % 

HCMV-specific T cells. To obtain a pure HCMV T 

cell culture we sorted the cells with CMV pp65specific 

streptamers by flow cytometry. Cytotoxicity 

was assessed in a 51-Chromium release assay 

using either pp65- loaded K562-A2 or HCMV-infected 

HLA-A*0201+ human fibroblasts as target 

cells. For distinct HCMV+ donors, minimal pp65 

peptide concentrations were in the range of as low 

as 0.03 nM to 100 nM triggering specific lysis at 

CD8+ HCMV+ to target ratios of 5:1 up to 20:1. 

They were indicative of high TCR affinities for the 

cognate antigen reflected by EC50-values ranging 

from 0.1 nM to 1.8 nM in peptide titration. Most importantly, 

HCMV T cells were efficiently capable of 

lysing CMV-infected human fibroblasts. After their 

antigen-specific expansion, HCMV-reactive T cells 

were equipped with a p53- or gp100- tumor anti- 

genspecific TCR by either RNA transfection or retroviral 

transduction. RNA electroporation yielded 

the generation of p53- as well as gp100- bispecific 

HCMV+ T cells: Both subsets proved efficient 

bifunctionality in CTL-assay and IFNg-Elisa after 

endogenous (pp65)- or exogenous (p53/gp100) 

peptide-specific stimulation. Preliminary data indicated 

that in retroviral transduction either TCR, 

the endogenous TCR CMV or the introduced TCR, 

are prone to be down-regulated dependent on the 

particular HCMV/TAA TCR combination. 

Our second approach was the simultaneous retroviral 

transduction of bulk human T cells with a 

HCMV- as well as p53 tumor antigen- specific TCR. 

Both retroviral TCR constructs contained drug selection 

cassettes which allowed for a normalization 

of TCR expression. In tetramer analysis, we 

were able to detect both, single TCR+ T cells and a 

substantial fraction of HCMV/TAA double TCR+ T 

cells. Endogenous TCRs were still present. Bifunctionality 

was corroborated in CTL-assays and in intracellular 

IFNg secretion stainings for specifically 

restimulated T cells. 

In summary, our data demonstrate two opportunities 

to generate virus (HCMV) and tumor antigen 

bispecific human T cells. Most importantly, the 

expression level of the HCMV- as well as tumor 

antigen- specific TCR is sufficient for specific 

antigen recognition. 

97

054 Bloetz | Cellular therapy 

Allo-reactivity to HLA class II mismatch alleles in CD4+ 

T-cell subsets 

Andrea Blötz 1 , Eva Distler 1 , Elke Schnürer 1 , Simone Thomas 1 , Ugur Sahin 1 , Matthias Theobald 

1 , Wolfgang Herr 1 

1 Dept. of Medicine III – Hematology & Oncology, University Medical Center, Mainz, Germany 

98 

In allogeneic hematopoietic stem cell transplan- 

tation (allo-HSCT), allo-reactive T lymphocytes 

of donor origin recognize antigens expressed on 

patient-derived leukemia cells, thereby mediating 

the beneficial graft-versus-leukemia effect. If alloantigens 

are expressed on non-hematopoietic recipient 

tissues, donor T cells also induce graft-versushost 

disease (GvHD). Since HLA mismatch alleles 

represent major targets of allo-reactive T lymphocytes, 

patient and donor are usually matched for 

the class I molecules HLA-A, -B, -C, and for the 

class II molecules HLA-DRB1 and -DQB1, in order 

do reduce the risk of GvHD. The HLA-DPB1 locus, 

however, is still ignored in donor selection. Interestingly, 

clinical studies have demonstrated that 

disparities at HLA-DQB1 and distinct HLA-DPB1 

alleles do not adversely affect the outcome of allo- 

HSCT. Because HLA class II is predominantly expressed 

on hematopoietic cells, allo-reactive CD4+ 

donor T cells recognizing HLA-DQB1 or permissive 

HLA-DPB1 mismatch alleles may primarily target 

patient leukemic and hematopoietic cells, while 

sparing non-hematopoietic recipient tissues. In this 

ongoing study we analyze the allo-reactive potential 

of CD4+ T-lymphocyte subsets by stimulating 

them in vitro against single HLA class II mismatch 

alleles. For that purpose, CD4+ peripheral blood T 

lymphocytes of healthy donors are sorted by flow 

cytometry according to the expression or absence 

of the differentiation markers CD45RA, CD45RO, 

CD62L and CCR7. As standard antigen-presenting 

cells for class II-mismatch stimulations, we use the 

HLA-deficient cell line K562 upon electroporation 

with in vitro transcribed RNA coding for the alpha 

and beta chains of single HLA class II molecules 

(HLA-DQ: DQA1-010201/DQB1-060201; HLA-DR: 

DRA1-0101/DRB1-0701). This procedure results in 

transient HLA expression for up to one week with 

strongest staining intensity (>80% positive cells) 

24h after electroporation. In addition, transfectants 

do not lose HLA class II expression upon freezing, 

thawing and irradiation cycles. In a first series of 

experiments, sorted CD4 T-cell subsets of 3 healthy 

donors were stimulated weekly with mismatched 

K562/HLA-DR or -DQ transfectants, respectively, 

and were tested for allo-reactivity 5 days after the 

first (d12) and second (d19) restimulation in IFN-γ 

ELISPOT assays. The strongest allo-HLA II reactivity 

was detected in the CD45RApos and CD62Lpos 

as well as in the CD45ROneg CD4+ subsets, in contrast 

to the corresponding counterpart fractions, 

confirming that allo-reactivity can be found mainly 

in naive and central memory T cells. Alloreactivity 

of the CCR7pos and CCR7neg cell fractions varied 

considerably. In all cases, anti-HLA class II reactivity 

of CD4 populations could be blocked by monoclonal 

antibodies to the respective HLA class II 

molecule used for stimulation. Proliferation did not 

show clear differences between individual CD4+ 

T-cell subsets. In summary, we show herein that 

the transient expression of HLA class II molecules 

in K562 cells by RNA electroporation is a rapid and 

efficient approach to detect and stimulate allo-HLA 

class II reactive CD4+ T-cell responses. The strongest 

class II mismatch reactivity was found in the 

naive and central memory CD4+ T-cell subsets. 

This approach based on K562 cells and HLA class 

II RNA as „off-the-shelf” reagents allows to rapidly 

generate CD4+ T cells with reactivity to single allo- 

HLA-DQ/DP alleles, which may be of potential use 

in adoptive immunotherapy of leukemias.

055 Stumpf | Cellular therapy 

Indirect presentation of HLA class II restricted minor histocompatibility 

antigens is a characteristic of HLA-DM resistant 

antigens 

Anita Stumpf 1 , Edith van der Meijden 1 , Roel Willemze 1 , Fred Falkenburg 1 , Marieke Griffioen 1 

1 Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands 

HLA-matched allogeneic stem cell transplantation 

is an effective treatment for haematological malignancies. 

The anti-tumor effect is mediated by donor 

T-cells recognizing polymorphic peptides in HLA 

molecules which are differentially expressed on 

patient and donor cells due to amino acid differences 

encoded by single nucleotide polymorphisms, 

the so-called minor histocompatibility antigens 

(MiHAs). Donor T-cells recognizing MiHAs 

with broad expression on hematopoietic as well as 

non-hematopoietic cells may not only induce beneficial 

antitumor immunity, but also detrimental 

graft versus host disease (GvHD). We recently identified 

5 HLA class II restricted MiHAs which were 

not directly recognized on nonhematopoietic cells 

even after HLA class II upregulation by cytokine 

treatment. However, it was recently shown in mice 

(Wang, ASH 2009, abstract no. 689) that indirect 

presentation of host antigens by donor antigen presenting 

cells (APC) contributed to CD4+ T cell mediated 

GvHD. Here, we analyzed whether this is a 

general phenomenon by analyzing processing and 

intercellular transfer of six HLA class II restricted 

antigens. 

As a model to investigate recognition by CD4+ T 

cells, we exogenously pulsed the nonhematopoietic 

HeLa cell line transduced with the respective HLA 

class II alleles with bacteria expressing six different 

recombinant proteins. Antigen processing was 

analyzed after retroviral transfer of invariant chain 

(Ii) with or without HLA-DM. Intercellular transfer 

of antigens was analyzed by co-incubation of HLA 

II+/MiHA- cells with HLA II-/MiHA+ cells or their 

culture supernatants. 

Recognition of all HLA class II restricted antigens 

was increased upon transduction of Ii. Additional 

transfer of HLA-DM, however, had divergent effects. 

Following HLA-DM transfer, 3 antigens were similarly 

recognized (DM-resistant), whereas recognition 

of the other 3 antigens was completely abolished 

(DM-sensitive). We observed intercellular transfer 

of DM-resistant, but not DM-sensitive, antigens to 

MiHA- /HLA II+ cells after co-incubation with 

MiHA+/HLA II- cells. Also in the absence of direct 

cellcell contact the antigen could be transferred by 

cell-derived particles as was shown by incubation 

with supernatant from MiHA+ cells. This transfer 

was not mediated by free peptides as 30kDa filtration 

of the supernatant abolished recognition. 

In summary, we show that some antigens can be 

intercellularly transferred leading to T cell recognition 

of MiHA-/HLA II+ cells. However, this phenomenon 

was restricted to a group of antigens that 

showed HLA-DM-resistant processing and presentation. 

These data suggest that late after allogeneic 

stem cell transplantation, when patient-derived 

APCs are replaced by the donor, only HLA-DMresistant 

antigens may be indirectly presented by 

donor APC, and therefore not every MiHA mismatch 

may evoke late onset GvHD. 

99

056 Hoyer | Cellular therapy 

Examination of T-helper cell / DC cross-talk by the transfection 

of T cells with RNA coding for TCRs 

Stefanie Hoyer 1 , Sabrina Schievelbein 1 , Gerold Schuler 1 , Jan Dörrie 1 ,*, and Niels Schaft 1 ,* 


* Contributed equally 

100 

The goal of immunotherapy of cancer is to initiate 

an adaptive immune response against the tumor. 

But up to now, cancer immunotherapy has mainly 

focused on the generation of tumor-specific CD8+ 

T cells, even though CD4+ T-cell help is also required 

for a long-lasting cytotoxic T lymphocyte 

response. However, the exact mechanism by 

which CD4+ T cells license dendritic cells (DC) 

and thereby modulate the priming and expansion 

of CTLs is not yet fully understood. For this 

purpose, we transferred melanoma-antigen-specific 

TCRs by RNA-electroporation into CD4+ T cells 

and co-cultured them with peptide-loaded DC to 

elucidate T-cell / DC cross-talk. We either introduced 

MAGE-A3/HLA-DP4-specific or gp100/HLA- 

A2-specific TCR-RNA into CD4+ T cells, which 

mainly resulted in antigen-specific Th1 cytokine 

secretion after co-cultivation with peptide-loaded 

DC. Besides, phenotypic changes were assessed in 

a time-dependent manner. We detected a clear antigen-specific 

up-regulation of maturation markers 

like CD25, CD40, CD80, CD86, and CD70 on both immature 

and mature DC after 20 hours of co-cultivation 

with T cells. In correlation, CD25 was antigenspecifically 

up-regulated as an activation marker 

on CD4+ T cells. The early activation marker CD69 

was antigen-specifically up-regulated already after 

2 hours, while CD27- and CCR7-expression on the 

T cells remained almost constant during the stimulation. 

Moreover, T-cell activation was completely 

cell-cell-contact dependent, while the maturation 

of the DC was in part mediated by soluble factors, 

as revealed by transwell experiments. These data 

indicate that DC / CD4+ T-cell cross-talk is a bidirectional 

process. Moreover, we currently examine 

the influence of pre-activation of CD4+ T cells on 

the antigen-specific cross-talk, and whether there 

is an antigen-specific cross-talk between DC and 

CD8+ T cells. Since TCR-transfected CD4+ T cells 

can induce DC maturation, they may be used to 

provide T-cell help to induce more efficient CD8+ 

T-cell responses for the immunotherapy of cancer.

057 Foerster | Cellular therapy 

Generation of multivirus-specific CD4+ and CD8+ T cells 

for adoptive immunotherapy 

Anna Foerster, Verena Lasmanowicz, Olaf Brauns, Sven Kramer, Petra Jekow, Wolfgang Rönspeck, 

Jürgen Schmitz, Mario Assenmacher, Anne Richter 

Department of Research & Development, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany 

Adoptive transfer of T lymphocyte populations 

with specificities for several antigens is an attractive 

strategy for the treatment of multiple infections 

in immunocompromised patients or of cancer. We 

have established a protocol for the generation of 

multiantigen-specific CD4+ and CD8+ T cells 

using newly developed pools of peptides for in vitro 

T cell stimulation and a subsequent magnetic enrichment 

of functional T cells according to IFN-γ 

secretion.Peptide pools consist of synthetic peptides 

of mainly 15 amino acid (aa) length with 11 

aa overlap covering the complete antigenic protein. 

We have analyzed the efficiency of peptide pools 

compared to recombinant proteins and immunodominant 

HLA-A2 and –B7-restricted peptides for 

short-term in vitro restimulation and induction of 

IFN-γ in cytomegalovirus (CMV) pp65 and IE-1 specific 

CD4+ and CD8+ T cells derived from CMVinfected 

healthy blood donors. 

The results show that the peptide pools as well as 

the recombinant proteins are useful for efficient 

activation of CD4+ T helper cells. In contrast, efficient 

stimulation of CD8+ T cells is achieved only 

using either the overlapping 15-mer peptide pools 

or the immunodominant peptide epitopes of 8-10 

amino acids. The latter are well defined only for 

a limited panel of antigens and are restricted to 

certain HLA alleles. 

To test the usability of peptide pools to generate 

multivirus-specific T cells for adoptive immunotherapy 

we stimulated PBMC from leukapheresis of 

healthy donors with a combination of four peptide 

pools selected from CMV pp65 and IE-1, adenovirus 

(AdV) hexon, and Epstein-Barr-Virus (EBV) EBNA-1 

and BZLF-1 for four hours. The Large Scale IFN-γ 

Secretion Assay Enrichment Kit was used to magnetically 

enrich IFN-γ secreting T cells to a purity of 

>90%. Antigen specificity and functionality of the 

enriched T cells were controlled after expansion. 

Cells expanded between 4 and 745 fold within 9-14 

days. Expanded cells contained high frequencies 

of pp65495-503/A2 tetramer+ and pp65417-426/B7 

tetramer+ CD8+ T cells. Additionally after restimulation 

of the expanded cells with the mixture 

of peptide pools and intracellular IFN-γ staining, 

21-53% of CD4+ T cells and 53-87% of CD8+ T 

cells produced IFN-γ. Moreover comparing the stimulation 

of PBMC with reactivation of the T cell 

lines with each peptide pool separately showed that 

the specificity for each antigen sustained during 

the enrichment and expansion phase. 

The concomitant addition of four peptide pools increases 

the competition of peptides to be loaded on 

MHC molecules, which might decrease the efficient 

activation of each antigen-specific T cell. Therefore 

we included in our study the separation of PBMC 

in four samples, separate loading of the samples 

with one peptide pool for two hours, and recombination 

of PBMC for T cell stimulation for four 

hours. We found comparable results in the number 

of enriched cells, expansion rate of T cells, and the 

antigen-specificity of the T cell lines for concomitant 

and separate antigen loading. 

In summary, we have established a protocol for 

rapid in vitro generation of multivirus-specific 

CD4+ and CD8+ T cells using a combination of 

peptide pools from several antigens for restimulation 

and subsequent magnetic selection of IFN-γ 

secreting T cells. 

101

058 Echchannaoui | Cellular therapy 

Evaluation of the safety of p53 TCR gene transfer in a 

humanized mouse model 

Hakim Echchannaoui 1 , Edite Antunes 1 , Carina Lotz 2 , Simone Thomas 1 , Ralf-Holger Voss 1 

and Matthias Theobald 1 


2 Actelion Pharmaceuticals Ltd, Allschwil, Switzerland 

102 

Adoptive cell therapy with T cells retrovirally trans- 

duced with tumor-associated antigen (TAA) specific 

TCRs is a promising approach for immunotherapy 

in patients with haematological malignancies. The 

TAA p53 is over-expressed in approximately 50% of 

human tumors. We have reported that HLA-A*0201 

(A2.1) transgenic mice can be used to circumvent 

self-tolerance to universal human TAA and to generate 

efficient tumor-reactive CTL. We used A2.1 

transgenic mice, in which the mouse CD8 molecule 

cannot efficiently interact with A2.1 to generate a 

high-affinity, CD8-independent p53(264-272) specific 

TCR. Retroviral expression of CD8-independent 

p53-specific TCR into T cells, allowed CD8+ T lymphocytes 

to acquire a broad tumor-specific CTL activity 

but also redirected CD4+ T cells into potent 

tumor-reactive, p53-specific T helper cells. However 

a particular safety concern with TCR gene transfer, 

is the formation of mixed TCR heterodimers 

between the introduced TCR α and β chains with 

the endogenous TCR chains, resulting in the potential 

generation of autoreactive T cells. To reduce 

the formation of TCR mixed dimers an additional 

inter-chain disulfide bond between the TCR α and β 

chain constant domains was introduced. We further 

improved the expression level of p53 TCR transgene 

using codon-optimization of the TCR sequence. 

Mouse T cells transduced with cysteine-modified 

(Cys) and codon-optimized (Opt) p53(264-272)A2.1 

TCR showed higher expression levels of the introduced 

TCR as compared to p53(264-272)-specific 

WT TCR. Importantly, p53(264-272)A2.1-Opt.Cys 

TCR transduced CTL recognized and killed a wide 

variety of malignant A2.1 tumor cells with altered 

p53 expression, but not p53-deficient A2.1 cells 

more efficiently as compared to CTL transduced 

with the WT p53(264-272)-specific TCR. 

However, when p53(264-272)A2.1-Opt.Cys TCR 

transduced mouse T cells are adoptively transferred 

into p53-deficient partially humanized (A2Kb) 

mice, under conditioning-induced lymphopenia, 

expansion of infused T cells following high dose 

IL-2 administration is associated with the development 

of lethal autoimmunity similarly to mice 

receiving p53(264-272)-specific WT TCR transduced 

T cells due to the formation of self-reactive 

TCRs. The so called off-target toxicity observed in 

our preclinical mouse model highlights the need 

for further improvement of TCR gene therapy to 

prevent TCR mispairing-induced autoimmunity. To 

address this concern, a novel single chain p53TCR 

construct was engineered and is currently under 

investigation.

059 Bourquin | Cellular therapy 

Efficient eradication of subcutaneous but not of autochthonous 

gastric tumors by adoptive T cell transfer in a SV40 T antigen 

mouse model 

Carole Bourquin 1 , Philip von der Borch 1 , Christine Zoglmeier 1 , David Anz 1 , Nadja Sandholzer 

1 , Cornelia Wurzenberger 1 , Robert Kammerer 2,3 , Wolfgang Zimmermann 2 , Stefan Endres 1 

1 Center of Integrated Protein Science Munich (CIPS-M) and Division of Clinical Pharmacology, 

University Hospital of Munich, 80336 Munich, Germany 

2 Tumor Immunology Laboratory, LIFE-Center, Grosshadern, Ludwig-Maximilian University, 

Marchioninistr. 23, 81377 Munich, Germany 

3 Institute of Immunology, Friedrich-Loeffler-Institut, Paul-Ehrlich-Straße 28, 72076 Tuebingen, Germany 

In stomach cancer, there is a need for new thera- 

peutic strategies, in particular for the treatment of 

unresectable tumors and micrometastases. We investigated 

the efficacy of immunotherapy in an autochthonous 

model of gastric cancer, the CEA424- 

SV40 T antigen (TAg) transgenic mice. Treatment 

efficacy against both the autochthonous tumors 

and subcutaneous tumors induced by the derived 

cell line mGC3 were assessed. In wild-type mice, 

a dendritic cell vaccine loaded with irradiated 

tumor cells combined with CpG oligonucleotides 

induced efficient cytotoxic T cell and memory responses 

against mGC3 subcutaneous tumors. In 

contrast, neither subcutaneous nor autochthonous 

tumors responded to vaccination in CEA424-SV40 

TAg mice, indicating tolerance to the SV40 TAg. 

To examine whether tumors in these mice were 

principally accessible to immunotherapy, splenocytes 

from immune wild-type mice were adoptively 

transferred into CEA424-SV40 Tag transgenic mice. 

Treated mice showed complete regression of the 

subcutaneous tumors associated with intratumoral 

infiltrates of CD8 and CD4 T cells. In contrast, 

the autochthonous gastric tumors in the same mice 

were poorly infiltrated and did not regress. 

Thus, even in the presence of an active antitumoral 

T cell response, autochthonous gastric tumors 

do not respond to immunotherapy. This is the first 

comparison of the efficacy of adoptive T cell transfer 

between transplanted subcutaneous tumors 

and autochthonous tumors in the same animals. 

Our results suggest that in gastric cancer patients, 

even a strong antitumor T cell response will not 

efficiently penetrate the tumor in the absence of additional 

therapeutic strategies targeting the tumor 

microenvironment. 

103

060 Schmitt | Cellular therapy 

WT1 and RHAMM specific CD8+ T cells can be isolated 

and purified by streptamers for adoptive transfer 

Anita Schmitt, Xu Xun, Xinchao Wang, Inken Hilgendorf, Kersten Borchert, Mathias 

Freund, Michael Schmitt 

Department of Internal Medicine III, University of Rostock, Rostock, Germany 

104 

Background: A positive selection of leukemia as- 

sociated antigen (LAA)-specific T cells would be 

highly desirable to amplify the GVL effect and to 

decrease the risk of GVHD. Wilms Tumor gene 1 

(WT1) and the receptor for hyaluronic acid mediated 

motility (RHAMM) are LAAs recognized by 

CD8+ T lymphocytes. Streptamers constitute a 

novel multimer technology to select specific CD8+ 

T cells, available at good manufacturing production 

(GMP) level. MATERIAL AND METHODS: Both tetramers 

and streptamers were used to detect the 

frequency of HLA-A2 restricted CD8+ T cells in 

the naïve peripheral blood (PB) from both healthy 

donors (HDs) and AML patients. RHAMM- and 

WT1-specific CD8+ T cells were further characterized 

for the expression of CD27, CD28, CD45RA 

and CCR7. In the next step, LAA-specific cells were 

positive selected by MACS columns after labeling 

with streptamers and thereafter immunophenotyped. 

Moreover, mixed lymphocyte peptide cultures 

(MLPCs) were performed to enrich WT1 specific 

T cells derived from the PB of HDs. RHAMM 

and WT1 specific cells were subjected to carboxylfluorescein 

succimidylester (CFSE) based proliferation 

and cytotoxicity assays. 

Results: 21 of 40 HDs showed naïve LAA specific T 

cell frequencies of 0.5 to 1.6% of all CD8+ T cells. 

In AML patients in complete remission significantly 

higher frequencies of LAA-specific T cells than 

in patients at diagnosis could be detected, up to 

6.0% of all CD8+ T cells. These cells revealed to be 

CD8+CD27-CD28+CD45RA+CCR7- effector T cells 

in flow cytometry. After positive selection by MACS 

columns a purity of 20-87% could be achieved for 

LAA-specific T cells. After a maximum of three 

rounds of MLPC, only a frequency of 2-5% could 

be achieved, thus demonstrating the power of the 

streptamer technology. Both tetramer- and streptamer-based 

selections yielded similar amounts of 

LAA-specific T cells. 

