Part C. TITLE PAGE

Quality of Life and Management of Living Resources

GENETIC PROGRAMMING OF HUMAN "CELL FACTORIES" FOR THE SPECIFIC TREATMENT OF CANCER AND VIRAL INFECTION

Acronym: CHIMERIC EURO-CELL

Table of Contents:

Page

1. C3: Management and Resources

3

        Partnership

5

        Partners

7

2. C4: Community Added Value

13

3. C5: Contribution to Social Objectives

14

4. C6: Exploitation and Dissemination

14

5. C7: Ethical Aspects

16

6. C8: Safety

17

7. C9: Ongoing Projects

17

C3. Management and Resources

C3.a. Project Management

Management capability of the Coordinator

The Department of Immunology of the Weizmann Institute of Science, will manage the co-ordination of the project, and will provide experienced administrative support. The coordinator, Prof. Zelig Eshhar, a previous head of the department will be assisted by a secretary who will dedicate 33% of her time to administering the project. Prof. Eshhar has broad experience as a research group leader, a committee member/head, service on the board of international professional societies including the European Society for Gene Therapy and Euregenethy, an European Network of Regulation of Gene therapy, member of editorial board of several scientific journals, and reviewer of many international grant proposals and journal manuscripts. He organized numerous scientific meetings and practical courses. Prof. Eshhar has extensive experience in the practical management of resources, finance and reporting from previous management of the Department of Immunology (1997-1999), past and present head of the Animal Users Committee and Animals Ethical Committee at the Weizmann Institute. He continuously manages large research grants. The completed projects have been exceptionally productive.

Organisation and management structure

The research project is made up of six partners from five different European countries (France, Israel, Sweden, The Netherlands, and United Kingdom). Two Partners are affiliated with universities or government funded research institutions, two are from University or Public Hospitals, one (Genethon) is a non-profit charity funded research institute and one (Got-A-Gene) is an industrial organization, clearly defined as an SME partner.

A steering committee will comprise the 6 partners responsible for the project and the coordinator will chair it. The steering committee will meet every 6 months, starting at month 0, and will decide on high level science, priorities of performance related to progress, technology implementation and management issues such as distribution of resources, exploitation, reports, etc. The coordinator will carry the overall responsibility for efficient planning, communication between partners and research teams, industrial observers and the EU. Communication will take place through e-mail, phone, fax and site visits. The project's secretary will persistently follow and monitor the budget spending and preparation of reports. The coordinator will take initiative when appropriate to initiate potential consultations for patent applications, and commercial exploitation and be assisted, if necessary, by experts in YEDA, the commercial liaison office at The Weizmann Institute. Where science permits, we shall adhere as closely as possible to the work plan and milestones outlined in the proposal, but reserve the right to respond flexibly to scientific developments in the field.

Communication and critical appraisal of progress

The present proposal builds in part upon existing collaborations (e.g. between partner 1&2, 1&4, 5&6). The aim of the proposal is to consolidate this productive European collaboration and to add and integrate new expertise in the form of vector development (partner 3), gene targeting (partner 6) clinical testing (partners 2, 4, 5) and biotechnological applications (partner 6). The goal of the concerted collaboration is the optimization of the chimeric approach towards its clinical application. Efficient and frequent interactions will be assisted by a common, closed website for all the partners which will enable on-line communication, discussions, display and analysis of results, and provide a portal and links to external sources of information. The project secretary will also serve as the website manager. Team headers are in charge of keeping a diary of progress and informing the co-ordinator of major results or problems. Short six month progress reports and a full annual report will be prepared for the coordinator who will edit and distribute them to the teams and other parties as agreed.

Diagrams no. 1 and 2 summarize the organization and planned activities of the network. Diagram no.1 shows the time-flow, diagram no. 2 shows the flow of materials and information between the different research disciplines and workgroups.

We shall begin with the preparation of several configurations of chimeric receptor genes, which will be tested, improved and selected according to their performance. Only a few, best performing configurations will end in clinical trials. We need intensive and constant communications and feedback between the working groups. Efficient communication will be assisted by appointment of project monitors (see table below). Monitors are in charge of keeping a diary of progress and informing the coordinator of major results and problems. Short 6 months reports will be prepared for the coordinator before each Meeting. The coordinator will edit and distribute the reports to the teams and other parties as agreed.

Project Partners Involved Monitor
Vector preparation 3, 4, 6 Dr. O. Danos, Genethon
Construction of CR Genes 1, 2, 4, 5 Dr. R. Hawkins, Manchester
Test in Mouse Models 1, 2, 4, 5 Dr. G. Gorochov, UPMC
Phase I Clinical Trial 4 Dr. R. Bolhuis, DDHCC

 

Planned Network Committee Meetings:

Progress, Milestones, and Deliverables will be monitored at biannual meetings and by a mid-term progress report (see below). The deliverables which consist of reports and publications in open peer reviewed literatures, will be reviewed by the Monitors (described above) before submission for publication to asses possible disclosure of patentable information/technology. In case of disagreement or conflict of interests, the coordinator (or somebody else, agreed by the partners involved) will serve as arbitrator. As a rule, preference will be given to the protection of novel information/technology by patent applications. Review of manuscripts and patent applications will be done promptly when needed. Results will be reviewed and discussed in the half yearly meetings.

