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Engineered o-glycosylation of a tumour antigen

Objective



Cell membrane mucins have a high potential as Cancer Vaccines. In particular the MUC1 gene product, PEM, is a candidate because this mucin is over expressed in many carcinomas, and cancer-associated epitopes have been identified, which are repeated 30-100 times, per molecule.
Both humoral and cellular anti-PEM immunity are found in cancer patients, and active immunisation with PEM based antigens is expected to improve this immunoresponse and show therapeutic benefits. In support of this we are aware that several US, Canadian and European Biotech companies are pursuing the development of a cancer vaccine based on PEM: The leadíng player in Europe is Transgene SA, Participant 5 in this proposal. A Concerted Action (BIOMED), coordinated by participant 1 (Grant No. BMH1/CT94/1462) is analysing the efficacy of existing MUC1 based immunogensfor Immunotherapy. The production and use of mucin molecules requires the establishment of novel strategies and analyses not yet introduced into the Biotech Industry. Mucins are normally covered by large O-glycans masking peptide and immature glycopeptide epitopes. Cancer cells generally produce mucins with incomplete O-glycosylation hence cancer-associated peptide and carbohydrate epitopes are exposed. The participants of this proposal originally identified and characterised these epitopes in Breast Cancers and have available a panel of reagents for defining their expression. PEM vaccine constructs based on non-glycosylated mucin peptides have been found to be inefficient in stimulating a potent anti-PEM response and it is clear that a vaccine construct should have O-glycans attached, at least in the form of sialylated GalNAc (sialyl-Tn), one of the major glycans added to the breast cancer mucin. This proposal aims at developing the necessary technology to produce such glycoproteins and to evaluate their potential as vaccines.
The Programme will involve the production of PEM with defined O-glycans in A. Wild type CHO cells, which, like breast cancers lack the core 2 enzyme known to be responsible for the extension of O-glycans added to PEM in normal breast cells. This cell line is expected to add sialylated T (SA alpha2,3 Gal beta1,3 GalNAc) as the major O-glycan.
B. A CHO cell engineered to add sialyl-Tn as the major O-glycan to those amino acids which are glycosylated in Breast Cancers. This cell line will be developed (i) by transfecting in cDNA coding for alpha2,6 sialyl transferase(T1 engineered to be positioned in an early compartment of the ER/Golgi and, (ii) by modifying the profile of expression of GalNAc transferases, if this is found to be necessary.
The project deals specifically with the area of Post Translation Modifications of Proteins listed in AREA 1 (Cell factories, 1.1.2. Animal Cell Biology). To develop the appropriate glycoforms, expertise from four different groups is required. The Biotechnology Company, Transgene SA, working with the Concerted Action group will analyse the products for their ability to react with antibodies from cancer patients and to stimulate immune responses in vitro. The Clinical Units associated with the CA group will test products in the Clinic. Thus the project will synergise with a programme funded by BIOMED, and with the BIOTECH Industry.
Patent applications from the ICRF covering the MUC1 gene are pending and options to license have been negotiated with Transgene.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Imperial Cancer Research Fund (ICRF)
Address
44 Lincoln's Inn Fields
WC2A 3PX London
United Kingdom

Participants (4)

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany
Address
Meyerhofstrasse 1
Heidelberg
GOETEBORG UNIVERSITY
Sweden
Address
9,Medicinaregatan 9
405 30 Goeteborg
TRANSGENE SA
France
Address
11,Rue De Molsheim 11
67082 Strasbourg
UNIVERSITY OF COPENHAGEN
Denmark
Address
20,Noerre Allee 20
2200 Koepenhagen