After positive selection, these T cells preserved an 

effector T cell phenotype and showed active proliferation 

in CFSE staining. Streptamer-selected T cells 

showed a trend towards higher CD28 expression 

thus indicating a more activated T cell status. 

Conclusion: In summary, the streptamer technology 

allows to select highly pure fractions of RHAMMand 

WT1-specific effector T cells with proliferative 

and cytotoxic properties. In analogy to DLIs specific 

for viral antigens such as CMVpp65, production 

of leukemia specific DLIs is feasible on a GMP level. 

Further leukemia antigens are currently evaluated 

by our group.

New targets & new leads 

105

061 Buchwald | New targets & new leads 

The ubiquitin-conjugase-8 targets active fms-like tyrosine 

kinase-3 for proteasomal degradation 

Marc Buchwald 1 , Thorsten Heinzel 1 , Frank D. Böhmer 2 , Oliver H. Krämer 1,3 

1 Friedrich-Schiller-University Jena, Center for Molecular Biomedicine Institute of Biochemistry and Biophysics 

2 

Friedrich-Schiller-University Jena, Center for Molecular Biomedicine Institute of Molecular Cell Biology, 

Hans-Knöll-Str. 2, D-07745 Jena, Germany 

3 

Friedrich-Schiller-University Jena, Center for Molecular Biomedicine, PD Dr. O. H. Krämer, 

Tel/Fax: 03641-949-362/949-352, e-mail: Oliver.Kraemer@uni-jena.de 

106 

The class III receptor tyrosine kinase FMS-like tyro- 

sine kinase 3 (FLT3) regulates normal hematopoiesis 

and immunological functions. Nonetheless, constitutively 

active mutant FLT3 (FLT3-ITD) causally 

contributes to transformation and is associated with 

poor prognosis of acute myeloid leukemia (AML) 

patients. Histone deacetylase inhibitors (HDACi) 

can counteract deregulated gene expression profiles 

and decrease oncoprotein stability, which renders 

them candidate drugs for AML treatment. However, 

these drugs have pleiotropic effects and it is often 

unclear how they correct oncogenic transcriptomes 

and proteomes. We report here that treatment of 

AML cells with the HDACi LBH589 induces the ubiquitin-conjugating 

enzyme UBCH8 and degradation 

of FLT3-ITD. Gain- and loss-of-function approaches 

demonstrate that UBCH8 and the ubiquitin-ligase 

SIAH1 physically interact with and target FLT3-ITD 

for proteasomal degradation. These ubiquitinylating 

enzymes though have a significantly lesser effect 

on wildtype FLT3. Furthermore, physiological and 

pharmacological stimulation of FLT3 phosphorylation, 

inhibition of FLT3-ITD auto-phosphorylation, 

and analyzing kinase-inactive FLT3-ITD revealed 

that tyrosine phosphorylation determines their proteasomal 

degradation. These results provide novel 

insights into anti-leukemic activities of HDACi and 

position UBCH8, which has been implicated primarily 

in processes in the nucleus, as a previously 

unrecognized important modulator of FLT3-ITD 

stability and leukemic cell survival.

062 Ashfield | New targets & new leads 

ImmTACs: bi-functional reagents for redirected tumour 

cell killing 

Rebecca Ashfield 1 , Linda Hibbert 1 , Nathaniel Liddy 1 , Giovanna Bossi 1 , Katherine Adams 1 , 

Anna Lissina 2 , Tara Mahon 1 , Namir Hassan 1 , Jessie Gavarret 1 Frayne Bianchi 1 , Nicholas 

Pumphrey 1 , Kristin Ladell 2 , Emma Gostick 2 , Andrew Sewell 2 , Nikolai Lissin 1 , Peter Molloy 1 , 

Yi Li 1 , Brian Cameron 1 , Malkit Sami 1 Emma Baston 1 , Penio Todorov 1 , Samantha Paston 1 , 

Rebecca Dennis 1 , Andy Johnson 1 , David Price 2 , Annelise Vuidepot 1 , Daniel Williams 1 , Bent 

Jakobsen 1 

1 Immunocore Ltd, 57C Milton Park, Abingdon, Oxfordshire, OX14 4RX UK 

2 Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, Wales, UK 

The human immune system can theoretically 

identify malignant cells by inspecting cell surface 

Class I HLA -peptide complexes for the presence of 

disease-associated epitopes. Indeed, many cancer 

patients generate CD8 cyto-toxic T cell responses 

to tumour-associated antigens; the majority of patients, 

however, fail to clear tumours since T cell 

avidity for self-antigens tends to be weak, and 

cancer cells employ escape mechanisms for avoiding 

destruction by T cells. To overcome these 

issues, we have engineered novel, bi-functional 

protein therapeutics termed ImmTACs (Immune 

Mobilising mTCR Against Cancer) which re-direct 

the immune system to target and destroy tumour 

cells with a high degree of potency and specificity. 

An ImmTAC comprises a high affinity ‘monoclonal’ 

T cell Receptor (mTCR) targeting a cancer-associated 

HLA-peptide complex, fused to an anti-CD3 

scFv domain which activates an anti-tumour T cell 

response. 

We demonstrate that ImmTACs against a number 

of different cancer-associated antigens can target 

and kill tumour cells expressing as few as 5-20 epitopes 

per cell with pico-Molar potency. ImmTACs 

preferentially activate effector memory CD8 T cells, 

resulting in secretion of multiple cytokines and 

tumour cell killing; a single activated T cell can kill 

multiple antigen positive tumour cells. Furthermore, 

we demonstrate that the reagents are able to 

inhibit tumour growth in mouse xenograft models. 

In vitro ImmTAC potency translates to a dose of 

less than 1mg in humans, representing a significant 

advance over existing targeted anti-cancer therapies 

including monoclonal antibodies. 

107

063 Domning | New targets & new leads 

Identification of acute myeloid leukemia-associated antigens 

recognized by allogeneic CD8+ T lymphocytes 

Sabine Domning 1 , Sylvia Köhler 1 , Eva Distler 1 , Christine Pohl 2 , Volker Lennerz 1 , Wolfgang 

Herr 1 , Thomas Wölfel 1 , Catherine Wölfel 1 

1 III. Medizinische Klinik, University Medical Center of the Johannes Gutenberg University, Mainz/Germany 

2 Institut für Pathologie, University Medical Center of the Johannes Gutenberg University, Mainz/Germany 

108 

CTL clones 1C6 and 4D7 against acute myeloid 

leukemia cells of patient MZ201 (AML FAB M5) 

were recently generated in allogeneic mixed lymphocyte 

leukemic cell cultures (allo- MLLC) using 

CD8+CD62Lhigh+ cells isolated from unrelated 

HLA class I-matched donor 332 (Distler, E., et al., 

2008). Both CTL clones were used for T cell-directed 

expression screening of a cDNA library constructed 

from MZ201-AML cells as previously described 

(Wölfel, C., et al., 2008). Library screening with 

4D7 led to the identification of a novel minor histocompatibility 

antigen, PLAUR-317P, encoded by a 

known polymorphic allele of the plasminogen activator 

urokinase receptor gene (PLAUR, syn.: uPAR, 

CD87; SNP rs4760) and restricted by HLAB* 56:01. 

Prolin (P) on position 317 generated an aggretope. A 

10mer carrying the polymorphic residue on its 2nd 

position (PLAUR-317P316-325: SPTITLLMTA) was 

recognized at lowest concentrations (half maximal 

lysis at 5nM) whereas the homologous PLAUR- 

317L316- 325 was not recognized. The frequency of 

PLAUR-317P was determined via LightCycler PCR. 

Seventy-four of 252 individuals (29,4%) carried one 

PLAUR-317P allele, which exceeds the frequency 

indicated for SNP rs4760 in public domain databases. 

Approximately 2% of individuals were homozygous 

for PLAUR-317P. PLAUR is known to be overexpressed 

in AML, especially in subtype FAB M5, 

in multiple myeloma and in various solid cancers. 

As the target of 1C6 lymphocytes we identified chemokine 

(C-X-C motif) ligand 3 (CXCL3). Its immu- 

nogenic peptide (RLLRVALLL, amino acids 14-22) 

was recognized in association with HLA-A*02:01 

and was derived from the signal sequence of CXCL3. 

The same peptide is also encoded by the related 

genes CXCL1 and CXCL2, that both induce recognition 

by 1C6 upon transfection. Due to its hydrophobicity 

a rather high concentration of the synthetic 

peptide was required in reconstitution assays (halfmaximal 

lysis induced at 0,3 µM). 1C6 recognized 

HLA-A*02:01+ leukemic cells of myeloid origin 

(AML and CML) (Distler, E., et al., 2008). Monocytes 

and endothelial cells, but not epithelial cells, 

isolated from HLA-A*02:01+ donors were weakly 

recognized, but their recognition could be strongly 

enhanced by pretreatment with proinflammatory 

molecules (IL1-ß, TNF-alpha and LPS). 

Using cDNA expression cloning with allogeneic 

leukemia-reactive T cells generated from a healthy 

donor we identified two antigens of distinct categories 

(mHag, structurally unaltered) that are 

overexpressed in myeloid leukemias and, among 

non-malignant cells, preferentially expressed in hematopoetic 

cells. Both antigens are candidates for 

GvL effects and GvHD. 

References: - Distler, E. et al. Acute myeloid leukemia (AML)reactive 

cytotoxic T lymphocyte clones rapidly expanded from 

CD8(+) CD62L(high)+ T cells of healthy donors prevent AML 

engraftment in NOD/SCID IL2Rgamma(null) mice. Exp. Hematol. 

36:451-463, 2008 - Wölfel, C. et al. Dissection and molecular analysis 

of alloreactive CD8+ T cell responses in allogeneic haematopoietic 

stem cell transplantation. Cancer Immunol. Immunother. 

57:849- 857, 2008

064 Hornig | New targets & new leads 

Combinatorial approach of a bispecific antibody and costimulatory 

antibody fusion proteins for targeted cancer 

immunotherapy 

Nora Hornig, Philipp Diebolder, Yvonne Eichinger, Roland E. Kontermann & Dafne Müller 

Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany 

Recombinant bispecific antibodies have shown to 

be able to retarget cytotoxic T cells to tumor cells 

in a MHC-independent manner, triggering effector 

cell activation and consecutive tumor cell killing. 

Considering that costimulation is an essential requirement 

not only to initiate T cell activation, but 

also for the regulation of a proper T cell response, 

we propose a combinatorial approach by a recombinant 

bispecific antibody and targeted costimulatory 

ligands in order to generate a highly efficient 

antitumoral immune response. In our model 

system we used a bispecific antibody recognizing 

the fibroblast activation protein (FAP) on tumor 

cells and CD3 on T cells. For targeted costimulation 

we generated antibody fusion proteins composed 

of a tumor binding antibody moiety and the extracellular 

domain of the costimulatory ligands B7.2 

and 4-1BBL, respectively. In view of the concomitant 

binding requirement of these constructs to the 

tumor cell, different antibody moieties, targeting 

the antigens FAP and endoglin on the tumor cell, 

were used. Costimulatory effects were assayed by 

incubating the fusion proteins with the bispecific 

antibody on tumor cells in presence of PBMCs and 

monitoring IL-2 and IFN-γ release. We showed that 

T cell activation induced by the bispecific antibody 

was significantly enhanced by targeted costimulation 

either with the B7.2 or the 4-1BBL fusion 

protein in a concentration-dependent and ligandspecific 

manner. 

Moreover, further signal enhancement was achie- 

ved by the combined application of both costimulatory 

fusion proteins, showing a synergistic effect. 

Thus, application of recombinant bispecific antibodies 

with combinations of tumor directed costimulatory 

fusion proteins of B7.2 and 4-1BBL might 

be a promising approach for efficient retargeting 

and activation of cytotoxic T lymphocytes in cancer 


109

065 Casares | New targets & new leads 

Suppression of Treg activity by a FOXP3 inhibitor peptide 

Noelia Casares, Laura Arribillaga, Francesc Rudilla, Diana Llópiz, José Ignacio Riezu-Boj, 

Pablo Sarobe, Francisco Borrás-Cuesta, Jesús Prieto and Juan José Lasarte 

Gene Therapy and Hepatology Area, University of Navarra, Center for Applied MedicalResearch (CIMA), 

Pamplona, Spain 

110 

Down-regulation of regulatory T (Treg) cell func- 

tion might be beneficial to enhance the immuno- 

genicity of viral and tumor vaccines or to induce 

breakdown of immunotolerance. Although the mechanism 

of suppression used by Treg cells remains 

controversial, it is already accepted that FOXP3 is 

the master gene in this subpopulation of cells. 

In this study, using a phage display random peptide 

library, we have identified a peptide, called P60, 

able to bind FOXP3 when measured by surface 

plasmon resonance. In vitro studies demonstrate 

that P60 inhibits murine and human Treg activity. 

In mice, it was found that P60 was able to restore 

the proliferation of murine spleen cells in response 

to anti-CD3 stimulation, which was inhibited by 

the presence of Treg. In human, P60 restored proliferation 

and IFN-g production of human peripheral 

mononuclear cells in a mixed leukocyte reaction 

inhibited by the addition of Tregs. Related to the 

mechanism of action, when we used a FOXP3-GFP 

transfection system in 293 cells, we found that P60 

is able to inhibit nuclear translocation of FOXP3. 

Moreover, P60 reduced the capacity of FOXP3 to 

inhibit NFAT and NF-kB transcriptional activity in 

Jurkat and 293 cells respectively. 

In vivo, selective inhibition of FOXP3 with P60 in 

newborn mice induced a lymphoproliferative autoimmune 

syndrome resembling the reported pathology 

in scurfy mice lacking functional FOXP3. 

However, P60 administration didn’t cause toxic 

effects in adult mice and, when given to BALB/c 

mice immunized with the cytotoxic T-cell epitope 

AH1 from CT26 tumor cells, it induced protection 

against tumor implantation. Similarly, P60 improved 

the antiviral efficacy of a recombinant adenovirus 

expressing NS3 protein from hepatitis C virus. 

These findings demonstrate that functional inhibition 

of Treg by the FOXP3-inhibitory peptide P60 

constitutes a strategy to enhance antitumor and 

antiviral immunotherapies.

066 Dettmar | New targets & new leads 

Identification of clinically useful combinations of trifunctional 

antibodies with chemotherapy using different preclinical 

models 

Kirsten Dettmar 1 , Petra Schroeder 2 , Carsten Lindemann 2 , Andrea Eberhardt 1 , Franziska 

Hirschhaeuser 3 , Diane Seimetz 1 , Judith Atz 1 

1 Fresenius Biotech GmbH, Munich, Germany 

2 EUFETS GmbH, Idar-Oberstein, Germany 

3 Institute of Physiology and Pathophysiology, University Medical Center of the Johannes Gutenberg-University 


The trifunctional bispecific antibodies catumaxo- 

mab (anti-EpCAM x anti-CD3) and ertumaxomab 

(anti-HER-2/neu x anti-CD3) exert their mechanism 

of action by simultaneous recruitment and activation 

of two different types of immune effector cells 

(T-cells and accessory cells) at the tumor site. Thus 

tumor cells and immune effector cells are brought 

into close proximity leading to physiological co-stimulation 

of T-cells, improved tumor cell elimination 

by different immunologic killing mechanisms 

and phagocytosis, as well as processing and presentation 

of tumor material by bound and activated 

accessory cells. 

Despite the development of different antibody therapies, 

chemotherapy (CTX) still represents the mainstay 

of cancer therapy. Therefore many antibodies 

are either used in combination with the established 

chemotherapeutic therapies or in short intervals afterwards. 

However, the commonly observed hematological 

toxicities of chemotherapeutics are regularly associated 

with some degree of immunosuppression. 

On the other hand, some of these chemotherapeutic 

agents are reported to support or even enhance the 

anti-tumor effect of immunotherapeutic drugs in 

certain doses. 

A largely intact immune system is the key for trifunctional 

antibodies to exhibit their full mode of action. 

Since chemotherapy might impact the number and 

/ or function of immune effector cells, we evaluated 

in different preclinical in vitro models whether the 

efficacy of trifunctional bispecific antibodies would 

be influenced when combined with chemotherapeutic 

drugs from different drug classes. 

The preclinical studies performed included in vitro 

cytotoxicity assays with established tumor cell lines 

evaluating potential synergy by using the method 

of Chou and Talalay. Furthermore, results from 3D 

tumor spheroids and from an autologous human 

ex vivo setting are presented. In addition, immune 

cells from cancer patients were obtained at different 

time points before, during and after chemotherapy 

and were assessed for their in vitro cytotoxicity mediated 

by trifunctional antibodies. 

So far the results from the different in vitro assay 

systems used indicate synergy for the combination 

of the trifunctional antibodies with 5-Fluorouracil 

(5-FU), cisplatin and epirubicin. Furthermore, no 

negative influence of these chemotherapeutic drugs 

was observed on the trifunctional mode of action 

in vitro. Catumaxomab and ertumaxomab were 

able to mediate an effective killing of tumor cells 

with immune cells from chemotherapy patients 

shortly after chemotherapy. The respective patients 

immune cells can be activated by these bispecific 

antibodies one week after chemotherapy with pyrimidine 

antagonists (5-FU) and alkylating agents 

(cisplatin) resulting in significant tumor cell killing 

in vitro. 

These results provide a basis for the possible combination 

of these drugs with trifunctional antibodies 

in the clinical setting. 

111

067 Hirschhaeuser | New targets & new leads 

Efficacy of catumaxomab in tumor spheroid killing is mediated 

by its trifunctional mode of action 

Franziska Hirschhaeuser 1 , Kirsten Dettmar 2 , Judith Atz 2 and Wolfgang Mueller-Klieser 1 



2 

Fresenius Biotech GmbH, Munich, Germany 

112 

Catumaxomab is an intact trifunctional bispecific 

antibody targeting human EpCAM (epithelial cell 

adhesion molecule) and CD3 with further binding to 

activating Fcγ receptor type I, IIa and III. We chose 

multicellular tumor spheroids (MCTS) of human 

EpCAM-positive FaDu tumor cells in co-culture with 

human peripheral blood mononuclear cells as an adequate 

three-dimensional in vitro model for pharmacological 

testing of catumaxomab. 

Besides catumaxomab, we used the F(ab`)2 fragments 

of catumaxomab and the parental antibodies 26/II/6 

(anti-human CD3) and HO-3 (anti-human EpCAM), as 

well as BiLu (anti-human EpCAM x anti-mouse CD3) 

which is not able to bind to human T cells. We assessed 

the spheroid volume growth, cytokine release (IL 2, IL 

6, IFN γ, TNF α), and immunohistological stainings for 

different immune cell types and for proliferation. 

After 6 days, spheroids cultured with the EpCAMtargeting 

antibody HO-3 showed almost the same 

volume as control spheroids without antibody. Combination 

of 2.5 ng/ml HO-3 with 2.5 ng/ml 26/II/6 

resulted in spheroid volumes which were reduced by 

61%; and approaches with 5.0 ng/ml 26/II/6 showed a 

70% volume reduction. Catumaxomab induced a 91% 

decrease in spheroid volume. Comparing the effects of 

26/II/6 and HO-3 to catumaxomab, demonstrates less 

efficacy of the parental antibodies either used alone or 

in combination. The importance of the CD3 binding 

site was emphasized by further control experiments 

with BiLu. Similar to HO-3, BiLu showed no tumor 

spheroid killing. The essential function of the intact 

Fc region was demonstrated in control experiments 

using the F(ab’)2 fragment of catumaxomab: The 

bispecific antibody fragment lacking this functional 

binding site resulted in a dose-dependent volume reduction 

of FaDu spheroids ranging from 54-81% for 

concentrations of 1.0, 2.5 and 10.0 ng/ml, while catumaxomab 

caused a decrease of 90-100% within the 

same concentration range. The highest concentrations 

of secreted IL 2 and IFN γ were observed in the setting 

with tumor spheroids, PBMCs and catumaxomab. 

Cultures of PBMC and 5.0 ng/ml 26/II/6 yielded the 

highest levels for IL 6 and TNF α. In contrast, incubation 

with BiLu did not induce cytokine secretion, 

and the incubation with F(ab’)2 resulted in increased 

cytokine levels only for 10.0 ng/ml, but several-fold 

less than induced by catumaxomab. The immunological 

stainings showed that the infiltrating lymphocytes 

are mainly CD8+ T cells in spheroids treated with 

catumaxomab and its F(ab’)2 fragment, whereas the 

total amount of CD45+ cells in catumaxomab treated 

spheroids was higher. 

In summary, the results show, that all binding partners 

of the postulated tricell complex have to be 

present to exert catumaxomab’s full mode of action. 

These distinct effects of catumaxomab are based on 

the unique composition of the trifunctional bispecific 

antibody. 

Acknowledgements 

The technical assistance of S. Dahms-Prätorius and R. Santos is 

greatly acknowledged. 

Supported by Fresenius Biotech GmbH and TRION Pharma GmbH 

(Munich, Germany).

068 Guthmann | New targets & new leads 

Cellular and humoral immune response to N-glycolyl-GM3 

elicited by racotumomab, an anti-idiotypic vaccine 

Marcelo D Guthmann 1 , Cecilia Venier 1 , Estrella Levy 1 , Mónica A Castro 2 , Gabriela Cinat 2 , 

Roberto Gómez 3 , Ana María Vázquez 4 , Leonardo Fainboim 1 

1 Hospital de Clínicas José de San Martín, University of Buenos Aires, Argentina 

2 Instituto de Oncología A. Roffo, University of Buenos Aires, Argentina 

3 Laboratorio Elea, Buenos Aires, Argentina 

4 Center of Molecular Immunology, Havana, Cuba 

Gangliosides are a family of sialylated glycolipids 

which are normal components of the cell membrane. 

They have been found to be important actors in 

multiple aspects of cellular interaction with its environment 

and with transmembrane signaling. As 

such, they are involved in cancer progression and 

have become the focus of several immunotherapeutic 

approaches. Not all gangliosides are equally 

immunogenic. N-acetyl-GM3, the main gangliosides 

on the cell surface and the most abundant 

ganglioside in normal serum, is one of the most 

immunologically tolerated member of the family. 

In contrast, N-glycolyl-GM3, is not expressed in 

human normal tissues due to a species-specific 

genetic mutation that abrogates the biosynthesis of 

N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has 

been reported, however, in human tumors, and its 

presence might be derived from dietary sources 

or a yet unknown alternate synthesis pathway. Nglycolyl-GM3 

is expressed in melanoma, breast and 

lung cancer cells, and is highly immunogenic. As a 

result, it has been considered as a target of choice 

for immunotherapy. 

We investigated with an extended vaccination protocol 

the immunogenicity and toxicity profile of racotumomab, 

an anti-idiotypic vaccine mimicking 

N-glycolyl-GM3. The year-long vaccination scheme 

consisted of six bi-weekly intradermal injections 

followed by 10 monthly boosters. Nineteen patients 

with high risk (stage III) or metastatic breast 

cancer were vaccinated with different dose levels 

of racotumomab (0.5, 1 and 2 mg). The humoral 

and cellular responses to racotumomab and to the 

targeted ganglioside were assessed at baseline and 

throughout the treatment. Anti-idiotype antibodies 

and anti-N-glycolyl-GM3 IgM and IgG antibodies 

were determined by ELISA. Serum antibody reactivity 

was also tested against P3X63 Ag8 653 murine 

myeloma cells and B16 murine melanoma cells. 