Meeting Time
(Month)
Major Issues
1 0 Project status and discussion of Workplan.
Choice of variants; Resource adjustments
2 6 Adjustment; Exchange of information
3 12 Progress Report
4 18 Critical Midway evaluation and adjustments
5 24 Setting priorities; Progress report;
Exploitation of results and tables
6 30 Progress report; Evaluation
7 35 Preparation of final scientific and financial report

The first and last meetings will take place at the Weizmann Institute-- then we shall rotate between the other institution-partners. At the first meeting, the Workplan will be updated with respect to the progress that has occurred between submission of the application, and initiation of research. This updated Workplan will be the reference point for the assessment of progress, consumption of resources, milestones and meeting of project objective. In addition to these meetings to which all partners will participate, there will be a number of bilateral meetings and short visits between interacting partners or members of their research team according to the circumstances, to ensure optimal collaboration on specific points. Expenses to cover travel cost and hotel accommodations for all partners are budgeted witthe travel component of each partner's budget

The midterm review after 18 months will assess the accomplishments of teams according to the following criteria:

Achievements of research goals, milestones and publications; consumption of resources.

Communication and collaborations/assistance to other teams.

Identification of aspects of research that have proved most successful; what contributed to this success.

Identification of changes which would have most significantly improved performance over the last 1 month.

 

C3.b. The Partnership

Realization of the clinical potential of the Chimeric Receptor (CR) approach is a complex endeavor resulting from the multiplicity of expertise and downstream technologies. Only multidisciplinary studies resolving this complexity can lead to significant progress that is needed to further develop the CR approach towards its clinical application. In the planning and organization of this concerted project, a special emphasis was placed on including partners, each a leading expert in his specific field, which will contribute and complement (but not overlap) the other partners in our integrated effort. Thus, the present highly integrated project ensures an optimal collaboration between the partners, with clearly identified contribution. The research project is made up of six partners from five different European countries (France, Israel, Sweden, The Netherlands, and United Kingdom). Two Partners are affiliated with universities or government funded research institutions (Eshhar, Hawkins), two are from University or Public Hospitals Debere/Gorochov and Bolhuis), one, (Danos) is directing a non-profit charity funded research institute (Genethon) and one, (Lindholm) is the CEO of Got-A-Gene an industrial organization, clearly defined as an SME.

 

The specific qualifications which each partner and institute bring to the project, and the partners knowledge, experience and detailed description of the principal scientific and technical personnel which will be involved in the project, is summarized in the following pages:

The unique contribution of each partner is summarized in the Table:

Partner

Activity

Contribution

1. Eshhar, WIS,

Rehovot

Basic Research

Institute

Project co-ordinator; management; molecular and cellular immunology; monoclonal &recombinant antibodies; construction of different designs of antibody and ligand-based CR; animal models for human tumor therapy

2. Debr@/Gorochov, CERVI,

Paris

Univ. Hospital

Clinical hematology and immunology; AIDS and lymphoma therapy; construction of novel MHC-peptide CR; animal models for HIV therapy

 

3. Danos, Genethon, Evry

Public Institute

Adeno-, Retro-and lentivirus vectors; vector production under GMP conditions; controlled transgene expression

4. Bolhuis, DDHCC, Rotterdam

Univ. Cancer

Center

Gene-immunotherapy of cancer; CR gene expression in T- cells; construction of TcR-VCR to tumor rejection antigens

5.Hawkins, UNIMAN,

Manchester

Univ. Hospital

Antibody engineering; phage antibody libraries; T cell activation and signaling pathways; co-stimulatory CR

6. L. Lindholm, GOTAGENE,

Goteborg

SEM

Targeted adenovectors; targeted gene delivery into lymphocytes and tumors;

 

C3. The Partnership List

Partner 1: Prof. Zelig Eshhar, Rehovot

Role and Contribution:

1 Coordinator

2 Responsible for WP. 1

3 Participates in WP. 4,6

Our laboratory in the Department of Immunology, The Weizmann Institute of Science pioneered the "T-body" approach ten years ago and innovated several configurations of chimeric receptors which proved functional in redirecting the specificity of both murine and human cytotoxic lymphocytes. We were granted a European Patent on the T body approach, and an additional one is pending in Europe. These two Patents are in an advanced phase towards allowance in the USA. We have broad experience in the generation of monoclonal and recombinant antibodies, their chemical and genetic manipulation and use for cancer treatment. We recently established human cancer xenografts in SCID mice as a model for cancer immunotherapy. Our lab was instrumental in disseminating the T-body approach throughout the world and we have established collaborations with several laboratories in Europe including a French-Israeli Research Grant with the laboratory of Prof. Debre and Dr Gorochov (Partner 2) in the early phase of construction of the anti-HIV scFvR and trained and collaborated with the laboratory of Dr Bolhuis (Partner 4) in the preparation of the first constructs of the chimeric TCR-Fv.

Principal scientific and technical personnel:

Prof. Zelig Eshhar: Will act as the project coordinator, will be responsible for the project management and Workpackage 1 and participate in Workpackage 5. In addition, he will supervise various aspects related to the chimeric receptor design and behavior of the genetically programmed cells. He pioneered the T-body approach, holds patents and patent applications on this technology, and has more than 10 years experience in the specific field. He has long-standing experience in experimental studies with monoclonal and recombinant antibodies as well as with T cell biology. He holds more than 140 publications in these fields. He just completed his tenure as Head of Immunology and is a member of the Board of the European Society of Gene Therapy as well as Euregenthy, an EC network of users of Regulation of Gene Therapy in Europe.

Dr. Dan Schindler: A senior research associate in the lab. Expert in molecular biology and genetic engineering who participated in the design and construction of the various chimeric receptors made in our lab. He has ten years experience in the field and holds about 35 related publications.

Dr. Yonathan Pinthus : A surgeon specializing in urology from Sheba Medical Center who is conducting his Ph.D. thesis in the lab in establishing the human tumor xenografts in the SCID mouse model for prostate cancer immunotherapy.

Ms. Tova Waks : An extremely experienced MSc laboratory assistant who is instrumental in all the studies done in preparation of mAb, in-vitro and in-vivo assays of chimeric receptor mediated T cell function, vector production and gene delivery. She has been a co-author of many of our publications in the field and is co-inventor on the patents.

