Apoptosis-related compounds and their use

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...

Reexamination Certificate

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C435S007100, C530S300000, C530S350000, C530S353000, C530S357000, C530S402000

Reexamination Certificate

active

06296850

ABSTRACT:

BACKGROUND OF THE INVENTION
Apoptosis is seen in all sorts of higher eucaryocytes from plants and insects to vertebrates (Kroemer G, Petit P, Zanzami N, Vayssiere J-L, Mignotte B: The biochemistry of programmed cell death. The FASEB Journal 9:1277-1287 (1995)). Apoptosis is a general phenomenon of vital importance. Decreased apoptosis leads to malformation, cancer and autoimmune disease, and enhanced apoptosis results in degenerative diseases, acute diseases such as infection by toxin-producing microorganisms and in rejection of transplanted organs.
Therefore, the detection, monitoring, measurement and regulation of apoptosis are important factors in the diagnosis and therapeutic treatment of the mentioned conditions.
Recently, Caulin C., et al (The Journal of Cell Biology, Vol 138, pp. 1379-1394) studied caspase cleavage of Keratin 18 and reorganization of intermediate filaments during epitelial cell apoptosis. Keratin 18 was cleaved in vitro by caspase-6, -3, and -7, and it was stated that the cleavage site common for the three caspases was the sequence VEVD/A (SEQ ID NO: 22), located in the conserved L1-2 linker region of K18.
DESCRIPTION OF THE INVENTION
During research work aiming at finding new useful specific monoclonal antibodies reacting with human cytokeratin 18 (CK 18), in addition to those already at hand, it was surprisingly found that a monoclonal antibody (MAb) (obtained after immunization of mice with a specified part of the amino-acid sequence of CK 18) recognized early apoptotic changes in cultured epithelial cells. This MAb was designated M30.
A detailed analysis, including synthesis and assay of a large number of amino-acid (aa) sequences revealed, that the specific epitope and binding-site for the MAb M30 consists of the aa sequence: EDFNLGDALD (SEQ ID No. 1). This 10 aa peptide sequence starts from the second coil of human CK 18 and represents aa No.383-392. This specific epitope is a neo-epitope liberated during apoptisis and it is not exposed in the intact CK 18 molecule. The complete aa sequence of CK 18 was published by Oshima et al. (Oshima RG, Millan JL, and Ceccena G. (1986). Comparison of mouse and human keratin 18: a component of intermediate filaments expressed prior to implantation. Differentiation 33:61-68.).
The different aspects of the present invention are based on the optimal binding site of the mAb M 30, i.e. the amino-acid sequence EDFNLGDALD,
SEQ ID NO: 1:

  Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp
   1                5                   10
or functionally equivalent sequences comprising the sequence Ala Leu Asp.
Initially it was believed that an important part for apoptosis is related to cleavage by caspase enzymes immediately after DALD (Asp Ala Leu Asp (SEQ ID NO: 5)), since this sequence occures in CK18 and caspases are known to cleave after DXXD wherein X stands for not defined aa.
However, the sequence DALD does not occur in such other intermediate filaments as CK 1-8, vimentin, desmin, different actins, neurofilaments, and lamins. In these the following sequences are found: LALD, MALD, MALD, VALD, MALD, and LALD (SEQ ID NO: 6, 7, 7, 8, 7, and 6), respectively.
Since the mAb M 30 recognizes apoptotic changes in non-epithelial cells such as cardiac muscle, mega—karyocytes, myeloblasts and neural tissues of fetus—which are not known to contain CK18—it is now believed that detection of protein fragments having the C-terminal sequence ALD indicates early apoptosis.
Thus, one aspect of the invention is directed to an apoptosis-related antigenic compound which specifically binds to an antibody which in turn specifically binds to the amino-acid sequence
SEQ ID NO: 1:
Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp,
1                5                   10
i.e. an apoptosis-related antigenic compound comprising an exposed antigenic site having the amino acid sequence SEQ ID NO: 1 or a functionally equivalent sequence. Further, the functionally equivalent sequence should comprise at least the sequence Ala Leu Asp.
It is the three-dimensional structure of the exposed antigenic site, or antibody-binding site, that defines the structure of the binding portion of an antigenic compound which specifically binds to the corresponding three-dimensional structure of an antibody. The antigenic compound of the invention therefore comprises compounds which are based on the structure of the SEQ ID NO:1, i.e. the sequence as such or a functionally equivalent sequence, i.e. homologous in function, comprising at least the sequence Ala Leu Asp, such as compounds having replacements of one or several amino acids in the above specified amino-acid sequence with other molecular parts, D- forms of the amino acids, non-natural amino acids and/or derivatives, as long as the three-dimensional structure of the SEQ ID NO:1 is mimicked. Therefore the common feature of the antigenic compounds of the invention is that they bind specifically to an antibody which in turn binds specifically to the aa sequence SEQ ID NO:1, i.e. antigenic compounds comprising an antigenic site having the amino acid sequence SEQ ID NO: 1 or a functionally equivalent sequence comprising at least the sequence Ala Leu Asp.
It can be mentioned that intact CK 18 molecules have the above-specified sequence hidden, and can therefore not react with antibodies specifically binding to said sequence, thereby making the recognition specific to apoptosis, i.e. those cases where the CK 18 molecule has been already cleaved after the C-terminal Asp of the sequence Asp Ala Leu Asp (SEQ ID NO. 5).
In a preferred embodiment of this aspect of the invention, the apoptosis-related compound is a peptide or a protein fragment comprising the C-terminal amino acid sequence Ala Leu Asp. The peptide may be a homologue to, an extension of or a truncation of the SEQ ID NO: 1, i.e. may have a homologous sequence having some amino-acid substitutions, extensions, truncations and/or deletions which do not lead to the elimination of the capability of the peptide to bind to the same antibodies as the amino-acid sequence SEQ ID NO: 1.
In another preferred embodiment of this aspect of the invention the antigenic compound of the invention is a peptide or a protein fragment comprising the C-terminal amino acid sequence Xaa Ala Leu Asp, wherein Xaa is selected from Asp, Leu, Met, and Val (SEQ ID NO. 5, 6, 7, and 8).
An example of a peptide according to the invention is an oligopeptide having the amino-acid sequence
SEQ ID NO: 1:
Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp.
  1              5                   10
Another example is an oligopeptide having the amino-acid sequence (EDGEDFNLGDALDSSNSMQT)(i.e. SEQ ID NO: 1 extended by 3 amino acids at the N-terminal and 7 at the C-terminal)
SEQ ID NO: 2:
Glu Asp Gly Glu Asp Phe Asn Leu Gly Asp Ala Leu
 1               5                   10

Asp Ser Ser Asn Ser Met Gln Thr
         15                  20
Yet another example is an oligopeptide having the amino-acid sequence (LEDGEDFNLGDALDSSNS)
SEQ ID NO: 3:
Leu Glu Asp Gly Glu Asp Phe Asn Leu Gly Asp Ala
 1                5                 &em

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