Frequency of N-glycolyl-GM3—reactive cells was 

calculated by IFNγ ELISPOT. For that purpose, cryopreserved 

PBMC and autologous DC were incubated 

with N-glycolyl-GM3—containing liposomes. 

Equivalent amounts of unloaded liposomes were 

added to control wells. After a 24-h culture, cells in 

each well were resuspended and transferred to triplicate 

wells in an IFNγ-precoated ELISPOT plate. 

Subsequent steps for detection of IFNγ-secreting 

cells followed standard ELISPOT procedures. Patients 

with no response at baseline and with a posttreatment 

two-fold increase in the number of spots 

in N-glycolyl-GM3 stimulated wells (versus unstimulated 

wells) were considered as responsive. 

All patients showed a strong antibody response to 

N-glycolyl GM3. In addition, ganglioside-specific 

IFNγ responses were recorded in 5 of 13 evaluable 

patients. 

113

069 Sørensen | New targets & new leads 

Cellular Immune Responses Against Indoleamine 

2,3-dioxygenase 

Rikke Bæk Sørensen, Inge Marie Svane, Per thor Straten and Mads Hald Andersen 

Center for Cancer Immune Therapy (CCIT), Department of Hematology, 54P4, University Hospital Herlev, 

Herlev Ringvej 75, DK-2730 Herlev, Denmark 

114 

Indoleamine 2,3-dioxygenase (IDO) is an immu- 

noregulatory enzyme that are implicated in sup- 

pressing T-cell immunity in normal and patholo- 

gical settings. Expression of IDO has been shown 

to induce T-cell anergy and/or the generation of 

adaptive regulatory T cells. In cancer patients IDO 

is expressed within the tumor itself as well as in 

antigen-presenting cells in tumor-draining lymph 

nodes, where it promotes the establishment of peripheral 

immune tolerance to tumor antigens. In the 

present study, we tested the notion whether IDO 

itself may be subject to immune responses. The 

study unveiled spontaneous cytotoxic T-cell reactivity 

against IDO in peripheral blood as well as 

in the tumor microenvironment of different cancer 

patients. We demonstrate that these IDO reactive T 

cells are indeed peptide specific, cytotoxic effector 

cells. Hence, IDO reactive T cells are able to recognize 

and kill tumor cells including directly isolated 

AML blasts as well as IDO-expressing dendritic 

cells, i.e. one of the major immune suppressive cell 

populations. Consequently, IDO may serve as an 

important and widely applicable target for anticancer 

immunotherapeutic strategies. Furthermore, 

as emerging evidence suggests that IDO constitutes 

a significant counter-regulatory mechanism 

induced by pro-inflammatory signals, IDO-based 

immunotherapy holds the promise to boost anticancer 

immunotherapy in general. 

Furthermore, we show that spontaneous cytotoxic 

T-cell reactivity against IDO exists in healthy 

individuals. Such IDO-specific T cells are able to 

boost immunity against CMV antigens by eliminating 

IDO+ suppressive cells. IDO-specific T-cells 

are induced in healthy individuals by non-specific 

inflammatory stimuli such as IFN-γ and IL-2. IDO 

specific T cells may play a vital role for the mounting 

or keeping of an effective adaptive immune 

response.

070 Andersen | New targets & new leads 

Identification of highly potent regulatory CD8+ T-cells 

specific for Heme Oxygenase-1 

Mads Hald Andersen 1 , Rikke Bæk Sørensen 1 , Marie Brimnes 1 , Inge Marie Svane 1 , Jürgen C 

Becker 2 , Per thor Straten 1 

1 Center for Cancer Immune Therapy (CCIT), Department of Hematology, Herlev University Hospital, 

Dk-2730 Herlev, Denmark 

2 Department of Dermatology, University of Würzburg, D-97080 Würzburg, Germany 

Regulatory elements are essential for the home- 

ostasis of the immune system. While regulatory 

CD4+T cells have received much attention over the 

past years, less is known about regulatory CD8+ T 

cells; moreover, potential antigens recognized by 

these cells remain elusive. In the present study we 

describe Heme Oxygenase-1 (HO-1) specific, HLA 

restricted, CD8 positive T-cells, which are able to 

suppress cells immune responses with an hitherto 

unseen efficacy. HO-1 has been defined as a “protective 

gene” with anti-inflammatory functions and 

is expressed at high levelt in malignant cells. Remarkably, 

HLA-restricted, HO-1 specific CD8 T-cells 

were readily detected directly ex vivo in peripheral 

blood lymphocytes obtained from cancer patients 

and in situ among tumor infiltrating T-cells. These 

HO-1 specific T cells were not only able to suppress 

IFN-γ release of antigen specific T cells, but also 

inhibited both the activity of CD4+ and CD8+ T 

cells in proliferation and cytotoxic assays. Notably, 

the inhibitory effect of HO-1 specific T cells was 

far more pronounced compared to conventional 

CD4+CD25+CD127- regulatory T-cells. The inhibitory 

activity of HO-1 specific T cells seems at least 

partly to be mediated by soluble factors. The identification 

of a new subset of highly potent, antigen 

specific CD8+ regulatory T-cells open new avenues 

for therapeutic interventions both in autoimmune 

diseases and cancer. 

115

071 Kleemann | New targets & new leads 

Identification of T-cell defined varicella-zoster virus proteins 

Patrick Kleemann 1 , Stefan Tenzer 2 , Eva Distler 1 , Eva Wagner 1 Ralf G. Meyer 1 , Sebastian 

Klobuch 1 , Simone Thomas 1 , Steffi Aue 3 , Bodo Plachter 3 , Wolfgang Herr 1 


2 Institute for Immunology, University Medical Center, Mainz, Germany 

3 Institute for Virology, University Medical Center, Mainz, Germany 

116 

Reactivated infection with varicella-zoster virus 

(VZV) causes herpes zoster. In immunodeficient 

cancer patients (e.g. after hematopoietic stem cell 

transplantation) dissemination of the virus can lead 

to life-threatening disease. Although an efficient 

live attenuated VZV vaccine for zoster prophylaxis 

exists, it is not approved in immunocompromised 

patients due to safety reasons. Knowledge of immunogeneic 

VZV proteins would allow designing a noninfectious 

nonhazardous subunit vaccine suitable 

for patients with immunodeficiencies. The objective 

of this study is to identify T-cell defined virus 

proteins of a VZV-infected Vero cell extract that we 

have recently described as a reliable antigen format 

for IFN-g ELISPOT assays (Distler et al. Biol. Blood 

Marrow Transplant. 2008, 14:1417-24). We first 

separated the VZV-infected/-uninfected Vero cell 

extracts by ultracentrifugation and reverse-phase 

HPLC. The collected fractions were screened for 

reactivity with peripheral blood mononuclear cells 

(PBMC) of VZV-seropositive healthy individuals 

by IFN-g ELISPOT assay. Using this strategy, we 

successfully identified bioactive HPLC fractions 

that contained immunogeneic VZV material. The 

immune reactivity was mediated by CD4+ memory 

T cells of VZV-seropositive healthy individuals as 

demonstrated by experiments with T-cell subpopulations. 

We next analyzed the bioactive HPLC 

fractions with MALDI and ESI mass spectrometry 

techniques and identified three (glycoprotein 

E; glycoprotein B and immediate early protein 62) 

different VZV derived proteins. In subsequent experiments 

we expressed these VZV-encoded proteins 

in dendritic cells (DCs) by in vitro transcribed 

RNA and screened transfected DCs with autologous 

T cells. Based on these experiments we established 

a protocol to perform these studies with PBMC of 

healthy donors and patients after hematopoietic 

stem cell transplantation. The work will hopefully 

lead to protein candidates of VZV to be included 

in a subunit vaccine, which can be safely used for 

zoster prophylaxis in immunocompromised cancer 

patients.

072 van Esch | New targets & new leads 

T cell epitope discovery through HLA class I peptide ligand exchange 

and combinatorial coding 

Wim van Esch 1 , Juk Yee Mok 1 , Annemieke Molenaar 1 , Arnold Bakker 2 , Mireille Toebes 2 , 

Sine Reker Hadrup 2 , Chengyi Shu 2 , Ernst Soethout 3 , Josine van Beek 3 , Huib Ovaa 4 and Ton 

Schumacher 2 

1 Dept. R&D, Sanquin Reagents, Amsterdam, The Netherlands 

2 Div. Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands 

3 Netherlands Vaccine Institute, Bilthoven, The Netherlands 

4 Div. Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands 

Human major histocompatibility complex class I 

(HLA-I) consists of beta2-microglobulin, a polymorphic 

heavy chain and a peptide ligand. Peptides 

in context with HLA-I on the cell surface are 

recognized by appropriate cytotoxic T cells. Our 

aim is to develop a platform for high-throughput 

identification of HLA-I ligands and T cell detection 

using flow cytometry in order to detect new 

disease-specific epitopes/T cells. To this end, conditional 

ligands that can be cleaved in the HLAbound 

state upon UV irradiation were designed 

for different HLA alleles. Cleavage of HLA-I-bound 

conditional ligand in the presence of another experimental 

ligand results in peptide exchange. 

Only those peptides that fit in the peptide binding 

groove will be able to maintain the integrity of the 

empty HLA complex, and the efficiency of HLA-I 

stabilization by experimental ligands can be determined 

by ELISA. Subsequently, HLA-I-binding 

peptides identified in this manner are tested for 

their immunological relevance. To this purpose, a 

combinatorial coding technology has been developed 

that allows the parallel detection of a multitude 

of different T cell populations in a single sample. 

Detection of antigen-specific T cells from peripheral 

blood by combinatorial coding is as efficient 

as detection with conventionally fluorescently-labeled 

HLA multimers and allows comprehensive 

screens to be performed. Using these technologies, 

several screens for HLA ligands have been performed, 

either manually or using robotics, using 

peptides potentially associated with infectious or 

non-infectious diseases. Large-scale screenings of 

their immunological relevance by analysis of T cell 

responses using combinatorial coding are ongoing. 

Together these techniques form a highly efficient 

platform for T cell epitope discovery in a broad 

range of human diseases and for the monitoring of 

disease- and therapy-induced T cell immunity. 

117

073 Suso | New targets & new leads 

Identification of novel CD4+ and CD8+ T cell epitopes from 

telomerase (hTERT) 

Else Marit Inderberg Suso 1 , Anne-Marie Rasmussen 1 , Sissel Trachsel 1 , Steinar Aamdal 2 , 

Svein Dueland 2 , Gunnar Kvalheim 3 , Gustav Gaudernack 1 

1 Section for Immunology, Oslo University Hospital-Radiumhospitalet, Norway 

2 Dept for Clinical Research, Oslo University Hospital-Radiumhospitalet, Norway 

3 Section for Cell Therapy, Oslo University Hospital-Radiumhospitalet, Norway 

118 

We have a number of patients who have been vacci- 

nated with the telomerase peptide vaccine GV1001 

or with dendritic cells (DCs) loaded with hTERT 

mRNA. 

Among these patients, several have had an extraordinary 

clinical course, involving disease stabilization 

over many years, partial and even complete 

remission of the disease. These patients offer a 

unique opportunity to study in detail the immune 

response against the vaccine. 

We have identified several novel hTERT epitopes by 

studying responses against a panel of 24 overlapping 

15-mer hTERT peptides as well as one 30-mer 

peptide. The results indicate that a profound epitope 

spreading within the hTERT antigen takes place in 

some patients following vaccination with a single 

T helper epitope (GV1001), and that many of these 

epitopes are recognized also following vaccination 

with hTERT transfected DCs. CD4+ T-cell clones 

specific for some of these peptides have been generated. 

In addition, CD8+ T cells specific for novel 

hTERT epitopes have been identified by pentamer 

staining. 

The recognition of these epitopes in a large number 

of patients further indicates a high degree of immunogenicity 

and HLA promiscuity. This also suggests 

that these peptides may be clinically relevant 

epitopes. Their identification is important for the 

development of the next generation of immunotherapy. 

We are now developing a second generation 

of hTERT peptide vaccines as well as T-cell receptor 

transfer based on “clinically validated” T cell receptors 

cloned from long term survivors.

074 Krug | New targets & new leads 

Transfer of mRNA encoding chimeric antigen receptors specific 

for MCSP into CD4+ and CD8+ T cells 

Christian Krug 1 , Katrin Birkholz 1 , Hinrich Abken 2 , Michael Schwenkert 3 , Georg Fey 3 , Gerold 

Schuler 1 , Jan Dörrie 1 ,*, and Niels Schaft 1 ,* 


2 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany 

3 Chair of Genetics, University of Erlangen-Nuremberg, Erlangen, Germany 

* contributed equally 

Adoptive transfer of bulk T cells with a tumoran- 

tigen-specific T-cell receptor (TCR) is an innova- 

tive and promising approach to treat malignancies. 

Chimeric antigen receptors (CAR), which consist 

of a scFv directed against a cancer surface antigen 

and the signaling domain of CD3-zeta and usually 

also of CD28 molecules, are an attractive alternative 

for normal TCR since MHC-restricted antigen 

presentation is not required. To avoid persistent 

auto-aggression, a reported life threatening risk of 

engineered T cells with constitutive CAR expression, 

we explored mRNA electroporation for transient 

receptor expression in human T cells. 

CAR, consisting of different antigen-binding and signaling 

domains, specific for melanoma-associated 

chondroitin sulfate proteoglycan (MCSP), which is 

expressed on melanomas, basal cell carcinomas, 

and some forms of childhood leukemias, were efficiently 

transfected into CD4+ and CD8+ T cells. 

Expression kinetics of the CAR were studied, and at 

day 9 after electroporation CAR-expression had disappeared. 

Upon specific stimulation with MCSP+ 

tumor cells, transfected CD4+ and CD8+ T cells 

secreted the cytokines IL-2, TNF-alpha, and IFNgamma. 

Moreover, the reprogrammed T cells were 

capable of killing target cells in an antigen-specific 

manner. The comparison of different CAR showed 

that using a binding domain with a higher affinity 

and stability improved the recognition of tumor 

cells. Furthermore, the incorporation of a CD28 

signaling domain sometimes improved the CAR 

surface expression, and always improved the lytic 

capacity and cytokine secretion of the T cells. 

In aggregate, this study shows for the first time a 

direct comparison of CAR with different scFv specific 

for the same antigen. Furthermore, RNA electroporation 

provides us with a technique to rapidly 

compare and choose CAR best suited for the immunotherapy 

of cancer. 

119

075 Lubojanski | New targets & new leads 

Identification of T cell-defined melanoma antigens 

S. Lubojanski 1 , M. Fatho 1 , A. Paschen 2 , D. Eberts 1 , C. Wölfel 1 , D. Schadendorf 2 , V. Lennerz 

1 , T. Wölfel 1 

1 III. Medizinische Klinik, University Medical Center Mainz 

2 Department of Dermatology, University Hospital Essen 

120 

CD8+ T lymphocytes can identify and eliminate 

cancer cells by recognizing peptides derived from 

tumor associated antigens (TAA) via HLA class I 

molecules. Although cancer cells are less immunogenic 

than pathogens, current knowledge predicts 

a high degree of individuality in tumor/T cell interaction. 

Even though some TAA and their epitopes 

have been identified, increasing knowledge of their 

identity should indicate the way to an effective strategy 

for specific immunotherapy [1]. 

The aim of this work is to identify and characterize 

T cell-defined antigens in the melanoma model 

MA-MEL-86. This model was derived from a female 

patient who had been first vaccinated with peptides 

and later on with tumor-lysate pulsed DCs. 

Three permanent melanoma cells lines had been 

established at different time points from resected 

lymph node metastases (MA-MEL-86A, -86B and 

-86C). These lines differ in their expression of 

the HLA I alleles. While MA-MEL 86A expresses 

HLA-A*01:01/-A*24:02, HLA-B*08:01/-B*15:01 and 

HLA-C*03:03/ -C*07:01; MA-MEL-86C had lost one 

haplotype (HLA-A*24:02, -B*15:01, -C*03:03), and 

due to a mutation in the ß2-microglobulin gene, 

MA-MEL-86B does not express HLA class I alleles 

at all. The enrichment of the melamoma-reactive T 

cells in autologous mixed lymphocyte-tumor cell 

cultures (MLTCs) was performed by weekly stimulation 

of peripheral blood lymphocytes (PBL) 

or PBL-derived CD8+ T cells against MA-MEL-86A 

and MA-MEL-86C, respectively. MLTC responders 

and T cell clones derived thereof were applied to expression 

screening of an antigen panel comprising 

39 known melanoma-associated antigens in association 

with the patient´s HLA class I alleles. They 

were also applied to expression screening of cDNA 

libraries constructed from cell lines MA-MEL-86A 

and -86C. For a detailed molecular analysis of the 

anti-tumor T cell repertoire. So far, three distinct 

antigens were identified in this model. MLTC responders 

stimulated with MA-MEL-86A were found 

to target a mutated neoantigen in association with 

HLA-B*15:01 (HERPUD1mut/B15). In addition, 

tumor-reactive T cells stimulated with MA-MEL- 

86C recognized known peptides from tyrosinase 

and gp100 both restricted by HLA-A*01:01. T cells 

against MA-MEL-86 cells predominantly recognize 

antigens not contained in the above mentioned 

panel. They are currently applied to further screening 

experiments. 

[1] Lennerz, V. et al. The response of autologous T cells to a 

human melanoma is dominated by mutated neoantigens. Proc 

Natl Acad Sci U S A. 102(44):16013-8, 2005

076 Staege | New targets & new leads 

Tumor antigens in Hodgkin lymphoma 

Martin S. Staege 1 , Carolin Winkler 1 , Daniela Max 1 , Wolfgang Altermann 2 , Gerald Schlaf 2 , 

Ursula Banning 1 , Dieter Körholz 1 

1 Department of Pediatrics, Childrens Cancer Research Center, Martin-Luther-University Halle-Wittenberg, 

06120 Halle, Germany 

2 University Medical Center, HLA-Laboratory, Martin-Luther-University Halle-Wittenberg, 

06120 Halle, Germany 

Hodgkin’s lymphoma (HL) is a lymphoproliferative 

disorder of unknown etiology. In most cases, molecular 

markers have suggested a B cell origin of the 

tumor cells. However, HL cells exhibit a characteristic 

gene expression profile which discriminates 

these cells from all other hematopoietic cells. Despite 

improvements in the therapy of patients with HL, a 

significant number of patients cannot be cured with 

current therapy strategies. In addition, toxicity of 

the currently used therapy is associated with high 

risk for secondary malignancies, cardiac complications 

or treatment-induced infertility. Cytotoxic T 

lymphocytes (CTL) can kill HL cells and have been 

used clinically for treatment of Epstein-Barr viruspositive 

HL. For patients with EBV-negative HL this 

strategy cannot be employed and alternative target 

structures have to be defined. In order to establish 

a system for the stimulation of HL-reactive T cells, 

we used dendritic cells (DC) as antigen presenting 

cells for autologous T cells. Because no specific antigens 

for EBV-negative HL are available, we transfected 

DC with RNA from established HL cell lines. 

After stimulation of peripheral blood mononuclear 

cells (PBMC) with RNA-transfected DC we assessed 

the reactivity of primed PBMC by interferon gamma 

ELISPOT. When we transfected DC (HLA-A*02,24; 

B*15; Cw*03,07; DRB1*04,11; DQB1*03) with RNA 

from HL cell line HDLM-2 (HLA-A*01,02; B*08,44; 

Cw*05,07; DRB1*13,15; DQB1*06) and used these 

DC for priming of the PBMC from the same DC-donor, 

we observed that the stimulated cells reacted 

with HDLM-2 cells but not with irrelevant tumor 

cells (TE671 sarcoma cells) or autologous PBMC. Interestingly, 

these HDLM-2-primed cells also reacted 

with HL cell line KM-H2 (HLA-A*11,24; B*52,15; 

Cw*04,12; DRB1*04,11; DQB1*03) but not with HL 

cell lines L-1236 (HLA-A*02; B*51; Cw*02; DRB1*14; 

DQB1*05) or L-540 (HLA-A*03,11; B*51; Cw*02,15; 

DRB1*04,11; DQB1*03). Because HDLM-2 cells and 

KM-H2 cells have completely different HLA haplotypes 

and KM-H2 cells share 6/10 HLA molecules 

with the used PBMC, these observations suggest 

the presence of HL-specific antigens expressed in 

both HL cell lines. These antigens are presented in 

the context of HLA class-I molecules of both cell 

lines leading to the T cell response. By analysis of 

Gene Expression Omnibus (GEO) microarray data 

sets from HL cell lines and primary HL samples 

in comparison with normal tissues und normal 

B cells we identified HL-associated transcripts. 

Among these transcripts we found known cancer 

testis (CT) antigens (e.g. PRAME (preferentially 

expressed antigen in melanoma) or CT45) and 

new antigens with CT-like expression pattern, e.g. 

ZBTB32 (zinc finger and BTB domain containing 

32). These antigens might be targets for future HLspecific 

immune therapy or for monitoring of HLdirected 

immune responses. 

121

077 Feger | New targets & new leads 

Analysis of HPV L1 specific T-cell immunity 

Thomas Feger 1 , Andreas Kaufmann 2 , Hans-Georg Rammensee 1 , Stefan Stevanovic 1 

1 Eberhard Karls University, Immunology, Tuebingen 

2 Charite Campus Benjamin Franklin, Gynecology, Berlin 

122 

There are over 200 different types of the human 

papillomavirus (HPV) described. Especially types 

16 and 18, but also types 31 and 45, are associated 

with cervical cancer. Types 16 and 18 alone are 

thought to be responsible for about 70% of all cervical 

cancer. Preventive vaccines based on L1 protein 

virus-like particles were licensed in 2007. Although 

the antibody response against L1 has been well documented, 

not much is known about the T-cell response 

to the viral L1 protein. Identification of class 

I and class II specific T-cell epitopes is essential for 

our understanding of HPV immunity, however. 

We used the epitope prediction algorithm SYFPEIT- 

HI to identify HPV 6, 11, 16 and 18 epitope candidates 

from the viral L1 protein. Class I and class II 

epitope candidates were synthesized and screened 

for their capability of memory T-cell activation with 

IFN-γ ELISPOT and intracellular cytokine staining 

using in vitro stimulated PBMCs of healthy donors. 

The identified specificities were used to select and 

clone epitope reactive T cells. 

Several novel class II epitopes from different regions 

of the L1 protein of the HPV strains 6, 11, 16 and 

18 were identified. T-cell responses against these 

epitopes are detectable in healthy donors. Analysis 

of the established T-cell clones showed cross 

reactivity against T-cell epitopes from strain 6, 11 

and 31, and against epitopes from strain 18 and 45, 

respectively. 

The knowledge of these L1 epitopes enables us to 

follow and compare T-cell responses elicited by 

vaccination or normal HPV infection. Furthermore, 

it explains the protection against additional HPV 

strains that are not included in but elicited by the 

preventive HPV vaccines.