Recent Relevant Publications

1. Eshhar, Z., Waks, T., Gross, G. and Schindler, D. Specific activation and targeting of cytotoxic lymphocytes through chimeric single-chains consisting of antibody-binding domains and the g or z subunits of the immunoglobulin and T cell receptors. Proc. Natl. Acad. Sci. USA 90: 720-724, 1993.

2. Fitzer-Attas, C.J., Schindler, D.G., Waks, T., and Eshhar, Z., Harnessing Syk-family tyrosine kinases as signaling domains for chimeric scFv receptors; optimal design for T cell activation. J. Immunol. 160: 145-154, 1998.

3. Mezzanzanica, D., S. Canevari, A. Mazzoni, M. Figini, M. Colnaghi, T. Waks, D.G. Schindler and Z. Eshhar. Gene transfer of variable regions from anti-human tumor antibody confers anti-carbohydrate antigen specificity to T cells. Cancer Gene Therapy, 5: 401-407 1998.

4. Bitton, N., Gorochov, G., Debre, P., and Eshhar, Z, Gene therapy approaches to HIV-infection: Immunological strategies: use of T bodies and universal receptors to redirect cytolytic T cells. Frontiers in Biosciences 4: March 30, 1999.

5. 0340793 - European patent - "Endowing cells with antibody specificity", and

6. 0638119 - European application - "Chimeric receptor genes and cells transformed therewith.

Partner 2: Drs. P Debre MD, Ph.D. and G Gorochov MD, Ph.D., Paris

Role and Contribution:

1 Responsible for WP. 3, 6

2 Participate in WP.1, 4,5,9

Our team is part of a large Department of Immunology (90 staff members) headed by Pr Patrice Debre. The department has access to all current state of the art laboratory equipment and technologies, including DNA sequencers, cell sorters, real time PCR, calcium imaging, protein purification, animal facilities, and P1 P2 and P3 labs. Pitie is the largest hospital in Europe and we have ample access to patient-derived material needed for the program. Facilities dedicated to gene therapy are created, human clinical programs headed by David Klatzmann have started and slots for our project already exist. In the past we have collaborated with Prof. Eshhar on the T body approach and will continue doing it under this project in WP.6. WP.3 is based on an original development we have recently patented and brings novel recognition element to the chimeric receptors that enable to redirect effector lymphocytes to eliminate specific T cells.

Principal scientific and technical personnel:

Patrice Debre M.D. Ph.D. (10% of his time on the proposed project)

Head of department of Immunology, in charge of the coordination with other departments of Pitie-Salpetriere hospital, legal issues, advisor to INSERM and CNRS.

Guy Gorochov M.D. Ph.D. group leader (30% of his time on the proposed project)

Responsible for the coordination and planning of experiments in Paris.

After a clinical training in hematology (U. of Paris) and a post-doc in the field of antibody engineering (Laboratory of Dr Greg Winter, MRC center, Cambridge, UK)

Dr Gorochov became associate professor of Immunology at Hopital Pitie-Salpetrie in Paris and leads a research group including 1 post-doc, 2 Ph.D. students, 1Msc student and two technicians. The current research interests of the team focus on the study and on the manipulation of the repertoires of antigen receptors.

Scientific and technical personnel under the supervision of Dr. Gorochov

Natacha Bitton, Ph.D. student. In vitro cellular studies, including infectious challenges with HIV. Optimization of gene transfer to human cells. NB will be in the laboratory until 07/2000, and then will be replaced by a postdoctoral scientist with similar tasks. NB has been working on the project for more than 3 years now and became an expert in the study of gene-modified T cell and in the manipulation of HIV-infected cells.

Karim Dorgham, technician. Construction of phage-displayed libraries of peptides and antibodies, for molecular evolution of receptors and optimization of function. Expert in DNA cloning, construction and screening of phage-displayed libraries.

Chistophe Parizot, technician. In vivo monitoring of transfected T cells after infusion. Cytofluorometry.

Jean-Pierre Eric, technician .In charge of the maintenance of animals.

Recent Relevant Publications

1. Expression of a peptide MHC complex displayed on bacteriophages. Le Doussal JM Piqueras B Debre P Gorochov G . Priority : French Application n� 9806213, May 15, 1998. PCT Application. CNRS.

2. Bachelez, H., F. Hadida, C. Parizot, B. Flageul, M. Kemula, L. Dubertret, P. Debree, and G. Gorochov . 1998. Oligoclonal expansion of HIV-specific cytotoxic CD8 T lymphocytes in the skin of HIV-1-infected patients with cutaneous pseudolymphoma. J Clin Invest 101, 2506-16.

3. Gorochov , G., A.U. Neumann, A. Kereveur, C. Parizot, T. Li, C. Katlama, M. Karmochkine, G. Raguin, B. Autran, and P. Debre. 1998. Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy [see comments]. Nat Med 4, 215-21.

4. Delhem, N., F. Hadida, G. Gorochov , F. Carpentier, J.P. de Cavel, J.F. Andreani, B. Autran, and J.Y. Cesbron. 1998. Primary Th1 cell immunization against HIVgp160 in SCID-hu mice coengrafted with peripheral blood lymphocytes and skin. J Immunol 161,: 2060-69.

5. Bitton, N., F. Verrier, P. Debre, and G. Gorochov . 1998. Characterization of T cell-expressed chimeric receptors with antibody- type specificity for the CD4 binding site of HIV-1 gp120. Eur J Immunol 28, 4177.

Partner 3: Dr Olivier Danos, Evry

Role and Contribution:

Responsible for WP 4,7.

