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
1999-12-08
2002-02-26
Bansal, Geetha P. (Department: 1692)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C530S388100, C530S387900, C435S069100, C424S185100
Reexamination Certificate
active
06350452
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of detecting and quantifying apoptosis. More particularly, the present invention relates to antibodies to the newly formed amino terminus resulting from cleavage of proteins during the process of apoptosis and to the use of such antibodies in detecting apoptosis in cells undergoing apoptosis or in cells that have undergone apoptosis.
BACKGROUND OF THE INVENTION
Most eukaryotic cells have the ability to self-destruct by activation of an intrinsic cellular suicide program referred to as programmed cell death or apoptosis. The process of apoptosis involves a cascade of cytoplasmic and nuclear events that result in a series of morphological changes, and eventually cause the demise of the cell. Apoptosis is characterized by distinct biochemical and morphological changes exhibited by cells undergoing programmed cell death, including DNA fragmentation, plasma membrane blebbing, and cell volume shrinkage. At the molecular level, activation of one or more aspartate-specific, cysteine proteases (caspases) is proposed to be the critical signal required to carry out apoptotic cell death (Yang et al.,
American Journal of Pathology,
152(2):379-389, 1998).
The caspases, also known as ICE (IL-1 &bgr;-converting enzyme)-like proteases, can be divided into three subclasses: ICE/CED3 family, CPP32/Yama family and the Ich/Nedd2 family (Duan et al.,
J. Biol. Chem.,
271:1621-1625, 1996). All family members share a high level of amino acid sequence homology with ICE, and contain a conserved QACRG pentapeptide in which the cysteine participates in catalysis (Nicholson,
Nature Biotech.,
14:297-301, 1996). Furthermore, all of these proteases are reported to require an aspartic acid residue at the substrate P1 position (Jänicke et al.,
The EMBO J.,
15(24):6969-6978, 1996).
CPP32 (Caspase 3) has been identified as one of the proteases that cleaves poly(ADP-ribose) polymerase (PARP) (Schlegel et al.,
J. Biol. Chem.,
271:1841-1844, 1996; Nicholson et al.,
Nature,
376:37-42, 1995). PARP is one of the enzymes associated with DNA repair. Cleavage of the approximately 116 kilodaltons (“kd”) PARP protein into fragments of about 89 kd and about 27 kd has been reported to contribute to the DNA fragmentation that is characteristic of apoptosis (Kayalar et al.,
Proc. Natl. Acad. Sci. USA,
93:2234-2238, 1996). Therefore, the identification of the about 89 kd or the about 27 kd fragments resulting from the cleavage of PARP within a cell is an indication that the cell is undergoing or has undergone apoptosis.
Proteins consist of macromolecules of amino acids linked by peptide bonds, to form polypeptide chains. Each amino acid in the chain consists of a carbon atom to which are attached four different groups (—R, —H, —NH
2
, and —CO
2
H), wherein the identity of R varies from one amino acid to another. The peptide bond links each amino acid to the next amino acid in the chain through a covalent bond formed between the —CO
2
H group of one amino acid and the —NH
2
group of the next amino acid, with H
2
O a byproduct of the reaction. Every polypeptide chain has two terminal amino acid residues, one at each end of the chain. The end of the chain with a —CO
2
H group which has not been linked to another amino acid is referred to as the “carboxy terminus” or “C-terminus”. The end of the chain with a —NH
2
group which has not been linked to another amino acid is referred to as an “amino terminus” or “N-terminus”. When an enzyme such as caspase cleaves PARP, it breaks a peptide bond in a polypeptide chain of the protein, creating one fragment with a new carboxy terminus and another fragment with a new amino terminus.
One reference, WO 98/21590, describes methods of detecting apoptosis by using antibodies that bind to the amino acids at the newly created carboxy termini of polypeptides generated by the cleavage of proteins by the caspase family of proteases. Because caspases cleave immediately carboxy-terminal of a characteristic four amino acid recognition site, the newly created carboxy terminus of a caspase cleaved protein consists of the last amino acid of the recognition site. WO 98/21590 describes the production of antibodies following immunization of rabbits with a polypeptide comprising a caspase recognition site (GDEVD) at its carboxy terminus. Although the antibodies of WO 98/21590 appear to recognize a cleaved fragment of PARP, these antibodies were cross-reactive with other proteins as well. This cross-reactivity is likely to result in inaccurate determinations of apoptosis in cells or cellular lysates.
In another attempt to produce antibodies that are specific to apoptotic fragments of PARP, Sallmann et al.,
Biochem. Cell Biol.,
75:451-456, (1997) immunized rabbits with synthetic polypeptides corresponding to the newly created carboxy terminus and amino terminus of PARP that are formed following cleavage of PARP by a caspase. Although the polyclonal antibodies produced by Sallmann et al. were able to distinguish between the two (carboxy terminal and amino terminal) apoptotic fragments of PARP, the antibodies were not able to distinguish between the cleaved fragment and uncleaved PARP. Therefore, the antibodies produced by Sallmann et al. are not specific to epitopes produced in apoptotic cells because they are immunoreactive with the uncleaved PARP present in non-apoptotic cells.
Therefore, there is a need for antibodies that are specifically able to distinguish apoptotic events in cells. These antibodies will enable more accurate results in methods for detecting apoptosis. Because apoptosis, or the inability of cells to undergo apoptosis, is associated with a number of disorders and diseases including cancer, neurodegeneration, autoimmunity, heart disease and others (reviewed in Hetts,
JAMA,
279(4):300-307, 1998), improved methods of detecting apoptosis will provide a better understanding of these diseases and will be useful in screening potential therapeutic agents that may induce or prevent apoptosis.
BRIEF SUMMARY OF THE INVENTION
The present invention seeks to overcome limitations in the prior art by providing methods and compositions related to antibodies that recognize specific epitopes that are indicators of apoptosis. Antibodies were produced that are specific to a new epitope (neoepitope) formed in apoptotic cells. The neoepitope is a product of cleavage of a protein at a specific cleavage site by a protease during apoptosis.
The present invention involves the use of antibodies raised against a neoepitope which comprises a polypeptide sequence which is homologous to only one end of one of the fragments of a protein cleaved during apoptosis. Every protein consists of a chain of amino acids. Each amino acid covalently linked to the next amino acid in the chain through a peptide bond. A peptide bond links the amine residue of one amino acid with the carboxyl residue of the next amino acid in the chain. The resulting chain has an unattached amine residue at one end, and an unattached carboxy residue at the other end. When the protein is cleaved, and the chain of amino acids broken at that point, two fragments of the protein are formed, one with a new carboxy terminus, and the other with a new terminal amine. In the present invention, the neoepitope comprises the new amino terminus at the beginning of the carboxy terminal fragment.
By immunizing animals with a polypeptide comprising a sequence of the amino acids of the newly formed amino terminus of a fragment of a protein formed by cleavage of the protein during apoptosis, antibodies were isolated that recognized the cleaved form of the protein but failed to recognize the uncleaved form. This result is surprising because attempts by others to produce specific antibodies to a new amino terminus of PARP formed after cleavage by a caspase failed because the antibodies were still immunoreactive with the uncleaved protein (Sallmann et al.,
Biochem. Cell Ciol.,
75:451-456, 1997). Furthermore, the isolated antibodies of the present invention show particular utility in
Bansal Geetha P.
Frenchick Grady J.
King Karen B.
Michael & Best & Friedrich LLP
Promega Corporation
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