L123 Kyzirakos | New targets & new leads 

Identification of novel EBV-specific MHC class-II epitopes 

Christina Kyzirakos 1 , Hans-Georg Rammensee 1 , Stefan Stevanovic 1 

1 Universität Tuebingen, Auf der Morgenstelle, Tuebingen, Germany 

Epstein-Barr Virus (EBV) persists in the vast majo- 

rity of adult individuals (>95%) as a lifelong latent 

infection of the host’s B-lymphocyte pool. Although 

chronic infections remain asymptomatic in most 

cases, immunocompromised patients can suffer 

from severe and lifethreatening EBV-associated diseases, 

such as post-transplant lymphoproliferative 

disorders (PTLD). Thus, immunotherapeutic strategies 

using adoptively transferred EBV-specific T 

cells are promising. One option is the generation 

and expansion of CD4+ and CD8+ T lymphocytes 

by using EBV-specific synthetic peptides for the stimulation 

of pre-existing memory T cells. Aim of 

our study was to identify a set of MHC class-II peptides 

which could be recognized by specific CD4+ 

T helper cells of basically every individual. 

15 amino acid long candidate epitopes of nine immunodominant 

antigens, three of the latent cycle 

(EBNA3A, LMP1 and LMP2) and six of the lytic 

cycle (BMLF1, BMRF1, BRLF1, BZLF1, BNRF1 and 

BLLF1) of the virus were predicted using the SYF- 

PEITHI epitope prediction programme (www.syfpeithi.de). 

For each antigen promiscuitive peptides 

with high SYFPEITHI scores were tested for immunogenicity 

using an IFN-γ-ELISPOT. PBMCs of 

at least 15 healthy, randomly chosen blood donors 

were cultured for 12 days in the presence of each 

candidate peptide. Functional and phenotypic analysis 

of T cells of several donors was performed by 

multicolor flow cytometry. 

Although 76 out of 107 tested peptides could be 

identified as T-cell epitopes, only five of them were 

defined as immunodominant, as more than 50% of 

tested blood donors showed peptide-specific T cell 

responses. So far, 19 of the tested peptides could be 

identified as MHC class-II epitopes. 

In conclusion, we could identify several new EBVspecific 

MHC class-II epitopes which can be used 

for promising immunotherapeutic approaches for 

the treatment of EBV-associated diseases like PTLD, 

as well as for diagnostic purposes. 

123

L125 Huijbers | New targets & new leads 

Vaccination against the extra domain-B of fibronectin as a 

novel tumor therapy 

Elisabeth J. M. Huijbers 1 , Maria Ringvall 1 , Julia Femel 1 , Sebastian Kalamajski 1 , Agneta 

Lukinius 2 , Magnus Åbrink 1 , Lars Hellman 3 and Anna-Karin Olsson 1 

1 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, SE-751 23 Uppsala, Sweden 

2 Department of Medical Cell Biology, Uppsala University, BMC, SE-751 23 Uppsala, Sweden 

3 Department Cell and Molecular Biology, Uppsala University, BMC, SE-751 24 Uppsala, Sweden 

124 

During physiological angiogenesis (e.g. wound 

healing and proliferation of the endometrium), 

embryonic development and tumor angiogenesis 

certain extra domains of the extracellular matrix 

molecules fibronectin and tenascin-C are inserted 

into the parent molecule by alternative splicing 

leading to the formation of different isoforms. 

Splice variants of both fibronectin and tenascin-C 

are often co-expressed specifically around neovasculature 

and tumor vasculature but have low or no 

expression in normal adult tissue, rendering them 

specific targets for anti-tumor therapy. 

In our approach we have targeted the extra domain- 

B (ED-B), which is a 91 amino acid domain alternatively 

spliced into fibronectin and highly conserved 

between species. It is identical in mouse, human, 

rabbit, dog, monkey and several other species. To 

evoke an immune response against the self-antigen 

ED-B we used a recombinant fusion protein consisting 

of a bacterial thioredoxin (TRX) part fused 

with the extra domain B (ED-B), termed TRX-EDB. 

This fusion protein has been injected together 

with Freund’s adjuvant, a strong immunostimulator, 

into eight-weeks old female wild type C57bl6 

mice. Mice were boostered twice in a period of five 

weeks before they were inoculated subcutaneously 

with T241 fibrosarcoma cells, a tumor type known 

to express ED-B. After a tumor growth period of 

three weeks animals were sacrificed and blood and 

tumors were removed. Nineteen out of 20 vaccinated 

mice responded with production of anti-ED-B 

antibodies and showed a 70% reduction in tumor 

size compared to control animals injected with 

Freund’s adjuvant and vehicle, lacking anti-ED- 

B antibodies. Staining of murine grade III glioma 

tissue, to see whether the serum from TRX-EDB 

mice could detect native ED-B, showed an extensive 

vascular staining pattern compared to normal brain 

tissue, which is devoid of ED-B. This shows that 

the anti-ED-B antibodies are able to detect native 

tissue ED-B. Quantification of tumor necrotic area 

revealed a tendency towards a greater necrotic area 

in TRX-EDB injected compared to control animals. 

Further investigation of the tumors with electron 

microscopy revealed morphological changes of the 

tumor vasculature of animals with anti-ED-B antibodies, 

which was consistent with an immune 

response towards the tumor vasculature expressing 

ED-B. Moreover in tumors from animals with 

anti-ED-B antibodies an increased number of infiltrating 

neutrophils was observed, compared to 

controls, confirming an immune attack of the vasculature. 

Furthermore we detected an increased 

amount of extravasated fibrinogen, indicative of 

vascular leakage, in tumors of animals with anti- 

ED-B antibodies compared to controls. 

Therefore ED-B and possibly other tumor vascular 

antigens such as the extra domain A of fibronectin 

or the extra domain C of tenascin-C alone or in 

combination are interesting candidate targets for 

treatment of solid tumors. Since the choice of adjuvant 

is of major importance for treatment success 

in humans, we addressed this issue in a second 

study and found a non-toxic biodegradable adjuvant 

as potent as Freund’s in inducing an immune 

response against a self-antigen (see poster by Julia 

Femel et al.)

125

126 

Tumor biology & interaction with the immune system

078 Maccalli | Tumor biology & interaction with the immune system 

Definition of the immunological properties of cancer stem cells 

isolated from human glioblastoma 

Cristina Maccalli 1 , Tiziano Di Tomaso 1 , Ena Wang 2 , Stefania Mazzoleni 3 , Gloria Sovena 1 , 

Soldano Ferrone 4 , Rossella Galli 3 , Francesco M. Marincola 2 , Giorgio Parmiani 1 

1 Unit of Immuno-Biotherapy of Solid Tumors, San Raffaele Foundation Scientific Institute, Milan, Italy 

2 Department of Tranfusion Medicine, National Institutes of Health, Bethesda, MD, USA 

3 Neural Stem Cell Unit, San Raffaele Foundation Scientific Institute, Milan, Italy 

4 Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, USA 

The main objectives of our study is to assess 

whether cancer stem cells (CSCs) isolated from glioblastoma 

multiforme (GBM) patients can be exploited 

as source of antigens to elicit T cell-mediated 

immune responses and their validation to design 

immunotherapeutic protocols for GBM. We have 

characterized, by immunofluorescence and cytofluorimetric 

or confocal microscopy analysis, the 

immune profile of 14 CSC and of 7 FBS tumor line 

pairs isolated from GBM samples. Moreover, PBMCs 

isolated from GBM patients were in vitro stimulated 

with autologous CSCs and the specific reactivity of 

T lymphocytes against GBM CSCs was evaluated by 

IFN-g, Granzyme B release (ELISPOT) or CD107a 

mobilization. We have assessed that both GBM CSC 

and their FBS-cultured non-CSC pairs showed a 

low immunogenic profile (MHC, NKG2DL and APM 

molecules). Up-regulation of most of these molecules 

was induced by IFNs or 5-Aza CdR, though 

more efficiently in FBS than in CSCs. Notably, T 

cell responses, both CD4+- and CD8+-mediated, 

against GBM could be raised in one GBM patient by 

stimulating in vitro PBMCs with autologous CSCs 

pre-treated with IFN-β. However, TH2-mediated 

responses with more efficient recognition of FBS 

tumor cell than CSCs, could be found in 3 additional 

GBM patients. One key finding of this study 

is that CSCs but not FBS cells inhibit allogeneic T 

cell proliferation. This immune-inhibitory activity 

mediated by GBM CSCs was not dependent on the 

secretion of TGFβ1 or 2, that were preferentially 

down-modulated in CSC lines, nor on IL-10 and 

IL1-3 that were undetectable in the supernatant of 

these cell lines. Expression of the indoleamine 2,3dioxygenase 

(IDO), a molecule implicated in the generation 

of immune tolerance, was detected in both 

CSCs and FBS tumor cell lines with high increase 

following IFN-g treatment of the cells. Furthermore, 

we found that both CSCs and FBS tumor cells 

expressed immune response inhibitory molecules, 

such as CTLA-4, PD-1 and PDL-1. Furthermore, a 

differential gene signature that was confirmed at 

the protein levels for some immunological-related 

molecules was also found between CSC and FBS 

lines. Moreover, this latter analysis provided information 

of some candidate molecules associated 

with CSCs that we are functionally investigating. 

Altogether, these results indicate a lower immunogenicity 

and higher suppressive activity of GBM 

CSC compared to autologous FBS lines and the need 

to identify immunomodulatory agents that can efficiently 

restore the expression of immunogenic molecules 

on CSCs. 

127

079 Poschke | Tumor biology & interaction with the immune system 

Immature immunosuppressive CD14+HLA-DR-/low cells in 

melanoma patients are Stat3hi and over-express CD80, 

CD83 and DC-Sign 

Isabel Poschke 2 , Dimitrios Mougiakakos 2 , Johan Hansson, Giuseppe V. Masucci 3 , 

Rolf Kiessling 3 

1 Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden 

2 I.P. and D.M. contributed equally to this study 

3 G.V.M. and R.K. contributed equally to this study as principal investigators 

128 

Myeloid derived suppressor cells (MDSC) have 

emerged as key immune-modulators in various 

tumor models and human malignancies, but their 

characteristics in humans remain to be unequivocally 

defined. 

In this study, we have examined CD14+HLA-DR-/ 

low MDSC in advanced 34 malignant melanoma 

(MM) patients. Their frequency in peripheral 

blood is significantly increased and associated with 

disease activity. Contrary to the common notion 

that MDSC are a heterogeneous population of exclusively 

immature cells, we find co-expression 

of markers associated with mature phenotype. 

We demonstrate for the first time over-expression 

of CD80, CD83 and DC-SIGN in human MDSC. 

Further, increased levels of Stat3, an important regulator 

in MDSC development and function, were 

noted in patient-derived CD14+HLA-DR-/low cells. 

Stat3 was altered towards an active, phosphorylated 

state in the HLA-DR- population of CD14+ cells 

and was more reactive to activating stimuli in patients. 

The described MM-MDSCs utilize arginase 

in conjunction with other undefined mechanisms 

to suppress CD4+- and CD8+-T-cells. Several observations 

suggest a redox imbalance in MDSC and 

indicate an important role of Stat3-dependent oxidative 

stress in MDSC-mediated T-cell suppression. 

These results emphasize the diversity of MDSC in 

human cancer and provide potential targets for therapeutic 

interventions.

080 Flores-Guzman | Tumor biology & interaction with the immune system 

Potential cancer stem cells in ret transgenic mouse model of 

spontaneous melanoma 

Fernando Flores-Guzman, Dirk Schadendorf and Viktor Umansky 

German Cancer Reserarch Center (DKFZ), Heidelberg, Germany 

The cancer stem cell (CSC) hypothesis suggests that 

neoplastic clones are maintained exclusively by a 

rare fraction of tumor cells with stem cells properties. 

CSC could represent disseminated dormant tumor 

cells without clinical signs of progression. We used a 

transgenic mouse spontaneous melanoma model, in 

which after a short latency, around 25% of all transgenic 

mice developed skin tumors with metastases 

in distant organs like liver and lungs. We found that 

CD133+ melanoma cells represent a small subset in 

primary skin tumors and lymph node metastases 

(< 1.5%). The amount of CD133+ melanoma cells 

both in primary skin tumors and lymph node metastases 

correlated with the primary tumor weight 

and the stage of tumor progression. We detected 

TRP2+CD133+ single melanoma cells in the bone 

marrow of tumor bearing mice suggesting thereby 

that these cells could be potential melanoma stem 

cells. We also found CD20, CD24, CD44 and CD166 

expression in primary tumors, which correlated 

with the tumor progression. Another potential melanoma 

stem cell marker nestin was express in most 

melanoma cells, whereas the multidrug resistance 

ABCB5 marker was expressed on less than 3% cells 

from primary skin tumors. 

In conclusion, our data demonstrate an existence 

of the subpopulation of CD133+ melanoma cells in 

primary skin tumors, metastatic lymph nodes and 

in the bone marrow of tumor bearing transgenic 

mice and suggest that these subsets could be potential 

CSC. 

129

081 Lion | Tumor biology & interaction with the immune system 

Poly(I:C)-electroporated myeloid leukemic cell lines become 

highly susceptible to NK cell-mediated killing and phagocytosis 

by immature DC 

Eva Lion 1 , Sébastien Anguille 1 , 2 , Evelien Smits 1 , Zwi Berneman 1 , 2 , Viggo Van Tendeloo 1 

1 Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp, Belgium 

2 Laboratory of Experimental Hematology, Antwerp University Hospital, Antwerp, Belgium 

130 

Natural killer (NK) cells and dendritic cells (DC) are 

proven to exert important functions in anti-tumor 

defence. Targeting both immune cells for immunebased 

therapy is currently advised. We recently 

showed that electroporation (EP) of acute myeloid 

leukemic (AML) cell lines with the synthetic double-stranded 

RNA TLR3-ligand poly(I:C) enhances 

their capacity to act on DC and NK cells. Aside from 

the fact that tumor cells themselves become apoptotic 

and start to secrete marked amounts of type 

I interferon (IFN), they are able to mature DC and 

promote NK cell IFN-γ. In this study, we explored the 

effect of poly(I:C)-EP AML cell lines on the killing 

capacity of NK cells and the phagocytic potential 

of DC. Additionally, we assessed these functionalities 

in three-party cocultures to see for functional 

cross-talk. We hypothesized that poly(I:C)-induced 

apoptosis and the presence of IFN-α would increase 

the NK cell cytotoxicity. In turn, this could lead to 

an increased tumor cell up-take by DC. 

All experiments were performed with fresh autologous 

monocyte-derived immature DC and purified 

NK cells of healthy donors. The K562 and U-937 

leukemic cell lines were used as targets. Flow cytometric 

detection of cytotoxicity and phagocytosis 

was acquired simultaneously. Cytotoxicity was determined 

based on the viability (annexin V- propidium 

iodide-) of PKH67-labeled AML cells. Phagocytosis 

of PKH67+ tumor cells by violet-labeled DC 

was expressed as the % PKH67+violet+ cells of all 

violet+ DC, selected for single cells. 

Our data demonstrate that poly(I:C)-EP of AML 

cell lines results in an increased susceptibility to 

NK cell-mediated killing (p=0.0023 for K562 and 

p

082 Bonertz | Tumor biology & interaction with the immune system 

Antigen-specific Treg in colorectal carcinoma control T cell 

responses against a limited tumor antigen repertoire 

Andreas Bonertz 1 , Jürgen Weitz 2 , Kim Pietsch 1 , Christoph Schlude 1 , Simone Jünger 1 , Yingzi 

Ge 1 , Kashayarsha Khazaie 3 , Moritz Koch 2 , Philipp Beckhove 1 

1 Translational Immunology Unit, The German Cancer Research Center, Heidelberg, Germany 

2 Department of Visceral Surgery, University Hospital of Heidelberg, Heidelberg, Germany 

3 Division of Gastroenterology, Northwestern University Feinberg School of Medicine, 

Robert Lurie Comprehensive Cancer Center, Chicago, Illinois, USA 

Previous studies suggest that regulatory T cells 

(Treg) may suppress a tumor-specific immune 

response against established tumors in patients. 

Here, we examined how a depletion of Treg influences 

the immune reactivity of colorectal carcinoma 

(CRC) patients against tumor associated antigens 

(TAA) and screened for the specificity of Treg 

in the same patients. 

We analyzed the T cell (TC) repertoires from the 

peripheral blood (PB) of 170 CRC patients for the 

presence and frequencies of spontaneously induced 

effector/memory TC against synthetic polypeptides 

spanning immunogenic regions from 9 different 

TAAs (CEA, EGFR, Heparanase, Her2/neu, Mage-3, 

Muc-1, p53, Survivin, Telomerase) before and after 

depletion of Treg by short-term IFNγ enzyme-linked 

immunospot analysis. In comparison, we assayed 

PB from 32 healthy donors for the presence of 

TAA-specific TC. Patients and healthy donors were 

divided into HLA-A2 positive and negative cohorts. 

Furthermore, we evaluated for the first time the 

specificities of Treg in the same patients for the respective 

TAAs. For this, dendritic cells were pulsed 

with TAA-peptides or IgG control antigen, coincubated 

with autologous Treg and analyzed for the 

Treg capacity to suppress in an antigen-dependent 

manner the proliferation of polyclonally activated 

conventional TC. 

In the majority of patients (60%), TAA specific TC 

were detected. The TAA recognition pattern was 

highly diverse within the CRC patients and was 

independent of the patients HLAtype. Healthy individuals 

contained significantly less TAA-reactive 

TC. After Treg depletion, the proportion of TAAreactive 

patients increased to 86%. The strongest 

increases of recognition were found for Muc-1 (14% 

vs. 39%), EGFR (13% vs. 38%), and CEA (18% vs. 

39%), which was also associated with an increase 

of the frequencies of TC specific for these antigens. 

Treg reacted specifically for individual TAAs in the 

PB of 40% of CRC patients. 

Treg-induced suppression of proliferation was most 

prevalently observed after Treg activation with 

Muc-1, HER2/neu, and CEA, whereas Treg specific 

for p53 were not observed. Interestingly, specificities 

of memory TC and Treg markedly differed 

in the majority of patients, leaving the possibility 

of using selected sets of TAA for tumor vaccinations 

that induce optimal effector TC responses but 

minimal Treg activity. 

131

083 Ge | Tumor biology & interaction with the immune system 

tumor-induced emigration of antigen-specific treg enables 

the bone marrow to foster spontaneous anti-tumor t-cell 

responses in breast cancer patients 

Yingzi Ge 1 , Christoph Domschke 2 , Helge Philipp Frebel 3 , Christopher Schnappauf 4 , Michael 

Hillier 1 , Florian Schuetz 2 and Philipp Beckhove 1 

1 Translational Immunology Unit, German Cancer Research Center, Heidelberg, Germany; 

2 Department of Obstetrics and Gynecology, Heidelberg University Women’s Hospital 

3 Institute of Microbiology, ETH Zurich, Zurich, Switzerland 

4 Heidelberg University Children’s Hospital 

132 

Adequate frequency and activity of tumor-specific 

T cells are crucial for successful cancer immunotherapy. 

Bone marrow from patients with breast 

cancer has been shown to serve as a reservoir of 

tumor-responding memory/effector T cells (Tmem/ 

eff). We detected that a depletion of regulatory T 

cells (Treg) unmasked a comparable population of 

anti-tumor T cells in the blood to that in the bone 

marrow, as determined with defined tumor-associated 

antigens (TAA) as well as tumor-cell lysates 

in IFN-γ ELISpot assay. The higher reactivity of 

TAA-specific Tmem/eff in the bone marrow is due 

to firstly, a remarkably lower frequency of resident 

Treg, as shown by FACS analysis, and secondly, 

a significantly reduced suppressive capacity of 

Treg upon TAA activation in vitro. Interestingly, 

we also found that T-cell tumor-responsiveness in 

the bone marrow inversely correlated to the frequency 

of intratumoral Treg, suggesting a mobilization 

of activated Treg into the blood. In support 

of this, we could identify a panel of tissue-homing 

receptors which are up-regulated on activated Treg 

derived from the bone marrow in comparison to 

those from the blood. The frequency of Treg expressing 

those breast tumor-relevant chemokine receptors 

was associated with the latency of Tmem/ 

eff anti-tumor reactivity in the circulation. Collectively, 

we propose that tumor-specific Treg in the 

bone marrow acquire migratory and suppressive 

capacity upon activation and sequentially home to 

tumor tissue. This recruitment in turn endows the 

bone marrow a unique environment devoid of TAAspecific 

Treg and capable of fostering anti-tumor 

Tmem/eff.

084 Chen | Tumor biology & interaction with the immune system 

Disturbed NK cell function in CML patients at diagnosis does 

not recover under treatment with imatinib mesylate 

Christiane I-U. Chen 1 , Steffen Koschmieder 2 , Linda Kamp 2 , Holden T Maecker 3 , Wolfgang 

Berdel 2 , Heribert Jürgens, 1 Peter P Lee 4 , Claudia Rössig 1 

1 Department of Pediatric Hematology and Oncolocy, University Hospital Muenster, Germany 

2 Department of Medicine, Division of Hematology and Oncology, University Hospital Muenster, Germany 

3 Human Immune Monitoring Center, Stanford University School of Medicine, USA 

4 Department of Medicine, Division of Hematology, Stanford University, USA 

Even though the tyrosine kinase inhibitor imati- 

nib mesylate (imatinib) has become the first-line 

therapy for patients with chronic myeloid leukemia 

(CML), allogeneic hematopoietic stem cell transplantation 

(HSCT) remains the only curative treatment. 

The important contribution of the immune 

system to curing CML is evidenced by the potent 

therapeutic effects of donor lymphocyte infusions. 

In addition to cytotoxic T cells, natural killer (NK) 

cells may be involved in immunologic control of the 

disease. Here, we used a transgenic tet-off inducible 

CML mouse model as well as peripheral blood 

samples from CML patients to explore the role of 

NK cells in CML. 

Splenic lymphocyte subpopulations from CML 

mice were analyzed by flow cytometry. Under continuous 

tetracycline treatment (tet-on), these mice 

have a bcr-abl-negative phenotype, whereas withdrawal 

of tetracycline induces bcr-abl transgene 

expression, resulting in initiation of leukemia and 

manifestation of a CML-like phenotype (tet-off, bcrabl+). 

Bcr-abl+ mice had significantly decreased 

relative numbers of NK cells compared to control 

mice (5.5% vs 13.8%, p = 0.045). While ex vivo expansion 

of purified NK cell populations from tet-on 

and tet-off mice demonstrated similar proliferative 

responses to stimulation with high-dose interleukin-2 

(IL-2), the cytolytic capacity of NK cells expanded 

from bcr-abl+ mice against K-562 cells was 

significantly reduced (10.7% vs. 27.3% at an E:T 

ratio of 1:1, p < 0.05). 

To address the functionality of NK cells in human 

CML, we quantified NK cells in peripheral blood 

samples from 13 patients at diagnosis and at several 

time points during imatinib-induced remission 

(range 10-59 months). Compared to age-matched 

healthy controls, NK cells were decreased at diagnosis 

(4,4% vs 13%, p < 0.05) and did not recover 

to normal levels under imatinib treatment (6.2% vs 

10%, p < 0.05). Functional experiments revealed 

reduced expansion of NK cells in response to coincubation 

with K-562/4-1BBL/mbIL-15 stimulator 

cells in CML patients versus healthy controls 

(26-fold expansion vs 48-fold expansion within 10 

days, p < 0.05). Moreover, the cytolytic activity of 

human CML NK cells was reduced both at diagnosis 

(8.3% lysis of K-562 target cells at an E:T ratio 

of 1:1) and under treatment with imatinib (11%) 

compared to control (18.9%, p < 0.05). 