Genethon is a non-profit Research Center created through the Association Francaise contre les Myopathies (AFM) and the Centre d�Etude du Polymorphisme Human (CEPH), with the support of the French Ministry of Research. Since 1997, Genethon has focused its efforts towards gene transfer and gene therapy research, under the scientific direction of Dr Olivier Danos. Genethon is located in a research facility of 3600 m2 in Evry, France. It includes research laboratories, associated with the Centre National de la Recherche Scientifique (CNRS / URA 1923), and a vector process development and production core facility. The goals of Genethon are to solve problems associated with the current gene therapy vectors and strategies and to facilitate access to high quality vectors for basic and clinical research in France and in Europe. Vectors derived from Retro / Lentiviruses, Adenoviruses, Adeno-Associated Viruses, as well as non-viral vectors are being studied and produced. Relevant to this project, scientists at Genethon have a long-standing expertise and recognition in retroviral vectors design, production, purification and use in gene therapy experiments.

Principal scientific and technical personnel involved in the project:

Olivier Danos, Ph.D. is the Scientific Director of Genethon and Director of Research at the CNRS. He completed his Ph.D. studies in Virology and Molecular Biology at the Pasteur Institute. Working on the molecular characterization of viruses. He was appointed by the CNRS in 1984. In his post-doctorate with Dr. Richard Mulligan at the Whitehead Institute he developed retroviral vectors for gene transfer. At the Pasteur Institute in 1989 he directed the Retrovirus laboratory studying strategies for gene therapy on animal models of genetic disease, as well as fundamental virology. He also served as Senior Scientific Director of Somatix Therapy Corporation He sits in several international peer review and expert committees on gene therapy and virology and holds over 85 publications in these fields. Corresponding member of the American Society for Gene Therapy and a funding member and member of the board of the European Society of Gene Therapy (ESGT). Elected president of ESGT in 1996. Dr Danos will actively supervise the overall organization of the project at Genethon, as Scientific Director. The project will involve participants from the development groups at Genethon (DNA constructs, cell cloning, bioprocess, assay development), the production service unit and the Quality Assurance group.

Ingrid Weber, Engineer: a specialist of cell technology and production, in charge of the vector production service, she has established routine state-of-the art procedures at Genethon for producing viral vectors on demand (MLV, Adeno, HIV and AAV)

Pascale Bouill@, Ph.D. : scientist in charge of the DNA construction development group , will be in charge of WP4, task 1. Dr Bouill@ has an expertise in DNA biochemistry, molecular biology and retrovirology

Otto-Wilhem Merten, Ph.D. : a senior scientist in charge of the bioprocess technology development group. He is an expert in the field of medium development and optimization, of process development in general, and of animal cell technology, in particular. He has 20 year of experience and recognized expertise in the field. He will be in charge of optimizing vector production and purification processes

Recent Relevant Publications

1. Danos, O., and R. Mulligan. 1988. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. U.S.A. 85, no. 17:6460-4.

2. Wilson, J.-M., O. Danos, M. Grossman, D.-H. Raulet, and R.-C. Mulligan. 1990. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 87, no. 1:439-43.

3. Davis, J.-L., R.-M. Witt, P.-R. Gross, C.-A. Hokanson, S. Jungles, L.-K. Cohen, O. Danos, and S.-K. Spratt. 1997. Retroviral particles produced from a stable human-derived packaging cell line transduce target cells with very high efficiencies. Hum. Gene Ther. 8:1459-1467.

4. Rendahl, K.G., S.E. Leff, G.R. Otten, S.K. Spratt, D. Bohl, M. Van Roey, B.A. Donahue, L.K. Cohen, R.J. Mandel, O. Danos, and R.O. Snyder. 1998. Regulation of gene expression in vivo following transduction by two separate rAAV vectors. Nat Biotechnol 16, no. 8:757-61.

5. Battini, J.L., O. Danos, and J.M. Heard. 1998. Definition of a 14 amino-acid peptide essenfor the interaction between the murine leukemia virus amphotropic envelope glycoprotein and its receptor. J. Virol. 72, no. 1:428-435..

Partner 4: Dr. R.L.H. Bolhuis, Rotterdam

Role and Contribution:

Responsible for WP.2,9.

Participates in WP.1,4,5,6,7

Dr. Bolhuis is the Chairman of the Department of Clinical and Tumor Immunology at the Academic hospital of Rotterdam DDHCC. The Department of Clinical and Tumor Immunology, has a proven track record regarding the study of receptors involved in Natural Killer cell and T lymphocyte killer cell activity with cancer cells and viral infected cells. Dr Bolhuis has originally described the T cell receptor gamma-delta in 1987 (Nature: 325; 683-688) and also identified the major target cell structure for the TCR gamma-delta (Science: 250; 1269-1273). The policy of the department is to translate basic scientific and technological research and developments into clinical applications and as such many gene-(immuno therapy) protocols have been developed in house. Several single- and multi-centre (international) clinical phase I and II studies have been performed under the co-ordination of principal investigators. During the past six years we have focussed on the construction of T cell receptor based chimeric receptors. We have filed a patent application on the TCR constructs. Another major focus is directed on the development of viral vectors for the efficient transduction of genes into primary human immune cells and specific targeting of the viral vectors towards them. We have established an international network of collaborations with industry and academia and are continuously involved in transfer of the know-how and technologies, especially within Europe. We have experience in collaborating within the framework of concerted action programs financed by the EU and in EORTC coordinated clinical studies.

Principal scientific and technical personnel:

Dr. Reinder L.H. Bolhuis: will act as the coordinator of the work described in the work package description on the generation of TCR based (chimeric) receptors and will co-coordinate the clinical feasibility study using single chain antibody based chimeric receptors. Dr. Bolhuis has more than 15 years experience in the implementation of clinical phase I and II studies according to WHO criteria. He served as a member of the Board of the European Society of Gene Therapy as well as Eurogenethy, an EC network of users on regulation of gene therapy in Europe.

