Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-01-22
2001-06-05
Hauda, Karen M. (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007400, C435S007920, C435S007940, C530S388800, C530S389100, C530S333000
Reexamination Certificate
active
06242203
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to tumour diagnosis and therapy, and to materials and methods for use therein.
More particularly, the invention is based on the identification of a cytochrome P450 form, specifically CYP1B1, in a wide range of tumours, with a high frequency of expression in each type, and proposes the use of this enzyme as a tumour marker, and as the basis of a selective therapeutic approach involving the design of drugs, eg which are activated to a cytotoxic form by the action of CYP1B1.
BACKGROUND TO THE INVENTION
The major goal of cancer chemotherapy is the development of anti-cancer drugs that are effective in a wide range of cancers and produce no toxic effects in normal tissues. The target of such drugs should be expressed only in tumour cells and not normal cells. However, to date, no such tumour specific target, general to all types of cancers, has been identified.
The cytochromes P450 are a multi-gene family of constitutive and inducible enzymes, which have a central role in the oxidative metabolic activation and detoxification of both a wide range of xenobiotics (2-4) and several groups of endogenous compounds active in cell regulation and cell signalling including arachidonic acid (5), steroid hormones (6) and fatty acids (7). The major families of P450 involved in xenobiotic metabolism each consist of several individual forms with different regulatory mechanisms and substrate specificities (2). The majority of P450s are primarily expressed in liver (2) although individual P450 forms are also expressed in specific extra-hepatic tissues (8) including small intestine, kidney and lung.
The human CYP1 gene family (individual P450 forms are identified by the prefix CYP in accordance with the current P450 nomenclature (3)), which is one of the major P450 families involved in the metabolism of xenobiotics, is now known to consist of three individual forms classified into two sub-families. The CYP1A subfamily contains two highly homologous and well characterised but distinct members. CYP1A1 (9) and CYP1A2 (10). CYP1A1 is an inducible P450 expressed primarily in extraheptic tissues (11) while CYP1A2 is a major form of P450 that is constitutively expressed in liver (12). Recently a second human CYP1 subfamily has been identified which to date contains one member, CYP1B1 (1). This P450 is dioxin-inducible, and sequence analysis of CYP1B1 shows 40% homology with both CYP1A1 and CYP1A2. Although CYP1B1 is assigned to the CYP1 family on the basis of its sequence, it appears to be structurally distinct from both CYP1A1 and CYP1A2.
Several forms of P450 are considered to have an important role in tumour development since they can metabolise many potential carcinogens and mutagens (13). Moreover, P450 activity may influence the response of established tumours to anti-cancer drugs; several cancer chemotherapeutic agents can be either activated or detoxified by this enzymes system (14). The presence of individual forms of P450 had previously been investigated in different types of cancer including breast cancer (15), lung cancer (16), colon cancer (17) and head and neck cancer (18) to determine if intra-tumour metabolism of anti-cancer agents by P450 could occur and thus influence the response of tumours to these agents. These studies have generally shown that the level of the P450 forms investigated is significantly reduced or absent in tumours when compared with the adjacent normal tissue in which the tumours have developed. However, our recent studies of several different types of cancer (19) including breast cancer, oesophageal cancer and soft tissue sarcomas have shown that there may be tumour-specific expression of a CYP1 form of P450.
Although CYP1B1 mRNA had previously been identified by Northern blotting in several normal human tissues (1), the presence CYP1B1 protein itself had not been demonstrated.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that CYP1B1 is a tumour-specific form of P450, present in a wide range of malignant tumours and not detected in normal tissues.
Accordingly, a first aspect of the present invention provides a method for the identification of tumour cells, which method comprises the use of a recognition agent, for example an antibody, recognising CYP1B1 protein to contact a sample of tissues, cells, blood or body product, or samples derived therefrom, and screening for a positive response. The positive response may for example be indicated by an agglutination reaction or by a visualisable change such as a colour change or fluorescence, eg immunostaining, or by a quantitative method such as in use of radio-immunological methods or enzyme-linked antibody methods.
The method therefore typically includes the steps of (a) obtaining from a patient a tissue sample to be tested for the presence of cancer cells; (b) producing a prepared sample in a sample preparation process; (c) contacting the prepared sample with a recognition agent, such as an antibody, that reacts with human CYP1B1 protein; and (d) detecting binding of the recognition agent to CYP1B1 protein, if present, in the prepared sample. The human tissue sample can be from for example the bladder, brain, breast, colon, connective tissue, kidney, lung, lymph node, oesophagus, ovary, skin, stomach, testis, and uterus.
A preferred sample preparation process includes tissue fixation and production of a thin section. The thin section can then be subjected to immunohistochemical analysis to detect binding of the recognition agent to CYP1B1 protein. Preferably, the immunohistochemical analysis includes a conjugated enzyme labelling technique. A preferred thin section preparation method includes formalin fixation and wax embedding. Alternative sample preparation processes include tissue homogenization, and preferably, microsome isolation. When sample preparation includes tissue homogenization, a preferred method for detecting binding of the antibody of CYP1B1 protein is Western blot analysis. Alternatively, an immunoassay can be used to detect binding of the antibody to CYP1B1 protein. Examples of immunoassays are antibody capture assays, two-antibody sandwich assays, and antigen capture assays. Preferably, the immunoassays is a solid support-based immunoassay. When Western blot analysis or an immunoassay is used, preferably it includes a conjugated enzyme labelling technique.
Although the recognition agent will conveniently be an antibody, other recognition agents are known or may become available, and can be used in the present invention. For example, antigen binding domain fragments of antibodies, such as Fab fragments, can be used. Also, so-called RNA aptomers may be used (36, 37). Therefore, unless the context specifically indicates otherwise, the term “antibody” as used herein is intended to include other recognition agents. Where antibodies are used, they may be polyclonal or monoclonal. Optionally, the antibody can produce by a method so that it recognizes a preselected epitope of said CYP1B1 protein.
A second aspect of the invention lies in the presence of CYP1B1 protein selectively in tumours, eg in kidney tumours and not normal renal tissue, combined with the absence of CYP1B1 protein expression in normal liver, which provides a mechanism for the selective targeting of anti-cancer drugs based on CYP1B1 metabolism in tumours. Drugs can be designed for, or screened for, specific metabolism by CYP1B1 in tumours whereby this metabolism converts a non-toxic moiety into a toxic one, which kills or inhibits the tumour or makes it more susceptible to other agents.
A third aspect of the invention provides for the targeting of cytotoxic drugs or other therapeutic agents, or the targeting of imaging agents, by virtue of their recognition of CYP1B1 epitopes on the surface of a tumour cell, whether as part of the complete CYP1B1 protein itself or in some degraded form such as in the presentation on the surface of a cell bound to a MHC protein.
Another aspect of the invention provides stimulation of the immune system of cancer patients, for example by ac
Burke Michael Danny
Greenlee William Frank
Melvin William Thomas
Murray Graeme Ian
Chen Shin-Lin
Fish & Richardson P.C.
Hauda Karen M.
University of Aberdeen
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