We conclude that patients with CML have both 

quantitative and qualitative defects within their 

NK cell compartment, which can be reproduced 

by transgenic bcr-abl expression in mice. NK cells 

do not recover to normal levels and normal function 

under imatinib treatment, even after obtaining 

complete remission. Further work will aim at 

identifying the underlying mechanisms of NK cell 

deficiency in CML, and the development of strategies 

to exploit NK cells for immunotherapy of the 

disease. 

133

085 Dubrot | Tumor biology & interaction with the immune system 

Treatment with anti-CD137 mAbs causes intense 

accumulations of liver T cells without selective antitumor 

mmunotherapeutic effects in this organ 

Juan Dubrot 1 , , Francisca Milheiro 1 , Carlos Alfaro 1 , Asis Palazón 1 , Ivan Martinez-Forero 1 , 

Aizea Morales-Kastresana 1 , María C. Ochoa 1 , Sandra Hervás Stubbs 1 , Maria Jure-Kunkel 2 , 

Lieping Chen 3 and Ignacio Melero 1 

1 Centro de investigación médica aplicada (CIMA) 

2 Bristol Myers-Squibb Pharmaceutical research institute, Princeton, NJ 

3 Sidney Kimmel Cancer Center. Johns Hopkins Medical School. Baltimore, BA 

134 

Background/Aims: Cancer therapy with agonist 

anti-CD137 mAbs has been shown to induce im- 

mune-mediated tumor rejections in mice and equi- 

valent agents of this kind are currently being tested 

in cancer patients. Previous reports indicated that 

CD137 stimulation induces polyclonal infiltrates of 

T lymphocytes in the liver. This study characterizes 

the liver infiltrates, the target-dependency of the 

phenomena and addresses the question of whether 

tumors nested in the liver are a more favourable 

target for CD137-based immunotherapy. 

Methods: Liver infiltrates were studied with conventional 

histology and multiple colour flow cytometry 

of total liver leukocytes. CD137-/- mice, mice 

with a single rearrangement of the TCR (OT-1 mice) 

and Rag-/- mice were used to clarify molecular requirements. 

Mice implanted with MC38 colon carcinomas 

either subcutaneously or inside the liver 

were used for comparative studies under treatment 

with agonist anti-CD137 mAbs. 

Results: CD137 treatment caused mononuclear 

inflammation in portal spaces of the liver, which 

gave rise to moderate increases in transaminases 

without signs of cholestasis. Marked increases in 

the numbers of CD8+ T cells were observed, including 

CD8+ T lymphocytes co-expressing CD11c. 

Infiltrates were absent in CD137-/- mice and mitigated 

in mice harbouring a single transgenic TCR on 

their CD8 T cells. Despite of the tumor-independent 

accumulation of T cells in the liver, immunotherapeutic 

effects were not more prominent against 

tumors located in this organ. 

Conclusions: Target-dependent effects of CD137 

stimulation lead to liver infiltration with T cells, 

but lymphocyte enrichment in this organ does not 

privilege this site for immunotherapeutic effects 

against transplanted tumors.

086 Chachibaia-Saginashvili | Tumor biology & interaction with the immune system 

Role of T & B-cells in malignant transition from chronic 

autoimmune inflammation during Sjogren ’s syndrome and 

treatment approach 

Tamar Chachibaia 1 , Ekaterina Aptsiauri 2 

1 Georgian Sjogren Association, Medical School AIETI, Department of Immunology, Tbilisi, Georgia 

2 Bristol Myers-Squibb Pharmaceutical research institute, Princeton, NJ 

3 Rare Diseases Alliance Georgia, Tbilisi 

Sjogren’s syndrome (SS) is a common systemic 

autoimmune disease, which was first described in 

1933 by Henrik Sjögren. It occurs as an isolated disorder 

(primary form), also known as the Sicca syndrome, 

or more often in association with another 

autoimmune disease (secondary form). Women are 

affected in 90% of cases, mostly in their peri/post 

menopause period of life. 

SS is characterized by dysfunction and destruction 

of the lachrymal and salivary glands associated 

with lymphocytic infiltration and immunological 

hyperreactivity. The decrease in tears and saliva 

(sicca syndrome) is the result of histo-pathologically 

evidenced lymphocytic infiltration and fibrosis 

of this exocrine glands. The infiltrate contains 

predominantly activated CD4+ helper T cells and 

some B cells, including plasma cells that secrete 

antibody locally. 

As might be expected from the presence of autoantibodies, 

a variety of functional abnormalities 

has been detected in the T cells, B cells, and macrophages. 

The development of parotid lymphoma 

in patients with SS are thought to represent endpoints 

of multi-step process of local antigen-driven 

chronic inflammation that is characterized by 

organ-specific B- or T-cell proliferation, polyclonality, 

oligoclonality and, eventually, monoclonality. 

Of interest are studies that indicate the presence of 

a monoclonal B-cell population within the salivary 

glands that gives rise to monoclonal immunoglobulins 

or light chains in the serum. 

About 5% of patients with Sjögren's syndrome 

will develop some form of lymphoid malignancy. 

(Tzioufas AG, Voulgarelis M (2007). Patients with 

Sjögren's syndrome have higher rate an approximately 

40-fold higher risk of developing lymphoid 

malignancies compared to both patients with 

other autoimmune diseases and healthy people. 

(Tzioufas AG & Voulgarelis M, 2007). Patients with 

severe cases are much more likely to develop lymphomas 

than patients with mild or moderate cases. 

(Smedby et al, 2006). The most common lymphomas 

are salivary extranodal marginal zone B cell 

lymphomas or non-Hodgkin lymphoma (Voulgarelis 

M. & Skopouli FN 2007) and diffuse large B-cell 

lymphoma (Smedby et al, 2006). 

The gain in knowledge regarding the cellular mechanisms 

of T and B lymphocyte activity in the 

pathogenesis of Sjögren’s syndrome (SS) and the 

current availability of various biological agents 

(anti-TNF-α, IFN-α, anti-CD20, and anti-CD22) have 

resulted in new strategies for therapeutic intervention. 

Currently, B cell-directed therapies seem to 

be more promising than T cell-related therapies. 

However, large, randomized, placebo-controlled 

clinical trials are needed to confirm the promising 

results of these early studies. 

135

087 Lutz-Nicoladoni | Tumor biology & interaction with the immune system 

Reinforcement of Cancer Immunotherapy by Adoptive 

Transfer of cblb-deficient Cytotoxic T Lymphocytes 

combined with a Dentritic Cell Vaccine 

Christina Lutz-Nicoladoni 1,2 , Patrizia Stoitzner 3 , Magdalena Pircher 1,2 , Stephanie Wallner 

1,2 , Thomas Gruber 4 , Anna Maria Wolf 1,2 , Günther Gastl 1,2 , Josef Penninger 5 , Gottfried 

Baier 4 , Dominik Wolf 1,2 

1 Tyrolean Cancer Research Institute, Tumorimmunology, Innsbruck, Austria 

2 Medical University Innsbruck, Internal Medicine V, Hematology and Oncology, Innsbruck, Austria 

3 Medical University Innsbruck, Department of Dermatology and Venerology, Austria 

4 Medical University Innsbruck, Department for Medical Genetics, Molecular and Clinical Pharmacology, Austria 

5 Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria 

136 

During the last decades rapid progress in immuno- 

logy facilitated the development of tumor therapy 

based on cell transfer. Autologous tumorinfiltrating 

lymphocytes (TILs) are transferred after ex 

vivo clonal expansion or ex vivo transduction with 

tumor antigen specific T cell receptors (TCR) and 

subsequent expansion into patients. Adoptive T 

cell transfers (ATC) are often combined with lymphodepleting 

therapies. However, the success of 

cancer immunotherapy is limited by potent endogenous 

immune-evasion mechanisms, which are 

centrally mediated by transforming growth factor 

beta TGF-β. The RING E3 ubiquitin ligase casitas B 

cell lymphoma (Cbl-b) is a negative key regulator 

of T cell activation. Cblb-deficient T lymphocytes 

are uncoupled from the requirement of CD28 costimulation, 

are anergy-resistent and resistant to inhibition 

by TGF-β. As a consequence, cblb-deficient 

animals reject tumors via cytotoxic CD8+ T cells 

(CTLs), which makes Cbl-b an ideal target in adoptive 

T cell transfer therapy. 

Here we provide evidence that cblb-/- CTLs are 

hyper-responsive to TCR/CD28-stimulation in vitro 

and protected from the negative effects induced by 

TGF-β. Nevertheless and unexpectedly, adoptive 

transfer of polyclonal, non TCR-transgenic cblb-deficient 

CTLs into tumor bearing immunocompetent 

wildtype (wt) mice is not sufficient to reject B16ova 

or EG7 tumors in vivo. Thus, cblb-deficient transferred 

CD8+ T cells were in vivo re-activated by a 

dendritic cell (DC) vaccine (i.e. SIINFEKL-pulsed 

DC). In strict contrast to ATC monotherapy, this approach 

now delayed tumor outgrowth and significantly 

increased survival rates, which is paralleled 

by an increased CTL infiltration rate to the tumor 

site as well as enrichment of tumor antigen-specific 

and interferon-gamma (IFN-g)-secreting CTLs in 

the draining lymphnodes. Moreover, in vivo cytolytic 

activity was increased in tumor bearing mice 

treated with DCs and cblb-deficient CTLs compared 

to those treated with DCs plus wt CTLs. 

In summary, we provide experimental evidence that 

genetic inactivation of Cbl-b in polyclonal, non-TCR 

transgenic adoptively transferred CTLs serves as a 

novel “adjuvant approach”, suitable to augment the 

effectiveness of established anti-cancer immunotherapy 

in immuno-competent recipients.

088 Schmid | Tumor biology & interaction with the immune system 

Development of a novel transgenic mouse model for melanoma 

Beate Schmid 1 , Danielle Arnold-Schild 1 , Mustafa Diken 2 , Christine Tertilt 1,3 , Markus Radsak 

4 , Hansjörg Schild 1 

1 Johannes Gutenberg University Medical Center, Dept. of Immunology, 

Langenbeckstr. 1, 55131 Mainz, Germany 

2 Johannes Gutenberg University Medical Center, III. Dept. Medicine, Hematology/Oncology, 

Obere Zahlbacher Str. 63, 55131 Mainz, Germany 

3 Johannes Gutenberg University Medical Center, Dept. of Pediatrics, 


4 Johannes Gutenberg University Medical Center, III. Dept. Medicine, Hematology/Oncology, 


Novel cancer therapeutics need to be evaluated 

in animal models before application in humans. 

Models using transplanted tumor cell lines may lead 

to tumor growth in mice, but their characteristics 

often substantially differ from autochthonously developing 

tumors in vivo. This may be important for 

the development of tumor tolerance and the establishment 

of anti-tumor immunity. Therefore, new 

animal models are needed where the endogenous 

development of tumors can be controlled and manipulated. 

To create an autochthonous tumor model 

we generated BAC transgenic mice with inducible 

expression of the melanoma oncogenes BrafV600E, 

Cdk4R24C and Mitf under the control of the melanocyte 

specific tyrosinase promoter. Furthermore 

we introduced the gene for firefly luciferase for monitoring 

of oncogene expression by bioluminescent 

imaging. Transcription of these genes is controlled 

by the tamoxifen inducible recombinase CreERT2 

also expressed specifically in melanocytes. In vitro 

studies of C22 cells transfected with the vector 

pcDNA3.1 containing the oncogene-luciferase expression 

construct indeed showed an increase in 

Braf and Cdk4 expression by western blot as well 

as luciferase activity indicating the functionality 

of the construct. The pronucleus injection of the 

final BAC construct resulted in three founders confirmed 

by PCR and southern blot analysis. Analysis 

of these mouse lines in vivo by bioluminescent 

imaging showed some base line luciferase activity 

in one mouse line indicating leakiness of the trans- 

genic construct. However, tamoxifen treatment 

greatly enhanced the luciferase activity indicating 

that our construct is operating as intended. Further 

analyses to characterize the tissue specific transgene 

expression and kinetics of tumor development 

are ongoing. After completion of these basic studies 

this novel mouse model will be a valuable tool for 

the preclinical assessment of novel cancer therapeutics. 

137

089 Sha | Tumor biology & interaction with the immune system 

Influence of activation of human immune effector cells 

on the penetration into tumor spheroids 

Weixiao Sha 1 , Franziska Hirschhaeuser 1 , Stefan Walenta 1 , Kirsten Dettmar 2 and Wolfgang 

Mueller-Klieser 1 



2 Fresenius Biotech GmbH, Munich, Germany 

138 

The trifunctional bispecific antibody catumaxo- 

mab (anti-EpCAM x anti-CD3) was designed for 

anti-tumor immunotherapy by targeting the tumor-associated 

antigen human EpCAM (epithelial 

cell adhesion molecule) on tumor cells and human 

CD3, expressed on T lymphocytes. In addition, catumaxomab 

recruits simultaneously Fcγ receptorpositive 

accessory cells, to promote the formation 

of a so-called “tri-cell-complex” which finally leads 

to the destruction of tumor cells by immune cells. 

MCTS (multicellular tumor spheroids) of the FaDu 

cell line (over-expressing EpCAM) were co-cultured 

with PBMCs (peripheral blood mononuclear cells) 

and incubated with the test agents in microtiter 

plates. With the objective to compare the influence 

of the different binding sites of catumaxomab we 

additionally used the F(ab`)2 fragments of catumaxomab 

lacking a Fc-region and BiLu (anti-human 

EpCAM x anti-mouse CD3) which is unable to bind 

to human T cells. Furthermore, LPS (Lipopolysaccharid) 

and con A (Concanavalin A) were applied 

to the test system. To asses the immune cell infiltration, 

rapidly frozen spheroids were cryosectioned 

and stained for immunohistology. Antibodies 

against CD45, CD14, CD2, CD4 and CD8 were used 

to discriminate different subtypes of infiltrating 

leukocytes in MCTS. Cytokine release (IFN γ, TNF 

α, IL 2, IL 6) gave further information on the type of 

immune response. Besides immunological parameters, 

the metabolic effects of the treatment with the 

different agents were analyzed by measurement of 

the extracellular lactate production in co-culture 

supernatants. 

Catumaxomab-mediated stimulation of PBMCs led 

to a massive penetration of spheroids by CD2+ T 

cells after 3 days and the volumes of the MCTS 

were significantly decreased. CD8+ T lymphocytes 

were identified as the dominating subtype of tumor 

infiltrating T cells. Higher levels of IL-2 through catumaxomab 

application were detected after 1 day, 

while no IL-2 was detectable after 3 days. Incubation 

with the F(ab`)2 fragment of catumaxomab resulted 

in similar results. Catumaxomab stimulation 

of co-cultures also resulted in increased concentrations 

of extracellular lactate. On the other side, IFN 

γ and TNF α secretion as well as lactate concentration 

was elevated in PBMC cultures stimulated 

with catumaxomab in the absence of tumor cells, 

whereas incubation with F(ab`)2 showed no detectable 

effect. These catumaxomab-mediated effects 

were enhanced in the presence of PBMC and tumor 

cells. Furthermore, BiLu did not reveal any effects 

in co-cultures of tumor cells with human PBMC. 

In summary, it was demonstrated that the catumaxomab-mediated 

stimulation of PBMCs in MCTS 

co-culture plays a pivotal role for tumor infiltration 

by cytotoxic T lymphocytes and secretion of antitumoral 

cytokines, thus underscoring the importance 

of the trifunctional mode of action for the 

initiation of an effective anti-tumoral response. 

Supported by Fresenius Biotech GmbH and TRION Pharma GmbH 

(Munich, Germany).

090 Strothmeyer | Tumor biology & interaction with the immune system 

Comparative Analysis of Predicted HLA Binding of 

Immunoglobulin Idiotype Sequences Indicates T Cell-Mediated 

Immunosurveillance in Follicular Lymphoma 

Anna-Maria Strothmeyer, Katja Zirlik, Marcus Dühren-von Minden, Marcelo Navarrete, 

Kristina Heining-Mikesch, Hendrik Veelken 

Department of Hematology/Oncology, University Medical Center Freiburg, 79106 Freiburg, Germany 

Active immunization against the individual B cell 

receptor ("Idiotype", Id) of B cell lymphomas can 

induce Id-specific immune responses (see accompanying 

poster by Navarrete et al.). Epitope mapping 

experiments with Id peptides show that these in vivo-induced 

T cell responses are directed preferentially 

against individual Id epitopes located in CDR, 

whereas FR and C region epitopes are ignored. We 

therefore speculated that Id-specific T cell immunity 

might occur naturally in lymphoma patients, 

and conducted a reverse immunology study of the 

Id in 39 follicular lymphoma (FL) patients. Clonal 

Id transcripts were identified from tumor biopsies 

by A-PCR. HLA-A and B alleles were determined 

by serotyping and high-resolution PCR genotyping. 

Potentially HLA-presentable nonameric peptides 

from all Ids and their corresponding germ-line (GL) 

VH and VL genes were identified for all analyzable 

HLA alleles of this cohort by reverse immunology 

(bimas.cit.nih.gov). Identified peptides were 

ranked according to their predicted score, and the 

sum of the scores for the 20 highest ranking peptides 

was calculated for each HLA allele. For every 

patient's HLA type, the sum scores of all Ids on 

his/her HLA alleles were added to yield a theoretical 

measure of every Id's immunogenicity. This 

"autologous" sum score was compared to the mean 

sum scores of all other 38 idiotypes in a matchedpair 

analysis. Separate analyses were performed for 

CDR peptides (containing at least 2 AA in any CDR) 

vs. non-CDR-peptides (as defined by imgt.cines.fr) 

and Id vs. corresponding GL sequences. 

The sum scores of Id VH sequences were lower for 

the patient's own Id than for the mean of the allogeneic 

Ids (p=0.0017). Every CDR (CDR1: p

091 Ramacher | Tumor biology & interaction with the immune system 

Immunosuppression in transgenic mouse melanoma 

model induced by myeloid derived suppressor cells 

Marcel Ramacher 1 , Michal Baniyash 2 and Viktor Umansky 1 

1 German Cancer Research Center (DKFZ), Heidelberg Germany 

2 Lautenberg Center for General and Tumor Immunology, Jerusalem, Israel 

140 

Melanoma is known for its poor response to current 

immunotherapies due to immunosuppressive cells 

in the tumor microenvironment like myeloid derived 

suppressor cells (MDSC). We performed the studies 

on the ret transgenic mouse model of malignant 

melanoma, which resembles human melanoma as 

regards to histopathology and clinical development. 

After a short latency, 25% of mice develop melanoma 

metastasizing to lymph nodes, lungs and liver. 

We found that MDSC from tumor bearing mice were 

characterized by increased numbers and higher 

nitric oxide (NO) production and arginase-1 (ARG1) 

expression than those from tumor free mice or nontransgenic 

littermates. This phenomenon could be 

due to exosomes released by melanoma cells in 

vivo. In fact, upon treatment of non-transgenic littermates 

with melanoma derived exosomes, MDSC 

amounts were significantly elevated. 

To investigate the effect of NO and reactive oxygen 

species produced by MDSC on CD4+CD25- conventional 

(helper) T cells and CD4+CD25+ regulatory 

T cells (Treg), these subsets were separated from 

spleens and treated with NO donor DETA-NONOate 

or hydrogen peroxide in vitro. Both factors induced 

much higher apoptosis in conventional T cells than 

in Tregs suggesting thereby the Treg resistance 

to the immunosuppressive microenvironment. To 

reduce the MDSC immunosuppressive effect and 

to improve antitumor immune responses, tumor 

bearing mice were treated orally with an inhibitor 

of phosphodiesterase (PDE)-5 sildenafil (Viagra). 

Lower NO production and ARG1 expression in 

MDSC was correlated with the inhibition of melanoma 

progression. We suggest that an effective 

immunotherapy should include the inhibition of 

MDSC immunosuppressive functions.

092 Tomsitz | Tumor biology & interaction with the immune system 

Establishment of a pre-clinical NOD/LtSz-scid IL2Rgammac null 

(NSG) mouse model to evaluate immunotherapeutic strategies 

for acute myeloid leukemia (AML) 

Dirk Tomsitz 1 , Ariane Brunk 1 , Marion Nonn 1 , Alexander Hohberger 1 , Shamsul A. Khan 1 , 

Eva Distler 1 , Matthias Theobald 1 , Wolfgang Herr 1 , Udo F. Hartwig 1 

1 Dept. of Medicine III - Hematology and Oncology, Johannes-Gutenberg-University Medical Center, 


Introduction: Acute myeloid leukemia (AML) is a 

cancer of the myeloid line of blood cells, characte- 

rized by the clonal expansion of immature mye- 

loblasts potentially initiating from rare leukemic 

stem cells. Xenotransplantation of human AML into 

immunodeficient mice is essential for establishing 

a preclinical model to i) assess the potency of modified 

donor lymphocyte grafts to induce immunotherapeutic 

graft-versus-leukemia (GVL)-reactivity 

and ii) to study the properties of leukemic stem 

cell biology. Here we report the successful engraftment 

of 8 out of 14 patient-derived, primary AML 

samples in NOD/LtSz-scid IL2Rgammacnull (NSG) 

mice within 2-12 weeks, with a mean of 0,2-40% 

human leukemic cells in the bone marrow (BM). In 

first studies, we also show the successful eradication 

of engrafted AML-blasts using AML-reactive, 

HLA-mismatched T lymphocytes. 

Methods: Mice were used unconditioned or irradiated 

with 150 cGy 4-16h prior to intravenous (i.v.) 

transfer of 5x104-1x107 primary AML cells. Engraftment 

kinetics and dose-dependencies were analyzed 

by means of clinicopathological criteria and 

flow cytometry. AML-reactive T cells were generated 

in a HLA-mismatched setting over 3 weeks from 

PBMCs isolated from buffy coats. One week after 

transplantation of 5x105 AML cells from patient 

MZ667, AML-bearing mice were i.v. injected with 

5x106 CD8+ leukemia-reactive T lymphocytes. 

Results: Stable AML-engraftment in NSG recipients 

was detected upon transplantation of 8 out of 14 

primary AML samples by determing the percentage 

of CD33+/CD45+ cells in blood, spleen and BM. 

Expression of Flt3 mutations appeared to promote 

engraftment of AML-samples. In addition to leukemic 

blasts, we frequently observed co-engraftment 

of human T cells present in the AML-graft, possib- 

ly facilitated due to irradiation of recipients. In 4 

cases, stable AML engraftment was inhibited by 

T cell outgrowth and induction of xenoreactivity. 

However, in general irradiation augmented the 

engraftment efficacy of AML. Thus, we attempted 

to circumvent T cell growth by a) depleting T cells 

from AML-samples using MACS-technology® or b) 

treating animals with immunosuppressiva such as 

Cyclosporin A, Mycophenolatemofetil and Tacrolimus. 