Dr. Jan Willem Gratama, a senior research and clinical associate in the department is an expert in clinical immunology, immuno-therapy and immune status monitoring who participates in the basic research and applied research activities of the department and is the first responsible clinician for the clinical diagnoses of leukemias and lymphomas as well as the immune status monitoring of patients undergoing gene-(immuno)therapy and cytokine treatment. He has over 10 years experience in this fields and holds over 80 related publications.

Dr. Ralph Willemsen, a molecular immunologist working at our Department of Clinical and Tumor Immunology has brought experience in vector constructions which he acquired working with a biopharmaceutical company. He is now conducting his Ph.D. thesis in our department. Dr. Willemsen is solely occupied with the development of TCR based receptors and gene transduction into human T lymphocytes.

Prof. Gerrit Stoter MD: the chairman of the Department of Medical Oncology has a proven track record in the conduction of clinical trials and will be the clinical coordinator of the feasibility study described in the project.

Cees Ronteltap: the department�s chief tissue culture - and cell processing- technician. Has trained and supervised the preclinical and clinical technical staff, for twenty years, in the department.

B. Luider: optimization of large-scale transduction of human immune cells, clinical scale preparation of cancer patient's immune cells.

Recent Relevant Publications

1. A retroviral vector system "STITCH" in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes.

2. M.E.M. Weijtens, R.A. Willemsen, E.H. Hart & R.L.H. Bolhuis. Gene Therapy, 9: 1195-1203 (1998) Genetic retargeting of T lymphocyte specificity. R.L.H. Bolhuis & J.W. Gratama Gene Therapy, 9: 1153-1155 (1998)

3. A T cell receptor g /CD3 complex found on cloned functional lymphocytes. J. Borst, R.J. van de Griend, J.W. vanOostveen, S.-L. Ang, C.J. Melief, J.G. Seidman & R.L.H. Bolhuis. Nature, 325: 683-688 (1987).

4. Recognition by Human Vg9/Vd2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells. P. Fisch, M. Malkovsky, S. Kovats, E. Sturm, E. Braakman, B.S. Klein, S.D. Voss, L.W. Morrissey, R. DeMars, W.J. Welch, R.L.H. Bolhuis, P.M. Sondel. Science, 250: 1269-1273 (1990).

5. Regression of advanced ovarian carcinoma by intraperitoneal treatment with autologous T lymphocytes retargeted by a bispecific monoclonal antibody. S. Canevari, G. Stoter, F.Arienti, G. Bolis, M.I. Colnaghi, E.M. di Re, A.M.M. Eggermont, S.H. Goey, J.W. Gratama, C.H.J. Lamers, M. A. Nooy, G. Parmiani, F. Raspagliesi, F. Ravagnani, G. Scarfone, J.B. Trimbos, S.O. Warnaar & R.L.H. Bolhuis. J. Natl.Cancer I. 87: 1463-1469 (1995).

Partner 5: Prof. Robert Hawkins, Manchester, UK

Role and Contribution:

1 Responsible for WP.5

2 Participates in WP.1, 4, 6

Professor Robert Hawkins has experience in antibody engineering, phage antibody libraries and both viral and non-viral gene transfer techniques. He developed and undertook the first UK trial of gene therapy for cancer (A Genetic Approach to Idiotypic Vaccination for B-cell Lymphoma) and is about to start a Phase II trial of this as an adjuvant therapy for patients with low-grade lymphoma in first remission. In addition to cancer vaccines, a major focus in his laboratories is on gene transfer to lymphocytes. A particular aim is to evaluate the potential benefits of providing antigen specific costimulation using chimeric T-cell receptors. Our group demonstrated the proof of principle of this approach and we are now developing improved constructs. In parallel, we are investigating improved methods of gene transfer and efficacy in tumour models with the plan to investigate the use of chimeric T-cell receptors in clinical trials. We have substantial grant support in related programmes and projects from the Cancer Research Campaign, the Kay Kendall Leukaemia Fund, the Leukaemia Society of America and the Neuroblastoma Society amounting to some �500K per annum. The laboratories are well equipped for this type of work and we have the necessary animal license and safety approvals. Our laboratories are a part of the Christie Hospital NHS Trust that is the largest specialist cancer hospital in Europe. A number of strategies in high dose chemotherapy and bone marrow transplantation have been pioneered here and there is a wide range of clinical trial activity from early Phase I trials through large multi-centre phase III trials. Thus there is access to patients with all tumour types, the resources and the facilities to undertake trials of the proposed type of cellular-gene therapy.

Within the framework of the proposal we will lead on the construction of chimeric receptors to provide costimulation and combinations of CD3z/g. Specifically we will generate optimal CD28 chimeric receptors and fusions of CD28-CD3z signalling domains. These will be assessed in vitro and in vivo for proliferation and cytotoxicity and conditions for efficient expression in patient-derived effector cells will be determined. Anti-tumor activity will be tested in SCID mouse models.

Principle Scientific and Technical Personnel:

Prof. Robert Hawkins: Director of Medical Oncology and Head of Cancer Studies at the University of Manchester. Expertise in antibody engineering and gene transfer technology. His clinical interest is in gastrointestinal cancers and lymphoma. He will thus be able to co-ordinate both clinical trials and lab aspects.

Dr Lez Fairbairn: is a Senior Scientist in the Paterson Institufor Cancer Research. He has expertise in retroviral vectors and SCID mouse models. He led the scientific aspects of the first UK Gene therapy trial for the metabolic disorder Hunters Syndrome.

Dr Said Dermime is a Senior Post Doctoral Scientist in the laboratory with expertise in T-cell immunology. He trained at the NIH with Prof. J Barrat and identified T-cell antigens in haemopoietic malignancies. He is in charge of our vaccine programme, which links closely with this work.

Dr Jim Embleton is a CRC Life Fellow who has twenty years experience of monoclonal antibodies and antibody therapy. He leads the development of human scFv fragments from large phage libraries.

