Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-03-23
1998-03-17
Green, Lora M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 721, 435 702, 435 7021, 4351722, 43524027, 436 64, G01N 33574, C12P 2108
Patent
active
057285373
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to tumour markers, more specifically cytokeratins, for cancer cells of epithelial origin. According to the invention a method for reproducible production of cytokeratin antigen/immunogen is provided.
There is a large need to be able to detect and diagnose cancer in an early stage before the patient has developed an inoperable tumour or metastases. Furthermore, it is desirable to be able to localize the tumour for localized treatment or prior to surgery.
An example of localized cancer treatment is where the tumour is killed with antibodies coupled to, for example, cytotoxin or radioactive isotopes according to known methods. These substances targeted against the tumour provide increased killing of tumour cells or an increase of the concentration of cytotoxin in the tumour and thereby decrease the side effects of the conventional cytostatic treatment.
From immunohistological and immunocytological tests it is known that certain carcinomas, i.e., tumour tissue of epithelial origin, contain tumour markers in the form of cytokeratins of different kinds. There are 19 different characterized cytokeratins, all being built of proteins. These cytokeratins form so called intermediate filament in the cells. The cytokeratin pair 8 and 18 are very frequent in simple epithelia.
The present invention is based on the discovery that the insoluble intracellular cytokeratins are released and fragmented in tumour tissues, whereby a large fraction of the cytokeratins becomes soluble. The soluble cytokeratin fragments leak out to surrounding body fluids, such as blood, urine, ascites and pleura.
2. Description of Related Art
In U.S. Pat. No. 4,774,620 cytokeratin fragments released in tissue culture medium of MCF-7 carcinoma cells are used to produce monoclonal antibodies. This method is not reproducible and is very unspecific since the actual tumour marker is not exactly known. Because unspecific cell material also is used as reference in tests, the latter become unreliable as regards quantification and specificity.
In EP A1 337 057 cytokeratins are chromatographically purified and enzyme digested to obtain "the alpha helical center portions thereof" which in turn are chromatographically purified and used as antigen in immunological tests and for production of monoclonal antibodies, respectively. Here the antigens/immunogen is obtained in a reproducible way. These known methods are sufficient for detecting cytokeratin fragments in body fluids and for characterizing to which cell category/type a tumour belongs to. However, the monoclonal antibodies obtained according to EP A1 337 057 require an initial solubilization of the sample to be tested.
SUMMARY OF THE INVENTION
Thus, there still exists a need to be able to detect and localize carcinomas in vivo and treat cancer patients with monoclonal antibody therapy. Also, there is a need of more simple and sensitive tests than those of the prior art for determining whether a tumour is progressive or not and following up monoclonal antibody therapy to evaluate the treatment. The present invention fulfils the above needs.
The cytokeratin fragments produced according to the present invention cause a stronger immune response of an animal injected therewith, and therefore higher reactivity and specificity of the antibodies, than those of the prior art. The present invention also gives a more appropriate reaction with several different sizes of cytokeratin fragments, although with retained specificity.
Below the invention will be described in a non limiting way. The abbreviations used are given at the end of the description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph showing the specificity and reactivity of 15 different monoclonal antibodies of the present invention in ELISA for cytokeratin 8 (left bar), and 18 (right bar).
FIG. 2 is a bar graph showing the specificity and reactivity of 15 different monoclonal antibodies of the present invention against cytokeratin 19 in ELISA, as in FIG
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Moll et al., Cell, vol. 31, (Nov. 1982), pp. 11-24, "The Catalog of Human Cytokeratins: Patterns of Expression in Normal Epithelia, Tumors and Cultured Cells".
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Silen .ANG.ke
Wiklund Bo
AB IDL Immunodeveloplab
Green Lora M.
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