Immunomagnetic T cell depletion resulted in 

stable but decelerated AML engraftment without 

T cell outgrowth, whereas preliminary results indicated 

that treatment of transplanted mice with 

immunsuppressiva abrogate engraftment of both 

AML and T cells. Finally, we started to explore our 

model for investigating GVL-responses of AMLreactive 

human T cell lines generated by repetitive 

stimulation of healthy donor lymphocytes with 

AML blasts. In first studies using HLA-mismatched 

AML MZ667-reactive CD8+ T cells we could demonstrate 

that following adoptive transfer of T 

lymphocytes into AML MZ667 bearing mice, leukemic 

blasts could be completely eradicated, whereas 

in controls stable engraftment was detectable in the 

BM. 

Conclusions: These results suggest that NSG mice 

represent a valuable tool for the establishment of a 

preclinical AML model to evaluate the immunotherapeutic 

GVL-reactivity of ex vivo modified T cellgrafts 

as well as to analyze properties of leukemia 

stem cell biology. 

141

093 Koslowski | Tumor biology & interaction with the immune system 

Tumor-associated genomic DNA hypomethylation 

effects positive autoregulation of HIF-1α 

Michael Koslowski 1 , Ulrich Luxemburger 1 , Özlem Türeci 2 , Ugur Sahin 1 

1 TRON gGmbH, Langenbeckstr., 55131 Mainz 

2 Ganymed Pharmaceuticals AG, Freiligrathstr.12, 55131 Mainz 

142 

Hypoxia-inducible factor 1α (HIF-1α) is frequently 

overexpressed in human cancers and controls the 

expression of several genes that have been implicated 

in tumor growth and progression. Activity of 

HIF-1α in cancer cells is regulated at the transcriptional, 

translational and posttranslational level by 

multiple inter- and co-acting molecular pathways. 

We show for the first time that tumor-associated 

genomic DNA hypomethylation facilitates positive 

autoregulation of HIF-1α, resulting in amplification 

of hypoxia-induced transactivation of HIF-1α target 

genes. Applying bisulfite sequencing, methylationspecific 

PCR, transcription factor binding assays, 

and chromatin immunoprecipitation we found that 

the HIF-1α promoter harbors a hypoxia-response 

element that is normally repressed by methylation 

of a CpG dinucleotide located in the core element. 

However, in colon cancer cell lines and in primary 

colon cancer samples we found frequent demethylation 

of this element, enabling binding and positive 

autoregulation of HIF-1α. Our results provide novel 

and highly unexpected insights into the complexity 

of HIF-1α regulation in cancer cells and implicate 

that tumor-associated genomic DNA hypomethylation 

is a positive factor for HIF-1α mediated effects 

on malignant cell growth.

094 Quaglino | Tumor biology & interaction with the immune system 

A miRNAs expression profiles during ErbB2 driven mammary 

carcinogenesis 

Elena Quaglino 1 , Maddalena Arigoni 1 , Elisabetta Ercole 1 , Federica Riccardo 1 , Guido Forni 1 , 

Raffaele Calogero 2 and Federica Cavallo 1 

1 Molecular Biotechnology Center (MBC), University of Torino, Italy 

2 Bioinformatics and Genomics Unit, Department of Clinical and Biological Science, University of Torino, Italy 

MicroRNAs (miRNAs) are noncoding RNAs that 

regulate global gene expression. They often act 

synergistically to repress target genes, and their 

deregulation can contribute to the initiation and 

progression of a variety of cancers. In view of the 

roles played by miRNAs in cancer progression, we 

performed a miRNAs microarray analysis aimed at 

identifying the modulation of miRNAs expression 

profiles during the progression of authoctonous 

mammary carcinomas arising in mice transgenic 

for the activated transforming rat ErbB2 oncogene 

(BALB-neuT mice). BALB-neuT mice constitute 

a suitable cancer-prone model, since inexorably 

the females develop an invasive and metastatic 

mammary cancer in all of their ten mammary 

glands with a step wide pattern and a systemic metastatic 

spread similar to that observed in human 

mammary cancer. Using Applied Biosystems 

Megaplex low density miRNA expression arrays 

miRNAs expression signature of diffused atypical 

hyperplasia (6 week-old BALB-neuT versus 6 weekold 

BALB-c females mammary glands) and invasive 

carcinoma (19 week-old BALB-neuT versus 19 

week-old BALB-c females mammary glands) were 

identified. PCA shows a wide difference in miRNAs 

expression between diffused atypical hyperplasia 

and invasive carcinoma. Moreover, rank product 

analysis allowed the detection of 6 and 4 miRNAs 

whose expression is correlated to invasive carcinoma 

and to diffused atypical hyperplasia respectively. 

Among those that were found to be upregu- 

lated in invasive carcinoma miR-135a and miR-135b 

were found to be expressed also in several lines 

derived from BALB-neuT mammary tumors, suggesting 

their role in mammary cancer progression. 

By contrast, the expression of miR-741 resulted 

undetectable in all the mouse cell lines derived 

from BALB-neuT mammary tumors, even if, its expression 

was upregulated in mammary BALBneuT 

tumors, suggesting its role in the tumor microenvironment 

phenotype. Modulation (overexpression 

or down-modulation) of miR-135b in breast cancer 

cells is under investigation in order to evaluate its 

role in the in vivo tumor growth, in vitro matrigel 

invasion as well as in vivo lung metastasis formation. 

143

095 Lanzardo | Tumor biology & interaction with the immune system 

Attenuation of PI3K/Akt-mediated tumorigenic signals through 

PTEN activation by DNA vaccine-induced anti-ErbB2 

antibodies 

Stefania Lanzardo 1 , Alessandra Porzia 2 , Arianna Citti 2 , Federica Cavallo 1 , Guido Forni 1 , 

Angela Santoni 2 , Ricciarda Galandrini 2 , Rossella Paolini 2 

1 Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, Italy 

2 Department of Experimental Medicine, Institute Pasteur-Fondazione Cenci Bolognetti, 

Sapienza University, Rome, Italy 

144 

Vaccination of BALB/c mice with a plasmid enco- 

ding for the extracellular (EC) and transmembrane 

(TM) domains of rat ErbB2 (EC-TMneu) protects 

from a lethal challenge of ErbB2+ tumor cells 

by eliciting both a cell mediated and a humoral 

immune response. ECTMneu vaccination also 

leads to the inhibition of spontaneous mammary 

tumors in rat ErbB2 transgenic mice mainly by eliciting 

anti-rat ErbB2 Abs capable of inhibiting in 

vitro the expansion of ErbB2+ cells. However, the 

molecular mechanisms underlying Ab-induced retardation/rejection 

of tumor growth have not been 

elucidated. 

By studying BALB/c mice deficient in immune components 

we show that the protective immunity to 

rat ErbB2+ tumors rests on the antibody response 

elicited by the electroporation of a DNA vaccine 

encoding the extracellular and transmembrane 

domains of rat ErbB2. In vivo the adoptive transfer 

of vaccine-elicited anti-rat ErbB2 antibodies 

protected against a challenge of rat ErbB2+ carcinoma 

cells (TUBO cells). In vitro such antibodies 

inhibited TUBO cell growth by impairing cell cycle 

progression and inducing apoptosis. To correlate 

intrinsic mechanisms of antibody action with their 

tumor-inhibitory potential, first we showed that 

TUBO cells constitutively express phosphorylated 

transgenic ErbB2/autochthonous ErbB3 heterodimers, 

and exhibit a basal level of Akt phosphorylation 

suggesting a constitutive activation of the 

PI3K/Akt pathway. The treatment with anti-ErbB2 

antibodies caused a drastic reduction of the basal 

level of Akt phosphorylation in the absence of an 

impairment of PI3K enzymatic activity. Notably, 

the same antibody treatment induced an increase 

of PTEN phosphatase activity that correlated with a 

reduced PTEN tyrosine phosphorylation. In conclusion, 

vaccine-induced anti-ErbB2 antibodies directly 

affect the transformed phenotype of rat ErbB2+ 

tumors by impairing ErbB2-mediated PI3K/Akt signalling.

096 Halama | Tumor biology & interaction with the immune system 

Natural Killer Cells are decreased in Human Colorectal Cancer 

Tissue and Liver Metastases despite High Levels of 

NK-Recruiting Chemokines 

Niels Halama 1,2 Monika Braun 3 , Christoph Kahlert 4 , Anna Spille 1 , Christian Quack 3 , Nuh 

Rahbari 4 , Moritz Koch 4 , Jürgen Weitz 4 , Inka Zoernig 1 , Peter Schirmacher 5 , Karsten Brand 2 , 

Niels Grabe 2 and Christine S. Falk 3 

1 Medical Oncology, National Center for Tumor Diseases, University of Heidelberg, Heidelberg, Germany 

2 Hamamatsu Tissue Imaging and Analysis (TIGA) Center, Institute for Medical Biometry and Informatics, 

University of Heidelberg, Heidelberg, Germany 

3 Immune Monitoring Unit, NCT, DKFZ and Institute for Immunology 

Im Neuenheimer Feld 305, 69120 Heidelberg 

4 Department of Surgery, University of Heidelberg, Heidelberg, Germany 

5 Institute of Pathology, University of Heidelberg, Heidelberg, Germany 

Background & aims Tumor infiltrating T lympho- 

cytes in colorectal cancer (CRC) have prognostic 

impact, but the role of NK cells in CRC tissue 

remains unknown. The contribution of intratumoral 

cytokines and chemokines in shaping the composition 

of the inflammatory lymphocytic infiltrate 

is also unclear. 

Methods: In this study, localization and densities 

of NK and T cells within primary CRC, CRC liver 

metastases, and normal tissues were analyzed on 

whole tissue sections. In parallel, the most important 

cytokines and chemokines were quantified in 

paired serum, primary CRC and adjacent mucosa 

samples and in CRC liver metastases and correlated 

with NK and T cell infiltration, respectively. 

Results: The different compartments displayed 

characteristic differences like significantly higher 

chemokine concentrations in CRC tissue. Most 

importantly, despite high local chemokine levels, 

NK cells were generally scarce within CRC tumor 

tissues, independent of HLA class I expression. 

Adjacent normal mucosa contained normal levels of 

NK cells. In contrast, corresponding T cell numbers 

varied substantially and were positively correlated 

with higher chemokine levels. 

Conclusions: Our findings indicate a distinct regulation 

of NK cells versus T cells in the CRC tumor 

microenvironment. NK cell migration into CRC 

tumor tissue is obviously impaired early during 

tumor development by mechanisms that do not 

affect T cell infiltration. 

145

097 Fraszczak | Tumor biology & interaction with the immune system 

Killer dendritic cells inhibit Treg differentiation 

Jennifer Fraszczak 1 , Malika Trad 1 , Nona Janikashvili 1,2 , Daniela Lakomy 1 , Maxime Samson 

1 , Sylvain Audia 1 , Julien Vinit 1 , Nicolas Larmonier 2 , Bernard Bonnotte 1 

1 CR Inserm 866, Faculty of medicine, 7 boulevard Jeanne d’Arc, 21000 Dijon, France 

2 Department of Paediatrics, Steele Children's Research Centre, University of Arizona, Tucson, AZ 85724, USA 

146 

Known for decades as the principal messengers of 

the immune system, dendritic cells (DC) play a critical 

role in the initiation and regulation of immune 

responses against tumor. We have recently reported 

on the non-conventional direct tumor killing activity 

of DC. We have demonstrated that mouse DC generated 

from bone marrow precursors are endowed 

with the capability to kill multiple types of cancer 

cells and have identified peroxynitrites as the main 

effector molecules underlying this cytotoxic activity. 

Importantly these killer DC (KDC) are capable 

of presenting tumor antigens from the cancer cells 

they had killed to specific T cells thereby inducing 

an efficient antitumor immune response. We here 

evaluate whether KDC may modulate regulatory T 

cells (Treg), critical contributors of cancer-induced 

immune tolerance and major obstacles for tumor 

immunotherapy. Our results indicate that KDC significantly 

impair the polarization of naive T cells 

into FoxP3-expressing immunosuppressive Treg 

but foster their differentiation into T bet-expressing 

Th-1 cells. The cellular and molecular bases 

responsible for this negative modulation of Treg by 

KDC is currently under investigation.

098 Trad | Tumor biology & interaction with the immune system 

Tumor infiltrating CD11c+ dendritic cells suppress T cell 

activation 

Malika Trad 1 , Jennifer Fraszczak 1 , Daniela Lakomy 1 , Maxime Samson 1 , Sylvain Audia 1 , 

Julien Vinit 1 , Emmanuel Katsanis 2 , Nicolas Larmonier 2 * and Bernard Bonnotte 1 

1 CR INSERM 866, Université de Bourgogne, Dijon, France 

2 Department of Pediatrics, Steele Children’s Research Center, University of Arizona, Tucson, USA 

* Equal contribution 

Many tumors, including breast cancers are infil- 

trated by dendritic cells (DC), known primarily for 

their capability to induce and sustain anti-tumoral 

immune responses. However, a significant deficit 

in the function of DC has been reported in cancer 

patients and in various animal tumor models, 

which may account for tumor escape from the 

immune system. Using the murine breast cancer 

model 4T1, we have investigated the phenotypical 

and functional characteristics of tumor-infiltrating 

DC (TIDC). Our results indicate that TIDC, isolated 

from 4T1 tumor-bearing Balb/c mice based on the 

expression of the marker CD11c, exhibit a mixed 

phenotype associating the expression of mature DC 

markers (co-stimulatory molecules) and the Gr-1 

molecule (a marker of immunosuppressive myeloid 

cells). Functionally, TIDC were unable to induce 

allogeneic T cell proliferation and produced low 

amount of the pro-inflammatory cytokine IL-12, 

required for the T cell activation. Conversely, TIDC 

secreted the immunosuppressive cytokine IL-10 

and were capable of suppressing the proliferation 

of T lymphocytes induced with anti-CD3 and anti- 

CD28. The mechanism(s) underlying T cell suppression 

by TIDC did not depend on nitric oxide, 

but may involve the enzyme indoleamine 2,3-dioxygenase 

(IDO) which was significantly upregulated 

in these cells. 

147

099 Hemmerling | Tumor biology & interaction with the immune system 

Human Langerhans cells reconstitute in skin xenografts 

Julia Hemmerling 1 , Joanna Wegner-Kops 1 , Diana Wolff 1 , Maria Sommer 1 , Eva M. Wagner 1 , 

Esther von Stebut 2 , Udo F. Hartwig 1 , Rudolf E. Schopf 2 , Matthias Theobald 1 , Wolfgang 

Herr 1 , Ralf G. Meyer 1 

1 Department of Hematology/Oncology and 

2 Department of Dermatology, at the University Medical Center of the Johannes Gutenberg-University 


148 

Dendritic cells (DC) of the skin, e.g. epidermal Lan- 

gerhans cells (LC), are potent regulators of T cells. 

They therefore are interesting targets for autologous 

T-cell stimulation in the context of vaccinestrategies 

as well as for the manipulation of allo-reactive 

T-cells in graft-versus-host disease (GVHD). 

In an attempt to study the biology of human LC in 

vivo, we transplanted human skin to immunodeficient 

NOD/SCID/γcnull (NSG) mice and studied the 

impact of xeno-transplantation on the distribution 

of LC in comparison to that of CD11c positive dermal 

DC. We analyzed skin xenografts at different time 

points after transplantation and found that during 

wound healing, LC were absent in grafts prepared 

4 to 6 weeks after transplantation. However, in 

many animals analyzed beyond week 6, LC were 

again present with almost normal distribution. 

These findings were re-confirmed by staining with 

antibodies against human CD1a, CD207/Langerin, 

S100 and HLA-DR. CD11c positive human dermal 

cells were also transiently reduced in numbers, but 

never vanished from the dermis after xenografting. 

In subsequent experiments, we analyzed the skin 

grafts by sequential biopsies and again demonstrated 

that more than 50 % of the skin grafts were 

devoid of LC in week 6. Three to 5 weeks later, 

LC were detected again in these transplants with a 

slightly reduced density compared to normal skin. 

In congenic mouse models of hematopoietic stem 

cell transplantation, murine LC have been shown 

to re-populate after local skin injury without the 

need of blood-derived precursors. Up to now, there 

are only few data supporting this hypothesis for 

human LC. By demonstrating the re-establishment 

of LC in the xenografts in the absence of any human 

hematopoiesis, our data confirm that human LC are 

able to re-populate the skin after local depletion. 

We further analyzed the proliferative activity of 

LC in the xenografts by double staining of CD207/ 

Langerin-positive cells with Ki67 and found a significantly 

increased proliferative activity of LC 

compared to healthy skin (31% vs. 5%). Proliferation 

is a unique feature of LC among DC. Our data 

suggest that this contributes to LC-reconstitution. 

In summary, we introduce a human skin xenograftmodel 

that allows studying the biology of LC and 

dermal DC. Our findings help to unravel the phenomenon 

of local LC-reconstitution in human skin. 

This model might also help to study the role of skin 

DC for inflammatory skin disease and skin GVHD 

as well as for vaccine-strategies in vivo.

100 Koop | Tumor biology & interaction with the immune system 

Downregulation of cancer/testis antigen 45 (CT45) in 

human HT1080 cells analysed by proteomic screening 

Anja Koop 1 , Hendrik Schmidt 1 , Melanie Nebendahl 1 , Christoph Gelhaus 2 , Matthias Leippe 2 , 

Ottmar Janssen 1 , Hans-Jürgen Heidebrecht 3 

1 Institute of Immunology, UK-SH, Campus Kiel 

2 Department of Zoophysiology, University of Kiel 

3 Institute of Pathology, UK S-H, Campus Kiel 

The cancer/testis antigen (CT) family is defined by 

its specific expression pattern. In most cases, CT 

antigens are exclusively expressed in germline cells 

and in some tumors. However, as for many other 

CT antigens, little is known about the function of 

CT45. Since various CT antigens induce antigen 

specific cellular or humoral immune responses, 

they are regarded as vaccine targets for anti-cancer 


To get an idea about the function of CT45, we 

looked for differentially expressed proteins in the 

CT45 positive human fibrosarcoma cellline HT1080 

after downregulation of CT45. Therefore five different 

cellpreparations of scrRNA and siRNA treated 

HT1080 were prepared 48h after treatment. Only 

cellpreparations with significantly downregulated 

CT45 expression were used for these experiments. 

In order to determine regulated proteins, lysates 

were analysed by 2D-DIGE and differentially expressed 

proteins were identified by peptide mass 

finger printing. Equal amounts of each sample were 

labelled with Cy3 or Cy5. After isoelectric focusing 

proteins were separated on 12,5% polyacrylamid 

gels (20x24cm). The in gel fluorescence corresponding 

to the protein concentration was detected by 

an fluorescence scanner. Differentially expressed 

proteins were picked, proteolytically cleaved by 

trypsin and then subjected to MALDI analysis. One 

of the differentially expressed proteins is Annexin1 

(ANXA1). In all five cellular lysates of 48h siRNA 

treated HT1080 cells it was significantly downre- 

gulated. ANAX1 regulates Phospholipase A2 activity 

and is considered to play an important role in 

the tumorgenesis of breast cancer. Further experiments 

will resolve the significance of the ANAX1 

downregulation. 

The work is sponsored by the Medical Faculty of the CAU Kiel 

and the Wilhelm-Sander-Stiftung. 

149

101 Karakhanova | Tumor biology & interaction with the immune system 

IFN-α up-regulates B7-H1 expression on dendritic cells 

and pancreatic tumor cells 

Svetlana Karakhanova 1 , Ralf Jesenofsky 2 , Susanne Serba 1 , Alexandr V. Bazhin 1 , Jan Schmidt 1 

1 Department of Surgery, University of Heidelberg, Germany 

2 II. University Hospital, Mannheim, Germany 

150 

Pancreatic carcinoma is one of the most aggressive 

human malignant tumors and most lethal cancers 

worldwide. Despite recent advances in surgery, radiation 

and chemotherapy, outcome of pancreatic 

cancer could be improved only partially. Therefore 

novel approaches are strongly needed. Immunotherapy 

represents such an option. Clinical data 

show promising results for approaches combining 

chemotherapeutics with activating cytokines, like 

IFN-α. However, in parallel with immune stimulating 

effects of this treatment, the presence and induction 

of immunosuppressive mechanisms should 

be considered in the course of such a pancreatic 

cancer therapy. Regulatory molecules of the B7-H 

family, especially B7-H1 (PD-L1, CD274), play an important 

role in the regulation of immune responses. 

In human pancreatic carcinoma tissue B7-H1 is upregulated 

and the patients, demonstrating increased 

B7-H1 expression have a poor prognosis. B7-H1 

expression in pancreatic tumors contributes to the 

tumor immune evasion and tumor progression and, 

as recently demonstrated, blocking of B7-H1 improves 

anti-tumor effects in a mouse pancreatic cancer 

model. 

In our study we aim to investigate the expression 

and function of B7-H1 molecules in the context of 

IFN-α therapy of pancreatic cancer. 

Using Flow cytometry, cytological methods as well 

as RT-PCR approaches, we showed that B7-H1 expression 

is up-regulated on human dendritic cells 

(DC) and some human pancreatic cancer cell lines 

upon treatment with IFN-α. Interestingly, in some 

cell lines we observed additive effect of 5-FU (chemotherapeutic 

agents used for pancreatic carcinoma 

treatment) and IFN-α on the increase of B7-H1 

expression. To confirm the inhibitory potential of 

IFN-α -induced B7-H1 molecule, we performed cocultures 

of T-Cells and IFN-α-treated DC with and 

without blocking of B7-H1 with specific antibodies 

and measured proliferation and cytokine production 

from T-cells. In ongoing study we are using an 

orthotopic mouse model of pancreatic carcinoma to 

demonstrate that additional blocking of B7-H1 molecule 

during 5-FU and IFN-α combinatory therapy 

may additionally improve the outcome of this treatment. 

Our data demonstrate that, despite the stimulation 

of anti-tumor effects, IFN-α up regulates the expression 

of immunosuppressive B7-H1 molecules, 

which could limit the effect of immunotherapy 

with IFN-α. Thus, it could be of advantage to reduce 

undesirable site effects of the IFN-α-induced B7-H1 

expression on the tumor and immune cells.

102 Zimmer | Tumor biology & interaction with the immune system 

Impact of endoplasmic reticulum aminopeptidases (ERAP) 

on T cell epitope generation in melanoma cells 

Christin Zimmer 1 , Silke Lubojanski 2 , Tanja Dannenberg 1 , Xhao Fang 1 , Ulrike Seifert 3 , Dirk 

Schadendorf 1 , Annette Paschen 1 

1 Department of Dermatology, University Hospital Essen, Essen, Germany 

2 Department of Medicine III, University Medical Centre, Mainz, Germany 

3 Institute for Biochemistry, University Medicine Charite, Berlin, Germany 

Tumor cells presenting antigen epitopes by HLA 

class I molecules can be specifically recognized and 

effectively killed by autologous cytotoxic CD8+ T 

lymphocytes (CTLs). Different proteolytic/peptidolytic 

components are involved in the intracellular 

generation of antigen epitopes. The cytosolic, multi-catalytic 

proteasome cleaves the tumor antigen 

into peptide fragments of different length, thereby 

defining the C-terminus for the majority of peptides 

presented by HLA class I molecules. While some 

of the proteasome products are of the correct size 

for direct binding to HLA class I molecules others 

are N-terminally elongated peptide precursors that 

require further trimming by aminopeptidases. In 

the endoplasmic reticulum of human cells two 

aminopeptidases, ERAP1 and ERAP2, have been 

identified. Their function in epitope generation by 

precursor trimming has been clearly demonstrated. 