Dr Raj Chopra: is a Senior Lecturer in Haematological Oncology and Bone Marrow Transplantation. Particular interest in allografts and donor lymphocyte infusions, areas we see as appropriate for our first trial of modified cellular therapy.

Dr Peter Stern : head of the Section of Immunology. Expertise in defects in antigen presentation by tumours.

There are many other clinical and scientific collaborators within the Christie CRC Research Centre.

Recent Relevant Publications

1. Chinnasamy N, Rafferty JA, Hickson I, Lashford LS, Longhurst SJ, Thatcher N, Margison GP, Dexter TM, Fairbairn LJ. Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase. Gene Therapy 1998; 5: 842-847.

2. Recombinant adenoviral delivery for in vivo expression of scFv antibody fusion proteins. Whittington HA, Ashworth LJ and Hawkins RE. Gene Therapy 1998; 5: 770-777.

3. Pharmacological Control of Antigen Responsiveness in Genetically Modified T lymphocytesAlvarez-Vallina L, Agha-Mohammadi S, Hawkins RE and Russell SJ. J Immunol. 1997; 159: 5889-5895.

4. Single-chain Fv antibody selected from a combinatorial library: clinical evidence of efficient tumour targeting. Begent RHJ, Verhaar MJ, Chester KA, Casey JL, Green AJ, Napier MP, Hope-Stone LD, Cushen N, Keep PA, Johnson CJ, Hawkins RE, Hilson AJW and Robson L. Nature Medicine 1996; 2: 979-984.

5. Antigen specific targeting of CD28-mediated T cell costimulation using chimeric scFv-CD28 receptors. Alvarez-Vallina L and Hawkins RE. Eur J Immunol. 1996: 26: 2304-2309.

Partner 6: Dr. Leif Lindholm MD, Ph.D., Goteborg, Sweden

Role and Contribution:

Responsible for WP. 8.

The biotechnology company Got-A-Gene was formed in 1992 by a group of 8 researchers as a holding company for the patent: "Targeted Delivery of Virus Vector to Mammalian Cells"(see below). Since 1997 the company has actively conducted research and development on retargetted adenovirus vectors. In 1999 the company made a financial deal with the Swedish Investment Company Novare Kapital AB who acquired 15% of the company�s shares against a capital investment in the company. The company is only now emerging as an entity in its own right and has acquired offices and laboratory facilities in a new "technique park" in Goteborg, Sweden.

Within the framework of the proposal, Got-A-Gene will concentrate on the construction of targeted vectors to provide specific transduction of T cells as well as tumor target cells. These will be of a great advantage for optimizing the in-vitro preparation of chimeric receptor expressing cells and it opens new avenue in vivo targeting of genes.

Dr Lief Lindholm MD Ph.D. (University of Goteborg) is a specialist in clinical immunology. He served as a research associate, in the department of microbiology and Immunology, University of Goteborg and professor of clinical immunology, Huddinge Hospital, Stockholm. He also worked as assistant chief physician at the Laboratory for Clinical Immunology, Sahlgrens hospital, Goteborg. Dr Lindholm has extensive experience in the Pharmaceutical industry, and has held senior positions in Stena Diagnostics AB, G@teborg, Sweden (1985-1987), was managing director of Pharmacia CanAg, G@teborg, Sweden (1987-1992), and Project leader, Immunotherapy programme, Pharmacia AB (1990-1992).He is currently Associate Professor in Immunology, at the University of Goteborg (part time)and CEO of Got-A-Gene AB

Principle Scientific and Technical Personnel:

Dr Lief Lindholm MD PhD is the CEO of "Got-a-gene", the SME partner in this proposal. He will direct the further development of targeted adenovirus vectors.

Maria Magnusson, PhD student (University of Goteborg). Associated with the project since 1996. Has been responsible for much of the construction work on recombinant fibers and has developed the method for rescuing of recombinant fibers into virus.

Petra Strand, Ph D student who has been working in a molecular biology project studying liver regeneration. She will join Got-A-Gene on May 31.

Elisabeth Pettersson Technician. She has extensive experience of molecular biology techniques and has worked in the Got-A-Gene project since 1995.

Recent Relevant Publications

1. Debinski W, Karlsson B, Lindholm L, Siegall CB, Willingham MC, FitzGerald D and Pastan I. Monoclonal Antibody C242-Pseudomonas Exotoxin A. A Specific and Potent Immunotoxin with Antitumor Activity on a Human Colon Cancer Xenograft in Nude Mice. J. Clin. Invest., 90: 405-411, 1992.

2. Johansson C, Segran S and Lindholm L. Tumor-growth suppression in nude mice by a murine monoclonal antibody: Factors hampering successful therapy. Int. J. Cancer, 48: 297-304, 1991.

3. Baeckstram D, Hanson GC, Nilsson O, Johansson C, Gendler SJ and Lindholm L. Purification and characterization of a membrane bound and a secreted mucin-type glycoprotein carrying the carcinoma associated epitope sialyl-Lewis a on distinct core proteins J. Biol. Chem., 266, 21537-21547, 1991

4. Baeckstr@m D, Nilsson O, Price MR, Lindholm L and Hansson GC. Discrimination of MUC1 mucins from other Sialyl-Lea-carrying glycoproteins produced by colon carcinoma cells using a novel monoclonal antibody. Cancer Res. 53: 755-761, 1993.

5. Johansson C, Nilsson O, Baeckstr@m D, Jansson EL, Lindholm L. Novel epitopes on the CA-50 carrying antigen: Chemical and immunochemical studies. Tumor Biol., 12: 159-179, 1991.

Patent: "Targeted Delivery of Virus Vector to Mammalian Cells". It was issued on Dec 9, 1997. US 5.695.991. Pending in Europe, Japan and Canada.

