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Antigen presentation by chaperon proteins and cd4+ t cell specific help for a new and efficient strategy of cancer vaccination.

Objective

To identify new HLA-classes I and II restricted antigens (Ags) from human tumours of different histology and to define the role of CD4+ T cells in induction and maintenance of effective cytotoxic T lymphocyte (CTL) responses.
To evaluate the role of chaperon proteins, including HSP, in inducing tumour specific T cell-mediated immune responses in human and murine cancers both in vitro and in vivo, comparing this approach with a different antigen-delivery system (recombinant filamentous phages).
To molecularly define processing and presentation pathways of chaperon proteins, including cloning of receptors involved in their specific uptake by antigen presenting cells (APC).
To engineer tumour cell and/or APC to overexpress chaperon proteins or their receptors as tools for effective immunization strategies.
To design pilot phase I-II studies for vaccine development in cancer patients.

Description of the work

We will identify new tumour-derived HLA-class I and II-binding peptides by several approaches (cDNA or expression libraries, differential screening by micro-array technologies, etc). These peptides will be complexed with chaperon proteins for in vitro and in vivo immunological studies. New Ags will be also identified from tumour-derived chaperon proteins either by direct biochemical approaches (peptide elution and mass-spectrometry sequencing) or by cDNA cloning using for the screening procedures chaperon-induced tumour-specific T cells.
The cellular interaction among dendritic cells (DC), CD4+ T cells and CTL will be studied in different in vitro systems. Attempts will be made to identify the signal involved in the induction of anti-tumour CTL in human system.
The role of chaperon proteins in Ag processing and presentation will be analyzed in tumour systems (in vitro and in vivo), and will include cloning of the receptors expressed by APC and mediating specific internalization of chaperon/peptides complexes. The ability of native tumour-derived chaperon proteins to induce and modulate T cell specificities and functions will be studied in different human cancers, including melanoma, colon, renal and ovarian carcinomas.
The assessment of the ability of antigenic peptide and chaperon proteins to function as natural adjuvant in potentiating tumour-specific immune responses, will be carried out as follows:
4a. engineering tumour cells to overexpress HSP in native form or covalently linked to immunogenic peptides. Evaluate their immunogenicity in vitro (human and murine systems) and in vivo (HLA transgenic mice)
4b. engineering APC to overexpress chaperon receptors in order to enhance their ability to process and present specific Ag.
On the basis of the acquired knowledge, vaccines will be developed in pre-clinical models; phase I-II pilot clinical studies in cancer patients will be designed.

Milestones

Availability of new human unique and shared tumour Ags presented by classes I and II HLA. Identification of immunogenic peptides bound to HSP and mediating an immune response in cancer patients.
Identification of the role of CD4+ T cells and definition of essential factors to induce an effective anti-tumour CTL response.
Identification of the pathway of Ag presentation by chaperon proteins: and of their receptor expressed by APC.
Engineered APC and optimization of the function of chaperon proteins in inducing an efficient T cell response: their use as vaccines in a preclinical model.
Design of phase I-II vaccination protocols for cancer patients.
The project outcome is the following:
1)new human tumor antigens (Ags) have been molecularly characterised. Few of them have already shown their immunogenicity in long-term disease-free cancer patients. These Ags can now be tested in vivo as vaccines. Less advanced is the discovery of Ags of renal cell carcinoma but experiments are ongoing to achieve such a task.
2)We have identified signal molecules that are crucial for induction of cytotoxic T cells by dendritic cells (DCs) that can now be applied together with HLA epitopes (see 1) for optimizing anti-cancer vaccines.
3)We showed that HSPs can bind specific receptors on DCs. We showed that HSP70/96 purified from human tumors can bind well defined tumor Ags.
4)We developed cellular systems that release, upon appropriate gene tranfection, large quantities of HSP complexed with a given tumor Ag. We are now scaling-up procedures to reach amounts of proteins suitable for patients' vaccination. Phages displaying human tumor epitopes can efficiently immunized transgenic mice.

Coordinator

ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI
Address
Via Venezian 1
20133 Milano
Italy

Participants (7)

ACADEMISCH ZIEKENHUIS LEIDEN
Netherlands
Address
Albinusdreef 2
2333 RC Leiden
CHRISTIAN DE DUVE INSTITUTE OF CELLULAR PATHOLOGY
Belgium
Address
Avenue Hippocrate 75, Icp 75.50
Bruxelles
KAROLINSKA INSTITUTE
Sweden
Address
Solnavagen 1
171 77 Stockholm
MOLECULAR MEDICINE SPA
Italy
Address
Via Olgettina 58
Milano
NATIONAL RESEARCH COUNCIL OF ITALY
Italy
Address
Via Marconi 10
80125 Napoli
THE UNIVERSITY OF TUEBINGEN
Germany
Address
Auf Der Morgenstelle, 15
72076 Tuebingen
UNIVERSITE DE PARIS-SUD XI
France
Address
Rue Georges Clemenceau 15
91405 Orsay