Thus, the aim of this study is to determine the influence 

of ERAPs on the presentation of specific 

tumor antigen epitopes by melanoma cells. At first, 

the expression level of ERAP1 and ERAP2 in several 

cell lines established from different metastases of 

melanoma patients was determined by quantitative 

real time PCR and Western Blot analysis. All cell 

lines tested expressed ERAP1 and ERAP2 at mRNA 

and protein level, albeit at different quantities. To 

gain further insight into the role of EARP1 in the 

generation of specific CTL epitopes, its expression 

was down-regulated either by transient transfection 

of melanoma cells with siRNA or stable 

transfection with a shRNA expression plasmid. In 

either case, knockdown of ERAP1 expression was 

verified by RT-PCR and/or Western Blot analysis. 

Interestingly, ERAP1 down-regulation increased 

the recognition of melanoma cells UKRV-Mel-15 

by CTLs specific for an epitope (Melan-A26/27-35) 

derived from the melanoma differentiation antigen 

Melan-A/MART-1. Conversely, overexpression of 

ERAP1 decreased the presentation of the Melan-A/ 

MART-126/27-35 epitope, pointing to a destructive 

role of ERAP1 in the processing of this epitope. In 

contrast, down-regulation of ERAP1 in Ma-Mel-86a 

cells did not affect the stimulation of autologous 

CD8+ T cells directed against a mutated neoantigen 

(please see abstract by Lubojanski et al.). 

Our results point to a more epitope-specific role of 

ERAP1 in antigen presentation: ERAP1 activity can 

either contribute to or interfere with the generation 

of specific epitopes while others might even be generated 

independently. 

151

103 Heidemann Krogh | Tumor biology & interaction with the immune system 

The phospho-regulated calcium-binding cancer-testis antigen 

cabyr, interacts with glycolysis-regulating enzymes in lung cancer 

cells 

Anette Heidemann Krogh, Allan Stensballe 2 , Anne Elbæk, Lotte Hatting Pugholm, Evo Kristina 

Søndergaard, Rikke Bæk, Malene Jørgensen, Kim Varming, and Søren Naaby-Hansen 

1 Department of Clinical Immunology, Aalborg sygehus, Århus University Hospitals, Aalborg, Denmark 

2 Department of Biotechnology, Aalborg University, Aalborg, Denmark 

152 

Background: CABYR was originally identified as a 

human sperm flagellum protein associated with the 

fibrous sheath structure and involved in capacitation-induced 

hyperactivated motility. Six variants of 

CABYR containing two coding regions (CR-A and 

CR-B) were cloned from human testis cDNA libraries, 

including five variants with alternative splice 

deletions. CABYR possesses several motifs and 

domains for self-assembly and protein interactions. 

Although CABYR transcripts corresponding to the 

entire CR-A are testis-specific, small amounts of 

minor splice variants are present in motile cilia of 

normal human bronchus and fallopian tubes. More 

interesting, CABYR expression has recently been 

demonstrated in a variety of solid tumours, including 

brain, lung, and head & neck squamous cell 

cancers. 

In testis and sperm CABYR seems to play a role in 

the regulation of calcium sequestration and episodic 

release, and to form a scaffold with other testisspecific 

AKAPs that serve to integrate PKA, Rho and 

calcium signalling. CABYR is finally thought to act as 

a Ca2+-shuttle in a high order fibrous sheath protein 

complex that mediate glycolysis-driven energy 

generation in the distal flagellum. While CABYR’ 

roles in spermatogenesis and gamete function are 

emerging, its function in cancer cells remains to be 

demonstrated. Here we report recent data from an 

ongoing study of CABYR in lung cancer. 

Results and Discussion 

To define CABYR’ interactions in the lung adeno- 

carcinoma cell line A549, immunoprecipitation (IP) 

with monoclonal antibodies against CABYR was 

combined with western blot (WB) and mass spectrometry 

(MS) analysis. Butyrate-induced protein 

1, a protein tyrosine phosphatase and the regulatory 

subunit of serine/threonine-protein phosphatase 

2A, were co-precipitated with CABYR. These 

putative interactions are in accordance with our 

previous findings of serine- and threonine-phosphorylation 

in testicular CABYR and that CABYR’ 

calcium-binding capacity is regulated by tyrosine 

phosphorylation. 

More notable, several enzymes involved in the regulation 

of glycolysis, including phosphofructokinase, 

pyruvate kinase, fructose-biphosphate aldolase, 

malate dehydrogenase, and the testis-specific 

subunit of lactate dehydrogenase, as well as hypoxia-inducible 

factor prolyl hydroxylase 3 and ADP/ 

ATP translocase 1, also co-purified with CABYR 

from A549 cells. The interaction between CABYR 

and lactate dehydrogenase was confirmed by bidirectional 

IP and WB. 

Taken together, these results suggest that CABYR 

participates in the regulation of anaerobic glycolysis 

and energy generation in lung cancer, and that 

CABYR expression is an indicator of aggressive 

tumour growth. The regulation of the CABYR interactome, 

and cancer cells ability to survive and proliferate 

under hypoxic conditions following RNAimediated 

downregulation of CABYR, are currently 

being investigated.

104 Hansmann | Tumor biology & interaction with the immune system 

Isolation of intact genomic DNA from FOXP3-stained and 

FACS-sorted regulatory T cells for epigenetic analysis 

Leo Hansmann, Christian Schmidl, Tina J. Boeld, Reinhard Andreesen, Petra Hoffmann, 

Michael Rehli, Matthias Edinger 

Department of Hematology & Oncology, University Hospital Regensburg, 93042 Regensburg, Germany 

The CD4+ T cell compartment contains cells of 

different lineages and differentiation states that 

include Th1, Th2, Th17 and natural regulatory T 

(Treg) cells. Due to the lack of exclusive surface 

markers for these T cell subpopulations, they can 

be differentiated from each other only by their cytokine 

secretion profile, their functional characteristics 

or, most reliably, their expression lineage defining 

transcription factors, including T-bet, GATA-3, 

RORC or FOXP3 for Th1, Th2, Th17 and Treg cells, 

respectively. We recently showed that stable expression 

of the Foxp3 gene in human Treg cells 

is regulated in parts by DNA methylation (Baron 

et al. Eur J Immunol 2007, Hoffmann et al. Eur J 

Immunol 2009, Schmidl et al. Genome Res 2009). 

Yet, due to the lack of methods for the isolation 

of intact genomic DNA from fixed and intranuclearly-stained 

cells we thus far had to rely on surrogate 

markers for the identification and isolation 

of human Tregs for epigenetic analyses. We now 

present a modified phenol-based DNA isolation protocol 

permitting the reliable purification of intact 

high-molecular genomic DNA for further downstream 

applications, such as MALDI-TOF MS based 

DNA methylation analysis, that includes bisulphite 

conversion and PCR amplification of the extracted 

DNA. This method enables us to investigate human 

Treg cell epigenetics with the same accuracy as in 

murine systems where transgenic reporter mice are 

usually used for the unequivocal identification and 

isolation of Foxp3+ Treg cells. We present the first 

methylation analyses of the FOXP3 locus in human 

conventional T- and Treg cells sorted for intranuclear 

FOXP3 expression. 

153

105 Lupp | Tumor biology & interaction with the immune system 

Mechanisms of Treg-mediated suppression 

of graft-versus-host disease 

Corinna Lupp 1 , Tobias Bopp 1 , Edgar Schmitt 1 , Hansjörg Schild 1 and Markus P. Radsak 1,2 

1 Institute for Immunology, Universitätsmedizin of the Johannes Gutenberg-University, Mainz, Germany 

2 III. Dept. of Medicine, University Hospital, Johannes Gutenberg-University, Mainz, Germany 

154 

The therapeutic efficacy of allogenic bone marrow 

transplantation for many hematological malignancies 

relies on the graft-versus leukemia effect by eliminating 

residual leukemic cells. The recognition of 

host alloantigens by donor T cells is most likely the 

basis of graft-versus-tumor effects, but is oftentimes 

also the cause of graft-versus-host disease (GvHD), 

a key contributor to the high morbidity and mortality 

rates in the context of bone marrow transplantation. 

Regulatory FoxP3+ CD4+ T cells (Tregs) are 

known to suppress the activation of conventional T 

cells (e.g. CD8+ or CD4+ T cells) and represent a 

therapeutic paradigm for the control of GvHD. 

In our present study we analyzed potential mechanisms 

to prevent or attenuate acute graft-versushost 

reactions in an allogenic MHC mismatched 

transplantation model. 

To study the suppressive effects of Tregs in detail we 

performed allogenic mixed lymphocyte reactions 

(MLR) in vitro and allogenic bone marrow/T cell 

transplantations in mice. In transplantation experiments 

we used BALB/c mice as recipients which 

became irradiated lethally and got a bone marrow 

and T cell transplant from C57BL/6 donor mice. 

Some mice additionally got Tregs from Interleukin 

10 deficient (IL-10 -/-) or the wildtype C57BL/6 

donors. The control group which did not get Tregs 

developed severe graft-versus-host disease symptoms 

after transplantation. 

Additionally transplanted Tregs from IL-10 -/- as 

well as from wildtype donors could attenuate cli- 

nical GvHD symptoms. And also in vitro the Tregmediated 

suppression of the alloreactive effector T 

cells’ proliferation occurred independent of IL-10. 

The analysis of the phenotype of dendritic cells 

(DCs) that had been cultured with Treg cells discovered 

a reduced expression of the costimulatory 

molecules CD 80 and CD 86. We could show that 

Tregs communicate with allogenic DCs via direct 

cell-to-cell contact, demonstrated by the transfer of 

the dye calcein from calcein-labeled Tregs to DCs. 

This calcein transfer could partially be prevented 

through the preincubation of the Tregs with the gap 

junction blocking peptide GAP 27. Only a slight dye 

transfer to the conventional T cells was detectable in 

the MLR indicating that the Tregs preferentially interact 

with the allogenic antigen presenting cells. 

In conclusion, our results demonstrate that Tregs 

interact with allogenic antigen presenting cells via 

gap junction intercellular communication. There 

is only a little cell-contact dependent interaction 

between the Tregs and the effector T cells and the 

cytokine IL-10 seems not to be indispensable for the 

inhibition of alloreactivity. 

These data provide the basis for future concepts to 

manipulate allogenic T cell responses to prevent 

GvHD.

106 Castle | Tumor biology & interaction with the immune system 

DNA copy number, including telomeres and mitochondria, 

assayed using next-generation sequencing 

John C. Castle* 1 , Christopher D. Armour 2 , Matthew Biery 2 , David Haynor 3 , Heather Bouzek 

3 , Tao Xie 4 , Ronghua Chen 5 , Kira Misura 6 , Stuart Jackson 5 , Jason M. Johnson 5 , Carol A. 

Rohl 5 , Christopher K. Raymond 2 

1 Institute for Translational Oncology and Immunology (TrOn), Mainz, Germany 

2 NuGEN Technologies, Inc., Seattle, Washington, USA 

3 University of Washington, Seattle, Washington, USA 

4 Pfizer, Inc., San Diego, California, USA 

5 Merck Research Laboratories, Boston, Massachusetts, USA 

6 Amgen, Inc., Seattle, Washington, USA 

DNA copy number aberrations are found in tumors 

and are potentially carcinogenic. We sought to 

develop an improved lab-automatable, cost-efficient, 

accurate platform using next-generation sequencing 

to profile nuclear, mitochondrial, and 

telomeric DNA copy number. This platform draws 

on the unbiased nature of next-generation sequencing 

and incorporates techniques developed for 

RNA expression profiling. To demonstrate this platform, 

we assayed tumor-derived cell line UMC-11 

using 5 million 33 nt reads and found tremendous 

copy number variation, including regions of single 

and homogeneous deletions and amplifications 

to 29 copies; 5 times more mitochondria and 4 

times less telomeric sequence than a pool of nondiseased, 

blood-derived DNA; and that UMC-11 

was derived from a male individual. The described 

assay outputs absolute copy number, outputs an 

error estimate (p-value), and is more accurate than 

array-based platforms at high copy number. The 

platform enables profiling of mitochondrial levels 

and telomeric length. The assay is lab-automatable 

and has a genomic resolution and cost that are 

tunable based on the number of sequence reads. 

155

156 

Enhancing immunity & adjuvants

107 Sevko | Enhancing immunity & adjuvants 

Paclitaxel in ultra-low doses reduces immunosuppression 

in ret transgenic tumor bearing mice 

Alexandra Sevko 1 , Michael Shurin 2 , Viktor Umansky 1 

1 German Cancer Research Center, Heidelberg, Germany 

2 Departments of Pathology and Immunology, University of Pittsburgh Medical Center, Pittsburgh, 

Pennsylvania, USA 

It has been recently shown that chemotherapeutic 

drugs such as paclitaxel, 5-fluoruracil or doxorubicin 

applied in ultra-low, noncytotoxic doses stimulate 

dendritic cell activity, and induce antitumor 

immune responses in mouse transplantable tumor 

models. 

We studied effects of ultra low-dose paclitaxel on 

tumor progression in ret transgenic mouse model 

of spontaneous melanoma, which closely resembles 

human melanoma regarding histopathology and 

clinical development. 

We showed that the survival of melanoma bearing 

mice upon the treatment was significantly increased 

as compared to non-treated group that correlated 

with a significant decrease in the tumor weight 

in mice from paclitaxel-treated group. To evaluate 

the mechanism of this antitumor effect, we focused 

on immunosuppressive cells like myeloid derived 

suppressor cells (MDSC) and regulatory T cells 

(Treg). Paclitaxel was found to decrease the number 

of MDSC infiltrating primary tumors and metastatic 

lymph nodes. Importantly, the amount of MDSC producing 

immunosuppressive agent nitric oxide was 

also decreased in these melanoma lesions and in the 

bone marrow. In addition, paclitaxel induced a reduction 

of Treg infiltrating primary tumors without 

affecting the total number of CD4+ T cells. 

We suggest that ultra low-dose paclitaxel therapy 

can neutralize tumor-induced immune suppression 

leading to the delayed tumor progression and prolonged 

survival of tumor bearing mice. 

157

108 Diaz-Valdés | Enhancing immunity & adjuvants 

Transforming growth beta 1 increases monocyte chemotactin 

protein-1 and interleukin-10 expression in A375 human 

melanoma cells enhancing monocyte migration and impairing 

dendritic cell function 

Nancy Díaz-Valdés 1 , María Basagoiti 1 , Javier Dotor 2 , Iñaki Monreal 1 , Jose Ignacio Riezu- 

Boj 1 , Francisco Borrás-Cuesta 1 , Pablo Sarobe 1 and Esperanza Feijoó 2 

1 Universidad de Navarra, Centro de Investigación Médica Aplicada, Area de Hepatología y Terapia Génica, 

Pamplona, Spain 

2 DIGNA Biotech, Madrid, Spain 

158 

Melanoma progression is associated to the expres- 

sion of different growth factors, cytokines and 

chemokines, which collaborate on processes such 

as cell proliferation, angiogenesis, metastasis and 

immunosuppression. Transforming growth beta 1 

(TGFβ1) is a pleiotropic cytokine involved not only 

in physiological processes but also in cancer development. 

Here we analyzed in melanoma cells 

the effect that TGFβ1 has on monocyte chemotactin 

protein-1 (MCP-1) and interleukin-10 (IL-10) expression, 

two known factors responsible for melanoma 

progression. TGFβ1 treatment of A375 human 

melanoma cells increased the expression of MCP-1 

and IL-10 and this effect was mediated by the crosstalk 

between Smad and the AKT and BRAF-MAPK 

signaling pathways. Conditioned medium from 

TGFβ1-treated A375 cells (TGFβ-MCM) enhanced 

MCP-1-dependent migration of monocytes, which 

expressed high levels of TGFβ, basic fibroblast 

growth factor and vascular endothelial growth 

factor mRNA. Concerning IL-10, it had an autocrine 

effect on A375 cells, enhancing their proliferation. 

Moreover, TGFβ-MCM induced a functional 

impairment of dendritic cells, characterized by the 

lower production of proinflammatory factors tumor 

necrosis factor-alfa and interleukin-12 and upregulation 

of immunoregulatory molecules programed 

death ligand-1, herpesvirus entry mediator and 

cyclooxygenase-2, mediated by IL-10. Additionally, 

TGFβ-MCM increased the expression of the inhibitory 

molecules indolamine 2, 3-dioxigenasa, and 

immunoglobulin-like transcript receptor in DC and 

significantly decreased the interferon gamma IFNγ 

production by T-cells in mixed leukocyte reaction 

assays. Finally, the effect of the 15-mer TGF-β1 inhibitor 

peptide P144 (Disitertide) was analysed in 

vitro and in vivo. In vitro, P144 inhibited TGF-β1dependent 

Smad2 phosphorylation and expression 

of MCP-1 and IL-10 in A375 cells. In vivo, treatment 

with P144 of A375-tumor-bearing nude mice significantly 

reduced tumor volume. These results show 

new effects of TGFβ1 on melanoma cells and their 

consequences on tumor progression and immunosuppression, 

strongly reinforcing the role of this 

cytokine as a molecular target in melanoma.

109 Bauer | Enhancing immunity & adjuvants 

The TLR9 ligand CpG reduces the suppressive function of 

myeloid-derived suppressor cells via interferon alpha 

Helen Bauer 1 , Christine Zoglmeier 1 , Daniel Nörenberg 1 , Georg Wedekind 1 , Philipp Bittner 1 , 

Stefan Endres 1 , Carole Bourquin 1 

1 Division of Clinical Pharmacology, Ludwig-Maximilian University Munich, Germany 

Tumor cells employ a wide range of mechanisms 

to evade recognition in tumor-bearing hosts. The 

induction and expansion of myeloid-derived suppressor 

cells (MDSC), a heterogenous population 

of immature, bone-marrow-derived myeloid cells, 

is one of these mechanisms. In mice, MDSCs are 

commonly defined as Gr1+CD11b+ and display 

a remarkable ability to suppress T cell responses 

in vitro via different mechanisms such as ROS-induction, 

arginase and iNOS activity. In our studies, 

we investigated the effect of a stimulation of the 

innate immune system with the TLR9 ligand CpG 

on the function and phenotype of MDSCs in tumorbearing 

mice. 

To investigate the effect of TLR9 activation on 

MDSCs, we treated C26 tumor-bearing mice with 

CpG and analysed the expression of a range of 

surface markers by FACS analysis. We show that 

a number of markers associated with MDSC maturation 

and activation is upregulated by CpG treatment. 

Investigation of the suppressive function of 

MDSCs revealed reduced suppressivity of MDSCs 

from CpG-treated mice. Similar results were obtained 

in an orthotopic model of gastric cancer. In 

vitro, CpG treatment of isolated MDSCs demonstrated 

that the reduction in suppressivity by CpG is 

not the result of a direct activation of MDSCs, but 

is mediated by cytokines released from other cell 

types. Both in in vivo and in vitro experiments we 

show that interferon alpha released by plasmacytoid 

dendritic cells following CpG treatment plays 

a major role in inducing MDSC maturation and reduction 

of suppressivity. 

In summary, our results show that MDSCs are 

strongly affected by TLR9 activation, resulting in 

drastic changes in MDSC phenotype and function. 

In addition, we provide evidence that interferon 

alpha induced by TLR9 activation is a major factor 

in promoting MDSC maturation and reduction of 

MDSC suppressivity in CpG-treated tumor-bearing 

mice. 

159

110 Femel | Enhancing immunity & adjuvants 

Identification of a potent, non-toxic adjuvant 

for use in therapeutic vaccines 

Julia Femel 1 , Elisabeth JM Huijbers 1 , Maria Ringvall 1 , Lars Hellman 2 , Anna-Karin Olsson 1 

1 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, SE-75123 Uppsala, Sweden 

2 Department of Cell and Molecular Biology, Uppsala University, BMC, SE-75124 Uppsala, Sweden 

160 

Monoclonal antibody-based therapies have made an 

important contribution to current treatment strategies 

for cancer and autoimmune disease. However, 

their cost-intensive production could possibly lead 

to limited availability of the therapy. Therefore obtaining 

antibodies produced by the body through 

therapeutic vaccination is an interesting alternative, 

due to the low amount of required recombinant 

protein. To circumvent the tolerance of the 

immune system against self-antigens, an efficient 

vaccination technology as well as a potent adjuvant 

is required. The lack of potent, but at the same time 

non-toxic and biodegradable adjuvants that can be 

used in the clinic, has been a major limiting factor 

for the development of therapeutic vaccines. 

We have identified the biodegradable squalenebased 

Montanide ISA720 combined with phosphorothioate 

stabilized CpG oligo 1826 (Montanide/ 

CpG 1826) as an adjuvant able to break tolerance 

against a self-molecule. A therapeutic vaccine 

against tumor angiogenesis has been developed in 

our group. This vaccine successfully breaks selftolerance 

against the extra-domain B (ED-B) of fibronectin 

using Freund’s adjuvant and the recombinant 

protein Trx-EDB (see poster by Elisabeth 

Huijbers et al.). To compare the immunostimulatory 

properties of Freund’s adjuvant and Montanide/CpG 

1826, we have vaccinated two groups of 

C57BL/6 mice with Trx-EDB in combination with 

either Montanide/CpG 1826 or Freund’s adjuvant. 

Blood samples were collected regularly and analy- 

zed for anti-ED-B antibodies. 

Comparing the ability of Montanide/CpG 1826 and 

Freund’s adjuvant to stimulate an antibody response 

against the extra-domain B of fibronectin revealed 

that Montanide/CpG 1826 induced higher anti-ED- 

B antibody titers, as well as less variation between 

individual animals, than Freund’s. Furthermore 

antibodies in animals treated with Montanide/ 

CpG 1826 were still detectable after eleven months, 

while antibodies in mice treated with Freund’s adjuvant 

had returned to base levels at this time point. 

Analysis of the IgG isotypes showed that IgG1 and 

IgG2b were the main isotypes in both treatment 

groups. However, preliminary data indicate that 

Montanide/CpG 1826 might have the capability to 

induce antibodies with higher affinity to ED-B than 

antibodies induced with Freund’s adjuvant. 

With Montanide/CpG 1826 we have identified a 

non-toxic alternative to Freund's adjuvant, which 

is at least as potent with respect to inducing an 

immune response against a self-antigen. This will 

facilitate introduction of therapeutic vaccines to the 

clinic.

