Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-24
2004-09-21
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06794139
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention concerns a pharmaceutical composition, its use in tumour diagnosis, therapy and prevention, methods for diagnosing, treating and preventing tumours as well as antibodies and their use.
The unregulated growth of tumour cells is caused by a new expression pattern of genes which regulate the cell cycle control, adhesion, angiogenesis, invasiveness and finally the formation of metastases (Pardee, Advances in Cancer Res. 65 (1994), 213-227; Ponta et al., Biochem. Biophys. Acta 1198 (1994), 1-10). The clinical course of tumour diseases such as breast cancer is characterized by several defined molecular processes such as oestrogen-independent growth, tamoxifen resistance, expression of vimentin, increase in invasiveness and finally cross-resistance towards a large number of chemotherapeutic agents which is often referred to as multi-drug resistance (cf. e.g. Clarke et al., J. Endocrinol. 122 (1989), 331-340; Sommers et al., Cancer Res. 53 (1992), 5190-5197; Sommers et al., Cancer Res. 49 (1989), 4258-4263 and Saceda et al., Mol. Endocrinol. 2 (1988), 1157-1162).
A key concern of tumour research is to identify genes which play an important role in the progression of tumour diseases and to provide new agents for tumour diagnosis, prevention or therapy on the basis of these genes.
SUMMARY OF THE INVENTION
The present invention describes the identification, cloning and characterization of the gene for a polypeptide which is named progression-associated protein “PAP”. This protein is expressed in the metastasising human mammary carcinoma cell line MCF-7
ADR
whereas no expression was found in the non-metastasising mammary carcinoma cell line MCF-7. Hence the PAP protein, a nucleic acid coding therefor as well as antibodies directed against the protein are suitable as agents for the diagnosis, therapy or prevention of tumour diseases and especially of breast cancer.
The invention provides a pharmaceutical composition which is characterised in that it contains the following active components:
(A) a nucleic acid which comprises
(a) the protein-coding nucleotide sequence shown in SEQ ID NO.1,
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code,
(c) a sequence hybridizing with the sequences from (a) or/and (b) under stringent conditions or
(d) an at least 20 nucleotide long section of the sequences from (a), (b) or/and (c),
(B) a polypeptide or peptide which is coded by a nucleic acid according to (A) or/and
(C) an antibody to a polypeptide or peptide according to (B).
In this connection stringent hybridization conditions are understood to mean that a hybridization still occurs after washing at 55° C., preferably at 62° C., particularly preferably at 68° C. in a low salt buffer (e.g. 0.2×SSC, 0.1% SCS) (see also Sambrook et al., (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press).
In a preferred embodiment the pharmaceutical composition additionally contains common pharmaceutical carrier substances, auxiliary substances or additives.
The protein-coding nucleotide sequence shown in SEQ ID NO. 1 codes for a protein with a length of 157 amino acids which corresponds to the B4B nucleotide sequence described by Ruegg et al., 1996 (B4B, a Novel Growth-Arrest Gene, Is Expressed by a Subset of Progenitor/Pre-B Lymphocytes Negative for Cytoplasmic &mgr;-Chain, Am.Ass.Imm., 1995, p. 72-80) and has a homology to already known proteins. In the experiments carried out by Ruegg et al., (supra) the expression of the B4B protein is claimed to arrest the growth of Cos-7 cells. Hence the use of the B4B protein is postulated as a potential tumour suppressor.
One protein which is homologous to PAP is the rabbit protein CL-20 which is induced in vitro during the differentiation of rabbit tracheal cells and occurs most frequently in squamous epithelial tissue (Marvin, J. Biol. Chem. 270 (1995), 28910-28916). The expression of CL-20 does not depend on the growth conditions but retinoids which inhibit the differentiation of squamous epitheleum repress the induction of CL-20.
A further protein which is homologous to PAP is the rat epithelial membrane protein EMP-1 which mainly occurs in the proliferation and differentiation zones of the outer gastric gland as well as in epithelial cells of the stomach region (Taylor et al., J. Biol. Chem. 270 (1995), 28824-28833).
PAP is also homologous to the peripheral myelin protein PMP22 from man, mouse and rat which is a myelin-associated transmembrane structural protein. PMP22 is a specific protein for growth inhibition which prevents cell cycle progression (Hayasaka et al., BBRC 186 (1992), 827-831; Edomi et al., Gene 126 (1993), 289-290; Welcher et al., Proc. Natl. Acad. Sci. USA 88 (1991), 7195-7198; Spreyer et al., EMBO J. 10 (1991), 3661-3668).
On the basis of the data of Ruegg et al. and the homology to the PMP22 protein responsible for maintaining the cellular dormant state, it was extremely surprising that the PAP protein is selectively expressed in the strongly degenerate metastisising cell line MCF-7
ADR
but not in the less degenerate cell line MCF-7.
Furthermore PAP has a high degree of homology to previously not yet published proteins whose sequences are stored in the gene library. These proteins are referred to as murine TMP or human TMP and have an amino acid identity of 76% or 95% to PAP.
The nucleotide and amino acid sequences of murine TMP are shown in SEQ ID NO. 3 and 4 and of human TMP are shown in SEQ ID NO.5 and 6. PAP and human TMP mainly differ in the region of the amino acids 32-48.
A further subject matter of the present invention is the use of a vector which contains at least one copy of a nucleic acid as previously defined or a section thereof for the transformation of a cell. This nucleic acid can for example be genomic DNA, cDNA or mRNA. It is preferably a recombinant DNA molecule.
The invention also concerns the use of a vector which contains an at least 20 nucleotide long section of the protein-coding sequence shown in SEQ ID NO.1. This section preferably contains a nucleotide sequence specific for PAP. These nucleic acids are especially suitable for preparing antisense nucleic acids that can be used therapeutically which preferably have a length of up to 50 nucleotides.
The vector containing the nucleic acid can be capable of replication in eukaryotes or prokaryotes. It can be a vector that can be integrated into the genome of the host cell e.g. bacteriophage &lgr;, or it can be a vector that is present extrachromosomally (e.g. a plasmid). The vector according to the invention can be obtained by subcloning the PAP-DNA into a base vector. Such base vectors and in particular vectors containing the elements necessary for protein expression are familiar to a person skilled in the art.
When cloning a nucleic acid coding for PAP, it is possible to prepare an expression vector that can be expressed in a suitable host cell to form the protein according to the invention. Preferred host cells are microorganisms such as
E. coli
or yeast and also higher cells such as mammalian or insect cells. Preferred expression vectors are for example plasmids, bacteriophage &lgr; for prokaryotes, yeast vectors or viral vectors for higher cells e.g. SV40, vaccinia, baculovirus. With regard to the expression of a PAP-coding nucleic acid reference is made in particular to the methods stated in Sambrook et al. (supra).
Systems suitable for expressing PAP contain suitable vectors e.g. the vector pcDNA1 (Invitrogen) in which the DNA to be expressed is under the control of the cytomegalovirus promoter.
A further subject matter of the present invention is a cell which is characterized in that it is transformed with a nucleic acid which comprises
(a) the protein coding nucleotide sequence shown in SEQ ID NO.1,
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or
(c) a nucleotide sequence which has a homology of more than 95% to a sequence from (a) or/and
Schiemann Sabine
Weidle Ulrich
Fredman Jeffrey
Johnston George W.
Rocha-Tramaloni Patricia S.
Roche Diagnostics GmbH
Strzelecka Teresa
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