Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Nucleoproteins – e.g. – chromatin – chromosomal proteins,...
Reexamination Certificate
2000-12-08
2004-02-24
Mosher, Mary E. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Nucleoproteins, e.g., chromatin, chromosomal proteins,...
C514S012200
Reexamination Certificate
active
06696547
ABSTRACT:
2. FIELD OF THE INVENTION
The invention relates to a protein for regulating apoptosis and to a nucleic acid encoding the protein. The protein and nucleic acids are useful in the regulation of apoptosis, and in methods for diagnosing or detecting apoptosis. The invention also relates to antibodies directed against the protein.
3. BACKGROUND OF THE INVENTION
Apoptosis is a type of cell death, which is thought to be genetically programmed, and which is distinct from the process of necrosis. During apoptosis, cells generally se their cell junctions and microvilli, the cytoplasm condenses and nuclear chromatin marginates into a number of discrete masses. Cory, S.
Nature
367:317-318 (1994).
Apoptosis can be induced via a variety of receptors, commonly referred to as “death receptors.” These receptors contain a “death domain” (DD). Examples include CD95, TNF-RI, DR3, DR4 or DR5, which induce apoptosis signal paths after binding their respective ligands. For example, after the CD95 ligand binds to the CD95 receptor, the receptor interacts with the adapter protein FADD/MORTI to induce the recruitment and activation of the protease FLICE/Caspase-8 at the DISC “death inducing signaling complex.” FADD and FLICE contain “death effector domains (DED).
Apoptosis can be inhibited by the transcription of anti-apoptotic genes, i.e. by the gene products thereof. For example, the protein FLIP “FLICE-inhibitory protein” inhibits the CD95 apoptosis signal path. German patent 19713434 of Deutsches Krebsforschungszentrum (the German Cancer Research Center). There is a need in the art for novel compounds for regulating of apoptosis, and for methods for using such compounds. It is thus an object of the present invention to provide such compounds and methods.
4. SUMMARY OF THE INVENTION
The invention relates generally to proteins for regulating and/or inducing apoptosis. The proteins of the invention generally include the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1) or an amino acid sequence which is substantially similar to the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1). Alternatively, the protein may consist of or consist essentially of the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1) or an amino acid sequence which is substantially similar to the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1).
In one aspect the invention relates to an amino acid sequence differing from the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1) by one or more amino acids, wherein the DNA encoding the amino acid sequence hybridizes with the DNA encoding the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1). Preferably, the amino acid sequence differs from the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1) by no more than 1, 2, 3, 4, or 5 amino acids. The amino acid sequence may also be an active fragment of the sequence of
FIG. 1A
(SEQ ID NO: 1) or of an amino acid sequence which is substantially similar to an active fragment. Examples of active fragments include amino acids 1-114 of
FIG. 1A
(SEQ ID NO: 1) or amino acids 109-318 of
FIG. 1A
(SEQ ID NO: 1). Additionally, the amino acid sequence may be a truncation of the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1), i.e., truncated at the C-terminus and/or the N-terminus, preferably by no more than 1, 2, 3, 4, or 5 amino acids at either terminus. The amino acid sequence may also comprise one or more conservative substitutions.
In a related aspect, the amino acid sequence differs from the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1) by one or more amino acids and hybridizes to the DNA encoding the amino acid sequence of
FIG. 1A
(SEQ ID NO: 1), preferably under stringent conditions.
The protein of the invention is suitably provided as a component of a pharmaceutical composition.
The invention also relates to a nucleic acid, such as a DNA or RNA encoding any of the proteins of the invention. In one aspect, the DNA comprises the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2) or a nucleotide sequence which is substantially similar to the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2). The DNA may differ from the DNA of
FIG. 1A
(SEQ ID NO: 2) by one or more degenerate codons. In a preferred aspect, the DNA comprises a nucleotide sequence corresponding to, or substantially similar to, nucleotides 28 to 369 of
FIG. 1A
(SEQ ID NO: 2), as provided in SEQ ID NO: 7, nucleotides 352 to 981 of
FIG. 1A
(SEQ ID NO: 2), as provided in SEQ ID NO: 8, or the nucleotide sequence of
FIG. 1B
(SEQ ID NO: 4). The nucleotide sequence is suitably provided as a component of an expression plasmid or an expression cassette, or as a component of a cell transformed by a nucleic acid comprising the nucleotide sequence.
The invention also relates to a process for preparing a protein of the invention. In one aspect, the method of production comprises culturing a cell transformed by an expression plasmid comprising a nucleotide sequence selected from the following: (a) the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2); (b) a nucleotide sequence which is substantially similar to the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2); (c) a nucleotide sequence which is substantially similar to the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2); (d) a nucleotide sequence which hybridizes to the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2), preferably under stringent conditions; (e) a nucleotide sequence which differs from the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 2) by one or more degenerate codons; (f) a nucleotide sequence corresponding to nucleotides 28 to 369 of
FIG. 1A
(SEQ ID NO: 2), as provided in SEQ ID NO: 7, or a nucleotide sequence substantially similar thereto; (g) a nucleotide sequence corresponding to nucleotides 352 to 981 of
FIG. 1A
(SEQ ID NO: 2), as provided in SEQ ID NO: 8; and (h) the nucleotide sequence of
FIG. 1B
(SEQ ID NO: 4) or a nucleotide sequence substantially similar thereto.
In another aspect, the invention provides an antibody which binds, preferably which specifically binds, to a protein of the invention. The antibody is preferably a monoclonal antibody. In a related aspect, the invention relates to a hybridoma cell line expressing an antibody of the invention.
The invention also relates to a method for detecting in a sample the presence a protein of the invention. In general, the method employs the following steps: (a) contacting the sample with a an antibody which specifically binds to a target protein of the invention; and (b) analyzing the sample for the presence of antibody specifically bound to target protein.
Similarly, the invention relates to a method for islolating a protein of the invention. The method generally employs the following steps: (a) contacting a composition comprising the target protein with an antibody which specifically binds to the target protein; (b) eluting the target protein from the antibody. The eluted protein may then be separated using known procedures, for example, standard cross-flow filtration techniques known in the art.
In another aspect, the invention relates to a method for inducing apoptosis in a cell. In this method, apoptosis is induced by contacting the cell with a protein of the invention. The cell may be contacted in vivo by administration of the protein of the invention to a subject in an amount sufficient to induce apoptosis. The method is useful, for example, in the treatment of diseases of the immune system and in the treatment of neoplasms. The administration may be direct, by administering the protein to the subject, or indirect, by administering to the subject a nucleotide encoding the protein for expression in the subject.
5. DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms ar
Krammer Peter
Peter Marcus
Deutsches Krebsforschungszentrum Stiftung des Offentlichen Recht
Fuierer Marianne
Hultquist Steven
Mosher Mary E.
Yang Mimi
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