111 Diekmann | Enhancing immunity & adjuvants 

mTOR inhibition by Rapamycin after intranodal 

RNA immunization improves the CD8+ memory 

T-cell response qualitatively and quantitatively 

Jan Diekmann 1 , Sebastian Attig 1 , Sebastian Kreiter 1 , Raouf Selmi 1 , Mustafa Diken 1 , Özlem 

Türeci 1 and Ugur Sahin 1,2 

1 Universitätsmedizin, Johannes Gutenberg-Universität Mainz, Germany 

2 Institute for Translational Oncoclogy (TrOn), Johannes Gutenberg-Universität Mainz, Germany 

Memory T cell formation is one of the most im- 

portant factors predicting the success of a cancer 

vaccine. By inducing a long lasting antigen-specific 

T cell memory, residual cancer cells and micro-metastasises 

can be targeted and thereby a possible 

relapse of the tumour can be prevented. Recent 

reports have suggested the key metabolic kinase 

mammalian target of rapamycin (mTOR) as an intrinsic 

regulator of memory formation in the CD8+ 

T cell compartment. The inhibitor of mTOR, Rapamycin, 

is used in the clinic to suppress immune rejection 

in the setting of organ transplants. Paradoxically 

it has been shown that Rapamycin treatment 

of mice during and after the course of an infection 

enhanced the formation and differentiation of the 

memory CD8+ T cell pool and also accelerated the 

transition from effector to memory cells. 

In order to investigate the effect of Rapamycin on 

the CD8+ T cell reponse in the setting of intranodal 

RNA immunization, we treated C57BL/6J mice 

with Rapamycin (Rapamune) for 3 weeks (d10 – 

d31) after a course of three RNA immunizations 

(d0, d3, d6) with IVT-RNA (20µg) coding for the 

OVA-derived SIINFEKL epitope (SIINFEKL-RNA). 

Immunized mice treated with the carrier of the drug 

served as a control group. During the treatment, the 

contraction of the SIINFEKL-specific CD8+ T cells 

was almost identical in both groups. However, the 

phenotype of the SIINFEKL-specific cells was markedly 

different: the Rapamycin-group developed 

significantly more CD127+/KLRG1- cells, potential 

precursors of long-lived central memory cells. Additionally, 

these cells showed a higher proliferative 

capacity after a rechallenge with the antigen (SIIN- 

FEKL peptide) at day +35. In this study, we show, 

for the first time, the beneficiary effect of Rapamycin 

on memory T cell differentiation in the setting 

of anti-cancer vaccination. 

These findings indicate that mTOR inhibitors could 

be used to improve the establishment of a longlasting 

and potent memory CD8+ T cell population 

after intranodal RNA-immunization, which 

could augment the outcome after tumour relapse/ 

therapy. 

161

112 Reinis | Enhancing immunity & adjuvants 

DNA methyltransferase inhibitors as modulators of anti-tumour 

immunity: implications for their use as adjuvants for 

immunotherapy 

Milan Reiniš, Jana Šímová, Marie Indrová, Romana Mikyšková, Veronika Polláková, Ivan 

Štěpánek, Jana Bieblová and Jan Bubeník 

Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 

162 

Epigenetic changes, such as aberrant DNA methyla- 

tion, play an important role in cancer cell deve- 

lopment. Notably, they can also be crucial for the 

development of tumour cell variants that escape 

the immune surveillance. Tumour cells frequently 

downregulate the expression of genes involved in 

antigen presentation and interactions with immune 

cells (e.g. MHC and costimulatory molecules). Expression 

of these genes can be silenced by DNA methylation 

in tumour cells and thus influence tumour 

surveillance. DNA methylation can be blocked by 

DNA methyltransferase inhibitors (DNMTi), promising 

anti-tumour agents. Recently, we have demonstrated 

on various murine MHC class I-deficient 

tumour cell lines that DNMTi (5-azacytidine 

and 2’-deoxy-5-azacytidine), used either alone or 

in combination with histone deacetylase inhibitors, 

upregulate expression of antigen-presenting 

machinery genes as well as cell surface expression 

of MHC class I and other costimulatory/inhibitory 

and adhesive molecules. Our further analysis 

revealed that DNMTi can have similar effects on 

the expression of selected immunoactive genes in 

tumour cells as IFNγ. 

Further, we have investigated the impact of in vivo 

administration of DNMTi on the growth of experimental 

MHC class I-deficient tumours, using a 

murine model for HPV-16-associated tumours. We 

have demonstrated their adjuvant/additive effects 

in anti-tumour immunotherapy either with unmethylated 

CpG oligodeoxynucleotides or with IL- 

12-producing cellular vaccines. Similarly to the in 

vitro treatment, administration of DNMTi upregulated 

MHC class I and other immune molecules 

on tumour cells, inducing their sensitivity to the 

CD8+-mediated immune response. 

Since in vivo administration of DNMTi can also 

affect differentiation and function of immune cells, 

we have tested changes in effector and regulatory 

cell populations in tumour-bearing animals upon 

the treatment. Our data indicate that, despite putative 

immunosuppressive effects (dendritic cell maturation 

impairment, block of cytokine production 

or induction of T regulatory cells), treatment with 

DNMTi combined with immunotherapy is an attractive 

anti-tumour therapeutic setting. 

This work was supported by grants Nos. 301/07/1410, 301/10/2174 

and 301/09/1024, Grant Agency of the Czech Republic, and in 

part by the Clinigene Network of Excellence for the Advancement 

of Gene Transfer and Therapy, EU-FP6 Project No. 018933.

113 Kermer | Enhancing immunity & adjuvants 

Antibody fusion proteins for cancer immunotherapy mimicking 

IL-15 trans presentation at the tumor site 

Vanessa Kermer, Volker Baum, Roland E. Kontermann & Dafne Müller 

Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany 

Cytokines driving the immune response are po- 

werful tools for cancer immunotherapy, but their 

application is generally limited by severe systemic 

toxicity. Targeted approaches by means of antibody-cytokine 

fusion proteins might enable to focus 

the cytokine activity to the tumor site, thereby 

reducing unwanted side effects. Here we investigated 

the possibility to improve the efficiency of 

IL-15 presentation in a targeted approach by the 

incorporation of an IL-15R chain fragment, mimicking 

physiological trans presentation. Therefor, 

antibody cytokine fusion proteins were generated 

composed of an antibody moiety targeting the 

tumor stromal fibroblast activation protein (FAP), 

an extended IL-15Rαsushi domain (RD) and IL-15. 

These fusion proteins exhibited antibody-mediated 

specific binding and cytokine activity. Comparing 

the cytokine effect of antibody fusion proteins 

with and without the RD component, we observed 

differences for the soluble and membrane-bound 

form in stimulating proliferation of cells expressing 

the intermediate (IL-15Rβγ) and high affinity (IL- 

15Rαβγ) IL-15 receptor. For the fusion proteins in 

solution, the presence of the receptor domain (RD) 

reduced the stimulatory activity on CTLL-2 (IL- 

15Rαβγ) cells and increased the stimulatory effect 

on Mo7e (IL-15Rβγ) cells and PBMCs. In the targeted, 

membrane-bound form of the fusion protein, 

the presence of RD did not interfere with the proliferation 

of CTLL-2 (IL-15Rβγ) cells and clearly enhanced 

the proliferation of Mo7e (IL-15Rβγ) cells. 

Thus, targeted presentation of IL-15 in combination 

with the RD in form of an antibody fusion protein 

appears as a promising approach to further improve 

the antitumor efficiency of IL-15. 

163

114 Lehmann | Enhancing immunity & adjuvants 

Modified vaccinia virus Ankara (MVA) - a safe vector virus 

and an adjuvant system for immunotherapy 

Michael H. Lehmann 1 , Ulrich Kalinke 2 , Gerd Sutter 1 

1 Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-Universität München, 

80539 Munich, Germany 

2 TWINCORE, Centre of Experimental and Clinical Infection Research, 30625 Hannover, Germany 

164 

Modified vaccinia virus Ankara (MVA) is a highly 

attenuated strain of vaccinia virus (VACV) being 

generated through serial passage in chicken 

embryo fibroblast cells [1]. Until 1988, MVA was 

used for safer vaccination against smallpox and has 

been administered to more than 100 000 humans 

without documentation of the severe adverse reactions 

associated with the use of conventional VACV 

vaccines. Analysis of the MVA genome revealed that 

during the attenuation process the virus had lost a 

substantial amount of genetic information including 

many viral genes regulating virus-host interaction. 

In consequence, MVA is unable to propagate 

in human and many other mammalian cells but 

can efficiently express viral and recombinant genes 

[2]. This phenotype strongly supported its use as 

viral vector and, by today, a variety of recombinant 

MVA vaccines are undergoing clinical testing in 

immune prophylaxis and immune therapy against 

infectious diseases and cancer. 

Here, we investigated the immune stimulatory properties 

of MVA. Surprisingly, MVA but not other 

VACV strains induced expression of type I IFNs [3]. 

We detected IFN-beta both in human monocytic 

cells and in the lungs of mice intranasally infected 

with MVA. Additionally, infection of human cells 

or mice with MVA but not with other VACV strains 

efficiently induced the expression of several chemokines 

(CCL2, CCL3, CCL4, CXCL1, CXCL10), 

which led to the fast attraction of leukocytes including 

monocytes, neutrophils, CD4+ and CD8+ 

lymphocytes to the site of infection [4]. Since active 

immigration of immune cells to the site of immunization 

is a prerequisite for induction of an effective 

immune response, MVA vaccines naturally comprise 

this hallmark of a potent vaccine adjuvant. 

In summary, the properties making MVA an ideal 

vector for immunotherapeutic protocols are (i) its 

clinical safety as replication deficient virus, (ii) its 

ability to easily transfer and express foreign genetic 

material in target cells of choice and, (iii) its capacity 

to activate type I interferons, chemokines and 

thereby a rapid immigration of leukocytes. 

References 

[1] A. Mayr, E. Munz, Veränderungen von Vaccinevirus durch 

Dauerpassagen auf Hühnerembryofibroblasten-Kulturen, 

Zentralbl. Bakteriol. Orig. A 195 (1964) 24-35. 

[2] G. Sutter, B. Moss, Nonreplicating vaccinia vector efficient 

ly expresses recombinant genes, Proc. Natl. Acad. Sci. USA 

89 (1992) 10847-10851. 

[3] Z. Waibler, M. Anzaghe, H. Ludwig, S. Akira, S. Weiss, G. 

Sutter, U. Kalinke, Modified vaccinia virus Ankara induces 

Toll-like receptor-independent type I interferon responses, J. 

Virol. 81 (2007) 12102-12110. 

[4] M.H. Lehmann, W. Kastenmuller, J.D. Kandemir, F. Brandt, 

Y. Suezer, G. Sutter, Modified vaccinia virus ankara triggers 

chemotaxis of monocytes and early respiratory immigration 

of leukocytes by induction of CCL2 expression, J. Virol. 83 

2009) 2540-2552.

115 Lladser | Enhancing immunity & adjuvants 

DAI (ZBP1/DLM-1) as a novel genetic adjuvant for cancer DNA 

vaccines that promotes CTL responses, overcomes tolerance and 

confers long-term tumor protection. 

Alvaro Lladser 1 , Dimitrios Mougiakakos 1 , Helena Tufvesson 1 , Andrew F.G. Quest 2 , Karl 

Ljungberg 1 *, Rolf Kiessling 1 * 

1 Immune and Gene Therapy Laboratory, Cancer Center Karolinska, Department of Oncology and Pathology, 

Karolinska Institutet. Karolinska Hospital R8:01, SE-17176 Stockholm, Sweden 

2 Laboratory of Cellular Communication, FONDAP Center for Molecular Studies of the Cell, Program of Cellular 

and Molecular Biology, Facultad de Medicina, Universidad de Chile, Santiago, Chile 

* KL and RK contributed equally to this work 

DNA vaccination is an attractive approach to induce 

antigen-specific cytotoxic T cell (CTL) responses 

capable of providing long-lasting protective immunity 

against cancer. Although effective in animal 

models, DNA vaccines against cancer have shown 

limited efficacy in clinical trials. Therefore, efficient 

delivery systems and powerful adjuvants are 

needed for DNA vaccines to overcome tumor-associated 

T cell tolerance. Indeed, the early activation 

of innate immune receptors and downstream signaling 

pathways are essential to promote effective 

adaptive immunity. Consequently, DNA vaccines 

benefit from adjuvants that boost innate immunity 

signaling. We have used the recently described cytosolic 

DNA sensor Z-DNA binding protein 1 (ZBP1 

also known as DLM-1), termed DNA-dependent activator 

of interferon regulatory factors (DAI) as a 

genetic adjuvant for DNA vaccines. In vivo electroporation 

(EP) of mice with a DAI-encoding plasmid 

(pDAI) promoted transcription of genes encoding 

type I interferons (IFNs), proinflammatory cytokines, 

chemokines and co-stimulatory molecules. 

Co-immunization of pDAI and antigen-encoding 

plasmids enhanced both in vivo antigen-specific 

proliferation and induction of CTLs. Moreover, codelivered 

pDAI overcame CTL-tolerance to a tumorassociated 

antigen (TAA) and conferred long-term 

anti-tumor protection. The DAI-adjuvanted CTL induction 

required NF-κB activation and intact type I 

IFN signaling, but not interferon regulatory factor 

(IRF) 3. This study demonstrates the potential of 

using intracellular innate sensors as genetic adju- 

vants to improve DNA vaccine potency. 

Acknowledgements: Research described here has 

been supported by grants to RK from the Swedish 

Cancer Society, the Swedish Medical Research 

Council, the Cancer Society of Stockholm, the European 

Union (Grant “EUCAAD” and “DC-THERA”), 

the Karolinska Institutet, “ALFProject” grants 

from the Stockholm City Council. AFGQ has received 

support from ICGEB (International Center of 

Genetic Engineering and Biotechnology, Trieste, 

Italy) grant CRP/CH102-01, Wellcome Trust award 

WT06491I/Z/01/Z and FONDAP grant 15010006. 

AL has been supported by a Fellowship for Postgraduate 

Studies “Presidente de la República” from 

CONICYT, Chile. DM was supported by a grant of 

the German Research Association (DFG). KL has 

been supported by a postdoctoral fellowship from 

the Swedish Society for Medical Research. 

165

116 Hotz | Enhancing immunity & adjuvants 

A rational protocol of repeated TLR7 stimulation 

circumvents induction of TLR tolerance and translates 

into efficient anti-tumor therapy 

Christian Hotz 1 , Andreas Völkl 1 , Daniel Noerenberg 1 , Nadja Sandholzer 1 , Cornelia Wurzenberger 

1 , David Anz 1 , Stefan Endres 1 and Carole Bourquin 1 

1 Center for Integrated Protein Science Munich, Division of Clinical Pharmacology, 

Department of Internal Medicine, Ludwig-Maximilian University of Munich, Munich, Germany. 

166 

Topical application of small molecule toll-like re- 

ceptor 7 (TLR7) agonists is often highly effective 

for the treatment of skin tumors, whereas their systemic 

application has been largely unsuccessful 

for the immunotherapy of cancer. One reason may 

be that repeated application of certain TLR ligands 

can induce a state of immune unresponsiveness, 

termed TLR tolerance. We show here that a single 

injection of the TLR7 agonist R848 in mice induces 

a short period of increased responsiveness followed 

by a state of hyporesponsiveness lasting several 

days. We demonstrate for the first time that TLR7 

tolerance occurs in plasmacytoid and myeloid dendritic 

cells, that play a critical role in the initiation 

and amplification of antitumor immune responses, 

and results in inhibited secretion of the key cytokines 

IFN-κ, IL-12p70 and IL-6 both in vitro and in 

vivo. We further show that TLR7 tolerance in plasmacytoid 

dendritic cells is accompanied by downregulation 

of the adaptor protein interleukin-1 receptor-associated 

kinase (IRAK-1). Based on these 

findings, we have designed a novel strategy for the 

treatment of murine CT26 tumors using cycles of 

repeated R848 injections separated by 5-day treatment-free 

intervals. We show that this protocol 

circumvents TLR7 tolerance and translates into 

efficient cancer immunotherapy whereas single injections 

given within shorter intervals do not.

117 Gonzalez-Carmona | Enhancing immunity & adjuvants 

Combining subcutaneous inoculation of AFP-expressing DC 

with intraperitoneal injection of IL-12-expressing DC increases 

survival in an established orthotopic HCC model 

Maria A. González-Carmona 1 , Annabelle Vogt 1 , Georges Decker 1 , Isabelle Bahadori 1 , Esther 

Raskopf 1 , Volker Schmitz 1 , Tilman Sauerbruch 1 and Wolfgang H. Caselmann 2 

1 Department of Internal Medicine I, University of Bonn, Bonn, Germany 

2 Bavarian State Ministry of the Environment and Public Health, Munich, Germany 

Background: Dendritic cells (DC) are a heterogene- 

ous population of professional antigen-presenting 

cells capable of priming CD4 and CD8 T-cell responses 

against tumor-associated antigens (TAA) 

such as alpha-fetoprotein (AFP). The presence of 

an immunosuppressive tumor-environment going 

along with the suppression of DC is one of the main 

reasons for a lack of immune responses after immunotherapy. 

IL-12 as a major Th1 cytokine is essential 

for the induction of cellular immune responses. In 

this study, the antitumoral effect of a vaccination 

with s.c. inoculated AFP-expressing DC combined 

with the intraperitoneal injection of IL-12-expressing 

DC in order to achieve an inflammatory tumor 

environment was analyzed in an orthotopic HCC 

model. 

Methods: Three adenoviral vectors were used: AdmIL12, 

Ad-mAFP and Ad-LacZ as a control. DC 

were obtained from the bone marrow of C3H-mice, 

cultured with GM-CSF and IL-4 and adenoviral 

transduced on day 6. 1x105 AFP-positive Hepa129cells 

were injected into the liver after laparotomy 

to induce orthotopic tumors. In a first experiment, 

tumor-bearing mice were treated twice with 1x106 

mIL-12-expressing or LacZ-expressing DC intraperitoneally. 

In a second experiment, mice were 

treated with the combination of 1x106 s.c. inoculated 

AFP-expressing DC followed by i.p. injection 

of 1x106 mIL-12-expressing DC. Survival time and 

occurrence of malignant ascites were closely monitored 

and documented. 

Results: In the first experiment 100% of mice in the 

control group (LacZ-expressing DC) developed mali- 

gnant ascites as a sign of tumor progression whereas 

only 30% of the mice treated with IL-12-expressing 

DC treated mice had developed. Furthermore, the 

mean survival after tumor induction was significantly 

prolonged in the mice group treated with 

Ad-mIL12-expressing DC (p

118 Trojandt | Enhancing immunity & adjuvants 

Effects of the anti-cancer agent topotecan on human 

monocyte-derived dendritic cells 

Stefanie Trojandt, Stephan Grabbe, Angelika B. Reske-Kunz and Matthias Bros 

Clinical Research Unit Allergology, Department of Dermatology, Medical Center of the Johannes Gutenberg-University, 

55101 Mainz, Germany 

168 

The camptothecin analogue topotecan has shown 

a remarkable activity against a range of cancers 

and tumors. Topotecan interacts with the enzyme 

topoisomerase I which is involved in DNA replication 

and repair. It forms a stable complex with this 

enzyme, thereby provoking the selective inhibition 

of topoisomerase I. This results in irreversible DNA 

double-strands breaks, giving rise to apoptosis and 

cell death. 

Due to the fact that dendritic cells (DCs) play an 

important role in anti-tumor immune responses, 

we raised the issue of whether topotecan may have 

an impact on DC activation. 

In this study we investigated the effect of topotecan 

on the molecular characteristics of DCs regarding 

the expression of DC-relevant molecules and their 

T cell stimulatory capacity. Human monocytes 

were differentiated to immature DCs and were 

stimulated with a maturation cocktail (TNFalpha, 

IL-1beta, Prostaglandin2). During stimulation with 

the cocktail the cells were treated with different 

concentrations of topotecan. At the concentrations 

used topotecan had no effect on the viability of the 

DCs. Contact of the DCs with this agent resulted in 

an impaired stimulatory capacity for allogenic T 

cells. Nevertheless the treated DC populations expressed 

high levels of HLA-DR and CD86. However, 

the expression of the costimulatory molecule CD80 

and the maturation marker CD83 was slightly impaired. 

In summary, we showed a modification in the func- 

tional activity of DCs after their treatment with the 

anti-tumor agent topotecan.

L126 Klier | Enhancing immunity & adjuvants 

Combined bacterial antibody therapy for colorectal carcinoma 

U. Klier 1 , C. Maletzki 1,2 , E. Klar 1 , Bernd Kreikemeyer 3 , M. Linnebacher 1 

1 Section of Molecular Oncology and Immunotherapy, Department of General Surgery, 

University of Rostock, Germany 

2 Division of Gastroenterology, Department of Internal Medicine, 

University of Rostock, Germany 

3 Department of Medical Micobiology and Hospital Hygiene, Institute of Medical Microbiology, 

Virology and Hygiene, University of Rostock, Germany 

Background & Aims: Here, we analyzed the po- 

tential of inactivated Staphylococcus aureus loaded 

with therapeutic monoclonal antibodies (mAb), for 

treatment of colorectal carcinomas in vitro and in 

vivo. The aim was to induce an inflammatory reaction 

first against bacteria followed by the activation 

of tumor specific immune responses. 

Material & Methods: Colorectal carcinoma cell 

lines, established in our laboratory, were treated 

with S. aureus, antibodies (Panitumumab (anti- 

EGFR), or Trastuzumab (anti- Her2neu)) or a combination 

of both for 24 and 48 hours respectively. 

Cell proliferation and viability were assessed by 

BrdU-incorporation assay and Calcein AM staining. 

Then, we tested the ability of these experimental 

agents to stimulate T cells and determined 

the changing in the pattern of Toll like receptors. 

Additionally, antitumoral effects were examined in 

co-culture experiments using peripheral blood mononuclear 

cells (PBMCs) from healthy donors. As 

in vivo model, Balb/c mice with established CT26 

tumors (stably expressing Her2/neu) received local 

injections of the therapeutics or saline as a control 

(total of 10 injections given twice a week, n=6). 

Tumor-infiltrating leukocytes were analyzed by 

immunohistochemistry. 

Results: Proliferation and survival of tumor cells 

was slightly affected by the therapeutical agents. 

However, antitumoral effects were boosted after 

co-culture with PBMCs in the presence of bacteria 

and mAbs. Flow cytometric analysis of PBMCs re- 

vealed an increase of CD4+/25+ and CD8+/25+ 

cells (24 h), indicative for activation of T cells by 

S. aureus. In vivo, S. aureus treatment induced a 

delay of tumor growth. These effects were accompanied 

by increased numbers of tumor-infiltrating 

CD11b+ granulocytes/macrophages, CD4+ as well 

as CD8+ T cells. Similar results were obtained after 

combination therapy. 

Conclusion: Our results demonstrate that S. aureus 

is capable to elicit direct antitumoral and immunostimulatory 

effects without the need of a targeted 

therapy with mAbs. Current investigations focus 

on testing specific antitumoral T cell responses 

subsequent to stimulation with S. aureus. 

169

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Postal Address 

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Center for Translational Oncology and Immunology 

Gutenberg University 

Mainz Langenbeckstr. 1, Building 708 

55131 Mainz Germany 

Telephone: +49-06131-17-5905 

Telefax: +49-06131-17-3490 

Website: www.cimt.eu 

E-Mail: office@cimt.eu 

Concept and Design 

Das LABOR - Büro für Gestaltung 

Rheinallee 88 / Geb. 25 

55120 Mainz, Germany 

Telephone: 06131 - 30 46 76 2 

Website: www.das-labor.net 

© Immunologische Krebs-Therapie e.V. (Association for Cancer Immunotherapy). All rights reserved. 

170 

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171

Abstract Book 2010 - CIMT Annual Meeting

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