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Antibodies specific for cyp1b1Antibodies specific for cyp1b1 description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20090023160, Antibodies specific for cyp1b1. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION
The present invention relates to tumour diagnosis and therapy, and in particular to antibodies specific for tumour specific cytochrome P450 CYP1B1. BACKGROUND OF THE INVENTIONCytochrome P450 (P450) CYP1B1 is the only known member of a recently identified sub-family of the CYP1 gene family. Human CYP1B1 was originally isolated from a dioxin treated keratinocyte cell line (Sutter et al., 1994) as part of a series of investigations to identify differential expression of genes caused by exposure to dioxin. The human CYP1B1 gene is located on chromosome 2p22-21 spanning 12 kb and is composed of three exons and two introns (Tang et al., 1996). The mRNA is 5.2 kb and encodes for a protein of 543 amino acids (Sutter et al., 1994). This is the largest known human P450 gene both in terms of mRNA size and number of amino acids and is also the simplest structurally. Both nucleic acid and amino acid sequence analysis shows that CYP1B1 displays only approximately 40% homology with CYP1A1 and CYP1A2. The low degree of similarity with existing members of the CYP1 family resulted in this P450 being assigned to a new CYP1 sub-family CYP1B which to date only contains the single member CYP1B1. Indeed hybridisation studies of human DNA suggests that there is only one member of the CYP1B gene family (Sutter et al., 1994). The CYP1B1 gene is transcriptionally activated by ligands of the Ah receptor including planar aromatic hydrocarbons (Sutter et al., 1994, Hakkola et al., 1997) and the most potent of these Ah receptor agonists for inducing transcription of the CYP1B1 gene appears to be dioxin (Hakkola et al., 1997). Orthologous forms of this P450 have also recently been isolated from a benzanthracene-induced cell line derived from mouse embryo fibroblasts (Savas et al., 1994; Shen et al., 1994) and adult rat adrenal cortex (Bhattacharyya et al., 1995; Walker et al., 1995). Although there is a high degree (greater than 80%) of both nucleic acid and amino acid sequence homology between the human, mouse and rat forms of CYP1B1 there also appears to be considerable species differences regarding tissue specific expression, regulation and metabolic specificity of CYP1B1 (Savas et al, 1994; Sutter et al., 1994; Bhattacharyya et al., 1995; Savas et al., 1997). Breast cancer is the commonest cancer to affect women and is an estrogen dependent tumour. Human CYP1B1 expressed in yeast (S. cerevisiae) shows high specific activity towards the 4-hydroxylation of 17β-estradiol (Hayes et al., 1996) converting it to 4-hydroxyestradiol and indeed human CYP1B1 is considered to be the most efficient 4-hydroxylase of 17β-estradiol. In contrast mouse CYP1B1 does not appear to act as an estradiol hydroxylase (Savas et al., 1997) indicating species differences in the metabolic capability of CYP1B1. Liehr and Ricci (1996) showed that there is a significant level of 4-hydroxylation of 17β-estradiol in breast cancer microsomes. They showed considerably higher 4-hydroxylation of 17β-estradiol in microsomes prepared from breast cancer compared with only a very low level of 4-hydroxylation present in normal breast tissue. Both immunoreactive CYP1B1 protein (Murray et al., 1997) and CYP1B1 MRNA (McKay et al., 1995) have been identified in breast cancer indicating that CYP1B1 is a major form of cytochrome P450 expressed in breast cancer. SUMMARY OF THE INVENTIONThe initial immunohistochemical studies of CYP1B1 in Murray et al. (1997) showed enhanced CYP1B1 expression in several types of human cancer including breast cancer and were performed using a polyclonal antibody to CYP1B1. The present invention relates to further developments in this area and concerns further antibodies specific for human CYP1B1, and in particular monoclonal antibodies raised using synthetic peptides based on CYP1B1 amino acid sequence as immunogens. The work described herein discloses the uses of the antibodies in immunohistochemistry and in the investigation of the expression of CYP1B1 by immunohistochemistry in a series of primary human breast cancers. Accordingly, in a first aspect, the present invention provides a method of making an antibody that specifically binds to cytochrome P450 CYP1B1, the method comprising raising the antibody using a peptide consisting of an amino acid sequence VNQWSVNHDPVKWPN or PExFDPARFLDKDGy, where x is D or N and y is L or F, or an antigenic fragment thereof. Typically, the antigenic fragments within these peptide sequences consist of 3 to 10 amino acids and more typically 3 to 6 amino acids. In a further aspect, the present invention provides a method of producing an antibody having obtained a hybridoma by the above method, the method comprising culturing a hybridoma producing the antibody and isolating the antibody thus produced. The method may comprise the further step of conjugating the antibody to an effector moiety such as a label, a toxin, a drug or a transport molecule. In a further aspect, the present invention provides an antibody which is capable of specifically binding to cytochrome P450 CYP1B1, wherein the antibody recognises an epitope in the cytochrome P450 CYP1B1 protein included within the amino acid sequence VNQWSVNHDPVKWPN or PExFDPARFLDKDGy, where x is D or N and y is L or F. Preferably, the antibody recognises an epitope of between 3 and 10 amino acids from the amino acid sequences, and more preferably an epitope of between 3 and 6 amino acids from the amino acid sequences. Preferred antibodies are monoclonal antibodies, for example as obtainable by:
(a) immunising an animal with the peptide conjugated to an immunogenic carrier;
(b) sacrificing the animal and fusing spleen cells obtained from the animal with myeloma cells to produce one or more hydridomas; and,
(c) screening the hybridomas for antibodies capable of binding the peptide.
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