DNA encoding variant CD44 surface proteins associated with metas

Details DNA encoding variant CD44 surface proteins associated with metas DNA encoding variant CD44 surface proteins associated with metas

1992-11-09

1996-04-09

Nucker, Christine M.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

4241841, 4241851, 4242771, 530350, 530300, 530395, 530828, 4351723, 4352523, 4353201, 536 231, 536 235, A61K 3512, C07K 14205, C12N 1512

Patent

active

055061192

DESCRIPTION:

BRIEF SUMMARY
The invention concerns variant CD44 surface proteins, antibodies against the variant determinants of these proteins, as well as processes for their production, furthermore the DNA sequences which code for these variant protein fragments, as well as the use of these proteins or parts thereof and the antibodies directed against them for the diagnosis and therapy of tumour metestases.


BACKGROUND OF THE INVENTION

The ability to metastase forms the actual life-endangering property of malignant tumour cells. The original primary tumour cells probably acquire this property by a whole series of changes in the course of the tumour progression. As a result of this process, cancer cell variants are continuously detached from the primary tumour mass, penetrate the extracellular matrix and migrate into the lymphatic system or the blood circulation. Often adhering to one another, the metastasing tumour cells are transported in the blood or lymph system, leave the vascular system at other places in order there to penetrate into secondary tissue and form daughter tumours (survey of Hart et al., 1989; Nicolson, 1987). The formation of metastases requires a whole series of interactions of the tumour cells with intercellular matrix and other cells. Almost all of these interactions require cell surface components, such as e.g. the receptors for matrix and lamina, surfacebound proteolytic enzymes, as well as cell adhesion molecules with inclusion of those which cause organspecific adhesion and thus organ preference of the metastasis, furthermore growth factors and growth factor receptors.
It is known that the membrane proteins differentiate non-metastasing and metastasing tumour cells of the BSp73 rat tumours, demonstrated by antibody reaction (Matzku et al., 1983 and 1989).


SUMMARY OF THE INVENTION

It has now been found that the metastasing BSp73ASML tumour cells contain a surface protein which, in part, corresponds to a known glycoprotein participating in the lymphocyte adhesion and cell-cell and cell-matrix exchange action (designation of the normal glycoprotein in humans: CD44, hermes-1, in the mouse: Ppg-1 and in the rat: HEBFln). However, the new variant CD44 surface protein differs from these known sequences by an extracellular region (ECR) of 154 amino acids which is introduced between the 220th and 237th amino acid of the human CD44 sequence (or 224th and 239th amino acid of the mouse sequence). This new glycoprotein appears to possess an important role for the cell/matrix or cell/cell binding in the case of the metastasis. Therefore, the production and characterisation of this protein region (ECR) forms one of the tasks of the present invention. By immunisation of mice with membrane proteins which have been obtained from BSp73ASML, spleen cells were produced which form antibodies against the ECR of the variant CD44 surface protein. According to the method of Ko hler (1981), these are fused by polyethylene glycol with myeloma cells in order to produce permanent cultures. By means of cloning and selection of those cultures which produce antibodies which react with BSp73ASML but not with the non-metastasing parent form and also not with other non-tumorigenic rat cells, there can be obtained specific antibodies against the new protein part ECR. For the further investigation, a monoclonal antibody was chosen which stains the BSp73ASML cells in the immunofluorescence test especially intensively, which has received the designation mAb1.1ASML (mAb: monoclonal antibody).
In the Western blot test, in a protein hydrolysate frown BSp73ASML, there can be determined 4 protein bands with molecular weights of 120,000; 150,000; 180,000 and 200,000 with mAb1.1.ASML, whereas extracts from rat fibroblast cells and non-metastasing rat tumour cells give no significant reaction. It has not yet been possible to determine whether these size differences are due to a different original amino acid sequence or to a differently strong subsequent protein modification. In any case, the epitope recognised by the antibody is contained in all 4

REFERENCES:
Lehmann, J. M. et al. Proc. Natl. Acad. Sci USA 86: 9891-9895 (1989).
Chan, B. M. C. et al. Science 251: 1600-1602 (1991).
Maniatis T. et al. Molecular Cloning pp. 382-389 Cold Spring Harbor Laboratory (1982).
Kugelman, L. C. et al. J. Invest. Dermotol. 99:381-385 (1992).
Bowvie, J. V. et al. Science 247: 1306-1310 (1990).
Kumar, V. et al. Proc. Natl. Acad. Sci. 87: 1337-1341 (1990).
Ellis, R. W. In: Vaccines, Protkin & Mortiner Eds. W. B. Saunders Co. (1988) pp. 568-575.
Young, R. A. et al. Proc. Natl. Acad. Sci. 80: 1194-1198 (1983).
Birnbaum et al., "Amplification, Expression and Localization of the c-myc Gene in BSp73 Rat Tumor Cell Lines," Anticancer Research, vol. 8, 1988, pp. 1185-1191.
Matzku et al., "Antigenic Differences between Metastatic and Nonmetastatic BSp73 Rat Tumor Variants Characterized by Monoclonal Antibodies," Cancer Research, vol. 49, Mar. 1, 1989, pp. 1294-1299.

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