C4. Community added value and contribution to EU Policies

The potential social, economic and clinical impact of the proposed project is of paramount importance because the developed technologies, proposed in this project, will enable the European Community to allow the generation of gene-immuno-therapeutic biopharmaceutical products that can critically affect the living conditions of its members: well over half a million Europeans die of cancer and virus-related diseases every year. The product that will be provided by the proposed project, by the use of state-of-the-art, cutting edge technologies, has the potential to advance the success rate of therapy of cancer and fatal viral infections, notably AIDS. Indeed, no effective therapies are available for the most frequent and common cancers. The gene-immuno-therapy strategy to be developed under this project will provide the necessary breakthrough, because of the inherent specificity of the produced, chimeric receptors, will allow genetically programmed immune cells, the �cell factories�, to identify and kill tumour cells and virally infected target cells. The gene immuno-therapy approach of this project may also be applicable in organ transplantation, bone marrow transplantation as well as autoimmune diseases, e.g., rheumatoid arthritis, over all, constituting a wide range of health prob.

European dimension :

This project is focussed upon five European research laboratories and one company. Each group has been assigned a separate task corresponding to their proven track record of success and expertise. Yet each participant is dependent on the others in achieving the overall demanding goals of the program. The chimeric receptor approach can truly be considered in its broad aspect to be a European invention, and most of the relevant European groups participate in this proposal. Tpioneering scientific work was done in the laboratory of Prof. Z. Eshhar, Rehovot, Israel, which holds the basic patent to the antibody-based chimeric receptor (T-body) and pending allowance of additional patents in the technology. The group of Dr R. Bolhuis in Rotterdam, The Netherlands assisted by Prof. Eshhar, innovated the TcR-V region based chimeric receptors and applied for a patent on the subject. Prof. P. Debre and Dr. G. Gorochov, Paris, France, invented and patented a novel recognition unit of the chimeric receptor based on a complex between the MHC molecule and its bound peptide. Prof. Hawkins, Manchester, UK, was the first to use the co-stimulatory CD28 molecules in the chimeric receptor context. These and the Genethon research center, directed by Dr O. Danos Evry, France, leader in vectorology and gene therapy in Europe, and the innovative technology of targeted delivery of adenovirus vector to mammalian cells, which is patented by our industrial partner, Got-A-Gene, led by Dr. L. Lindholm, Goteborg, Sweden, are central to the project and emphasize the innovative, multi-disciplinary and multi-national dimension of this integrative European project. Th research we propose can not be performed on a national level of each of the participating groups, simply because the core expertise needed for the delivery of its objectives and to realization of its therapeutic potential are in the domain of several separate research centers in Europe.

Altogether, within the area of chimeric receptor technology, Europe has a very strong position based on the previous work as described above. The proposed project will make use of this favorable European position. The combination of leading European research groups in the field, ability to integrate the core multi-disciplinary technologies in the field together with the industrial participation in the project, will significantly strengthen European competitiveness within an important health area.

C5. Contribution to Community social objectives

The major contribution of the proposed project is in improving the quality of life and health of Citizens of the Community. One in every five people worldwide is diagnosed with cancer sometime in their lifetime, and cancer is the second leading cause of death in the developed world. In Europe alone, well over half a million die of cancer and virus related diseases every year. Despite significant advances in both cancer detection and therapy, the incidence of many forms of cancer is rising, and only small increases have been achieved in survival for most forms of cancer. Thus, it appears that the classical triad of surgery, chemotherapy and radiation, while effective against many forms of cancer, is insufficient to control or cure many malignancies.

Much effort has been directed at immunotherapy of malignant fatal viral disease such as AIDS, but success has so far been elusive. The chimeric receptor approaches represents a novel therapeutic modality combining the advantages of humoral and cell mediated immunity, and harnessing them towards tumor and HIV elimination. Our groups have made much progress in the development of chimeric T cell receptors of several different configurations, and in the development of novel vectors, for efficient transduction of these genes into effector cells.

During the course of this contract, we will use an integrated, problem solving approach to optimize several key aspects of the chimeric receptor methodology. We will further develop the technology and demonstrate pre-clinical feasibility. By the end of the grant period, it is our intention to complete pre clinical trials, and initiate phase I clinical studies. Thus, the chimeric receptor methodology will be developed into a clinical strategy for the benefit of society.

We view the benefits of our research to the European Community as two-fold. As an effective approach to cancer therapy as well as for the elimination of infectious viruses, we anticipate that the chimeric receptor methodology will enable the control and possible cure of tumors and AIDS, which are not responsive to classical therapeutic modalities. We also aim at developing a novel recognition domain to the chimeric receptor that will offer a new treatment for some autoimmune diseases and enable non-matched tissue transplantation. We anticipate the chimeric receptor approach to be less toxic than aggressive chemotherapy and irradiation, yielding a great advantage in the quality of life of cancer patients and their families.

Secondly, many aspects of the chimeric receptor modality have already been patent protected, and are suitable for commercialization. This includes the chimeric receptor constructs themselves, for which we already hold patent rights in Europe, and the targeted viral delivery system which Got-A-Gene, our industrial partner, brings to the project. The further optimization and testing of the chimeric receptor approach by a consortium of European laboratories will enable the commercialization of this bioprocess by the European biotechnology community.

C6. Economic development and scientific and technological prospects; exploitation and dissemination plans

The industrial application of this research project is the implementation of an integrated �cell factory� bioprocess for the genetic engineering of programmed cells for therapeutic use. The project aims at the development and optimization of a generic technology which will allow to engineer effector lymphocytes from a patient, using a defined, pre-existing set of vectors harboring chimeric receptor genes. The downstream technologies include:

1. Construction of chimeric receptor genes specific to a wide scope of malignancies, infectious viruses and potentially, antigens involved in autoimmune diseases and graft rejection.

