Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
1999-09-03
2003-10-07
Ungar, Susan (Department: 1642)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S007100, C435S325000, C530S387100, C530S403000
Reexamination Certificate
active
06630350
ABSTRACT:
BACKGROUND AND SUMMARY OF THE INVENTION
The present invention concerns monoclonal antibodies (MAB) which specifically bind a complex of &agr;1-antichymotrypsin (ACT) and a serine protease, in particular prostate-specific antigen (PSA) and have essentially no cross-reactivity with non-complexed ACT and non-complexed serine proteases. These monoclonal antibodies can be used to detect ACT-serine-protease complexes and in particular to detect PSA-ACT.
The prostate-specific antigen is a glycoprotein with a molecular weight of 33 kDa. It is formed in the prostate epithelial cells and is a component of seminal fluid. PSA has the enzymatic activity of a neutral serine protease.
The main function of PSA is to cleave the seminogelins I and II and fibronectin that are gel-like proteins which, as the main component of the ejaculate, block the mobility of the sperm. PSA liquefies the seminal coagulum by hydrolysing these proteins and thus enables the sperm mobility.
Enzymatically active PSA is inactivated in the serum by various inhibitors so-called serpins (=serine protease inhibitors), by the formation of covalent complexes. Most of the immunologically detectable PSA is bound in the serum to &agr;1-antichymotrypsin (60-95%). Further complexes are formed with &agr;2-macroglobulin, &agr;1-antitrypsin, inter-&agr;-trypsin inhibitor and protein C inhibitor. In addition an enzymatically inactive PSA also occurs which no longer complexes with serpins.
&agr;1-antichymotrypsin is a glycoprotein with a molecular weight of ca. 69 kDa and a carbohydrate moiety. ACT plays an important role in the control of inflammations as one of the main inhibitors in the acute phase. ACT also forms complexes with chymotrypsin, cathepsin G and glandular kallikrein hK2. ACT is present in human serum in a 10,000-fold higher concentration than PSA (on a molar basis).
Apart from free PSA, the PSA-ACT complex is the main form of the immunologically detectable total PSA in serum. Prostate cancer leads in many cases to an increase in the serum PSA level. However, since slightly increased PSA serum values are also found in benign prostate hyperplasia, PSA is not a cancer-specific marker especially in the low concentration range. The previously available screening tests for the possible presence of a prostate carcinoma in a patient have always been tests for detecting total PSA. Since PSA normally occurs in very low concentrations in the serum of male persons, a so-called cut-off has to be defined for such a test. PSA values which are above this cut-off are evaluated as an indication for the presence of prostate carcinoma. Since the PSA concentration increases with increasing age of the patients, cut-off values of 4 to 6 ng/ml have previously been used for the test for the detection of total PSA. As a result some patients which had a prostate carcinoma in the early stage were not detected in these screening tests.
Already in the Japanese unexamined laid-open patent application 62-46263 it was found that increased values of complexed PSA occurred in patients with a malignant prostate tumour compared to patients with benign prostate hyperplasia. In this unexamined laid-open patent application an immunoassay was described which detects using a combination of an antibody against &ggr;-seminoprotein (&ggr;-seminoprotein is identical with PSA; see Schaller et al., Eur. J. Biochem. 170, 1987, 111-120 and Nakamura, Cancer 74, 1994, 1655-1659) and an antibody against &agr;1-antitrypsin.
A method for the detection of PSA-ACT is described in WO 92/01936 in which a combination of the antibody 2E9, which binds uncomplexed PSA and also binds PSA in the complex with ACT, and an antibody against ACT is used.
In addition there are diagnostic tests for the detection of free, non-complexed PSA and total PSA i.e. the sum of free and complexed PSA. All these tests contain antibodies which in the case of a detection of free PSA only recognize PSA in a non-complexed form or in the case of a detection of total PSA only recognize PSA in a complexed and free form.
The detection of ACT in a complexed form with serine proteinases and in particular the detection of PSA-ACT was, as described above, only previously possible with the aid of a sandwich test using two antibodies of which one of the antibodies was directed against PSA and the other antibody was directed against ACT. Since ACT occurs in human serum in a ca. 10,000-fold excess compared to PSA and thus the complex composed of PSA and ACT also occurs, it is not possible to rule out negative test interference by this high excess of ACT. In particular it is essential in these previously known tests for detecting PSA-ACT to include at least one wash step to remove excess ACT before adding the ACT-specific antibodies in the test procedure. Thus a one step test procedure which is desirable for many automated diagnostic tests is not possible.
Therefore the object of the present invention was to provide an improved test for the detection of PSA-serine proteases, in particular PSA-ACT which should if possible not have interference by the presence of high ACT concentrations in the serum and which allows a screening which is as sensitive as possible for the detection of a prostate carcinoma.
The object is achieved by a monoclonal antibody against a complex of human ACT and a serine protease which has essentially no cross-reactivity with free, non-complexed human ACT and free, non-complexed serine proteases.
In particular the object was achieved by an MAB against a complex of ACT and a serine protease which has essentially no cross-reactactivity with free human ACT and free serine proteases and which has a substantially higher affinity and specificity for PSA-ACT than for other serine protease-ACT complexes in particular chymotrypsin-ACT and cathepsin-G-ACT.
The monoclonal antibody can be used in all tests familiar to a person skilled in the art for the detection of a protein. In a preferred test procedure using two antibodies (sandwich test) the test can be carried out in one step i.e. without an additional wash step to remove the excess ACT. This is a decisive improvement compared to the previously possible tests which all included a wash step to remove the excess ACT, especially for screening tests in which numerous samples have to be tested as rapidly as possible.
The monoclonal antibodies according to the invention against a complex of human ACT and a serine protease essentially have no cross-reactivity with free human ACT and free serine proteases. Essentially no cross-reactactivity is understood as a cross-reactivity which in a test for the detection of the ACT-serine protease complex does not result in an influence by free ACT or free serine protease. The level of cross-reactivity with individual components that can still be tolerated depends on the concentration at which these components can occur in human serum. Since ACT occurs in a very large excess, the cross-reactivity must be infinitesimal in this case i.e. substantially below 1%. It was not possible to detect any cross-reactivity of the monoclonal antibodies according to the invention using the available methods. The BIACORE® system from the Pharmacia company was used to detect the cross-reactactivity. Antibodies with an affinity constant of less than 10
5
l/mol for the tested substances exhibited no significant binding and thus no detectable cross-reactactivity in this system.
The monoclonal antibodies according to the invention exhibited no cross-reactivity towards non-complexed PSA, chymotrypsin and cathepsin G in the BIAcore®. In order to check cross-reaction with all potentially interfering substances that occur in human serum, human serum was added in this case to the screening test. A human female serum was used so that no ACT-PSA complexes are present. The monoclonal antibodies according to the invention exhibited no detectable cross-reactivity with other components occurring in this serum.
Since PSA-ACT represents the most clinically relevant serine protease-ACT complex, the monoclonal antibodies according to the inventio
Deeg Rolf
Gallusser Andreas
Hubner-Parajsz Christa
Kaufmann Martin
Kientsch-Engel Rosemarie
Amick Marilyn L.
Davis Minh-Tam
Roche Diagnostics GmbH
Ungar Susan
Woodburn Jill L.
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