2. Vectors for efficient gene delivery (preferentially, targeted) into human lymphocytes.

3. Cell processing.

Exploitation of the project results requires the consolidation of several biotechnological sectors. Got-A-Gene, the Biotech Company that will participate in the project (Partner # 6) and play an active part in targeted gene delivery, is certainly a preferred candidate for exploitation and commercialization of the project results. The potential market for this immuno-gene therapy is very large and with optimization, is of a great economic value, estimated in hundred million Euro per year.

Partner 2 has developed and protected by patent an original technology that allows selecting from phage libraries P-MHC complexes that bind a specific T cell receptor. A start-up company (Antigene) originating from the lab is being established and its role will be to construct large libraries of T cell antigens to be used in different contexts. In the collaboration with Antigene, the role of the academic group of Guy Gorochov is to identify and isolate Targets cells, for instance T cell lymphoma cells, and to select from the phage libraries the corresponding P-MHC complex that binds the targets.

As described before, Europe has a strong position. This includes our patents in the field, vector producing companies and companies for cell processing. This can be foreseen to have a favorable economic impact, both directly on the company that will process and supply the product and industries that will produce the intermediate elements. Increased employment opportunities will be created.

Dissemination of the RTD results

All the results of this cost shared RTD project will be published in reports to the European Commission and eventually, in open peer-reviewed literature. All partners have their own organizations securing patents and or granting licenses for technology and intend to secure new information from this RTD project in joint patents if applicable. Decisions will be taken on patent filing by the participants. The cost of patents will be covered by the institutions involved; it should be pointed out that clinical procedures in general are not patented in Europe (in contrast to the USA).

All the partners have a long and established track of publishing their work in the open literature and present their workat major international meetings. Additionally, they all are inventors in patents and their intellectual property rights are committed to their parent institutions. The deliverables that consist of reports and publications in open peer reviewed literatures, will be reviewed by the Monitors (described in C3) before submission for publication to assess a possible disclosure of patentable information/technology. In case of disagreement or conflict of interests, the Partners agreed to handle the case in a good fate and that an arbitrator agreed by the involved partners will resolve the conflict. As a rule, preference will be given to the protection of novel information/technology by patent applications. Review of manuscripts and patent applications will be done promptly when needed to allow publications in top competent journals. We shall take the necessary precautions to secure our communication when needed. Nevertheless, we shall take all the measures to encourage the participating groups to interact freely within the general framework of the project. Such an enhanced mobility of scientific and technical staff, as well as free exchange of materials and protocols, constitute a major goal of the project and guarantees its success.

The target groups that will influence the future implementation of the Chimeric Receptor technology consist of oncologists, hematologists, infectious disease experts, immunologists, transplantation surgeons, biotechnology companies, the pharmaceutical industry and national health bodies and insurance companies. These target groups will be addressed by publications in open high impact scientific journals and in lectures by the participants in professional meetings and conferences. These activities are essential for scientists and part of their routine activities, there is no need to specify these dissemination activities as a separate task of the project.

C7. Ethical aspects

Specify if your project involves:

  • @ Human embryos or foetus No
  • @ Use of other human tissue Yes
  • @ Research on persons No. Yes for phase I clinical trials- all appropriate permissions will be obtained before initiation of studies.
  • @ Use of non-human primates No
  • @ Use of trans-genic animals No
  • @ Use of other animals Yes
  • @ Genetic modification of animals No
  • @ Genetic modification of plants No

This study involves the use of small rodents (SCID mice). These mice enable the modeling of the human immune system, to allow experimental manipulation. In our study, we will inject these mice with human tumors, which we will attempt to cure using the Chimeric Receptor approach to immunotherapy. These animal studies are essential in order to demonstrate the efficacy of this approach, and to optimize treatment protocols, with the ultimate goal of developing a therapeutic modality for use in humans.

Human samples will consist of tumor tissue removed during therapeutically required surgery, and small blood samples (50 ml), obtained with the informed consent of patients.

Human cells will be transfected with chimeric receptor genes, using vectors approved for use in humans. These transfections DO NOT involve any germ line modifications to the genome, and transfected cells will only be returned to patients in Phase I trials, planned for the end of this grant period, and only after specific permissions are obtained form all relevant organizations.

In preliminary in vitro studies and experiments in mice, all biomaterials and patient samples will be incinerated after use. No drugs or biological materials will be administered to patients, except in the context of phase I clinical trials.

All appropriate permission for animal experimentation has been obtained by each of the participating laboratories. Prof. Eshhar serves as Chairman of the Animal Experimentation and Ethical Committee at the Weizmann Institute of Science.

We shall supply copies of all institutional and national ethical and safety approvals from all partners pending approval of this proposal, before signing of the final contract.

C8. Safety provisions

The laboratories of all the partners operate under strict institutional controls, and conform to national safety regulations of each country.

All permissions for use of potentially infectious materials have been obtained.

Permission for work with HIV virus:

Animal License: "Therapeutic Applications of Monoclonal Antibodies" PPL 30/1101

GMAG: 98/559 "Gene Therapy with Retroviral Vectors"

99/569 "Gene Therapy with Lentiviral Vectors"

Ethical approval for Clinical Trials will be obtained when appropriate.

There is no risk of transmission of infected material among species and across species and to the environment, since HIV-infected samples are dealt with in restricted and strictly confined areas. Furthermore, there is no risk of release into the environment of genetically modified organisms, as cells, media, and materials are incinerated after use.

Vector manufacturing will be performed within a dedicated service facility (www.genethon.fr/gvpn) which operates under a Quality Assurance system and follows standard operating procedures. Vectors are released only after strict quality control.

C9.Ongoing projects and previous proposals

No similar proposal has been submitted by any of the applicants