Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-07-12
2002-03-26
Brusca, John S. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S358000
Reexamination Certificate
active
06362317
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to antibodies, antibody fragments and fusion proteins thereof, and their use in determining DNA double-stranded breaks.
BACKGROUND OF THE INVENTION
In a normal human cell, about 2 meters of DNA are folded and packaged into a nucleus less than 10 microns in diameter. Much of this compaction is performed by wrapping the DNA around nucleosomes, which are complexes composed of histone proteins. Compaction and packaging into the nucleus provides a level of protection for fragile strands of DNA. However, despite the compaction of DNA into chromosomes, double-stranded breaks in DNA are common.
DNA double-stranded breaks jeopardize a chromosome's physical integrity essential for its correct segregation during mitosis and meiosis as well as its informational redundancy critical for maintaining accurate encoding of cellular components. Given that DNA double-stranded breaks have the potential to cause a great deal of damage to the cell, it is not surprising that multiple cellular mechanisms exist for dealing with this serious lesion, including non-homologous end joining, homologous recombination, and apoptosis. On the other hand, DNA double-strand cleavages are necessary in several important cellular processes, including recombination during meiosis and mitosis, V(D)J recombination during immune system development, and mating type switching in
S. cerevisiae
. Aside from natural processes, DNA double-stranded breaks also appear following exposure to radiation, magnetic fields, toxins, mutagenic chemicals, various medications, and the like.
While much is known about the causes and actual rejoining of DNA double-stranded breaks, much less is known about how these breaks are initially recognized. Elucidation of an organism's initial response mechanisms to double-stranded breaks would provide a means to detect DNA double-stranded breaks. Currently, DNA breaks are detected using pulsed-field electrophoresis, filter elution, sucrose gradient sedimentation, single cell gel electrophoresis or the TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) assay. The former three methods are useful for only gross measurements of DNA damage in a large sample.
Single cell gel electrophoresis, also known as the comet assay, capitalizes on the movement of fragmented DNA out of a cell body into the agarose gel toward the anode during electrophoresis. The resulting shape resembles a comet, the cell body being the comet head and the fragmented DNA being the comet tail. The comet assay, frequently used in the study of apoptosis, is an improvement over the aforementioned techniques in that it allows measurement of DNA cleavage in an individual cell. However, this technique requires a great deal of time and effort to study DNA breaks in larger samples and lacks sensitivity.
The TUNEL assay employs terminal deoxynucleotidyl transferase to incorporate modified nucleotides onto free 3′-hydroxyl ends of DNA fragments. In most cases, DNA breaks are visualized by differential staining of intact and fragmented DNA. However, the TUNEL assay, as well as the comet assay, require many hundreds of DNA double-stranded breaks to yield a positive signal.
In that only a few double-stranded breaks can result in phenotypic change and 40 double-stranded breaks result in cell death, the currently available assays clearly do not have the sensitivity to be useful in all situations, particularly with non-lethal amounts of breaks. In view of the above, there exists a need for a sensitive means of determining DNA double-stranded breaks. It is an object of the present invention to provide such a means. This and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an isolated or purified antibody or antigenically-reactive fragment thereof that specifically binds to a C-terminal phosphorylated serine in an H2A histone protein. The present invention further provides a fusion protein comprising the isolated or purified antibody or an antigenically-reactive fragment thereof. The present invention further provides a product useful for the determination of DNA double-stranded breaks. The product comprises the present inventive isolated or purified antibody or antigenically-reactive fragment thereof.
Also provided by the present invention are a method and a kit for determining double-stranded breaks in DNA. The method comprises contacting a sample comprising H2A histone proteins with the isolated or purified antibody or antigenically-reactive fragment thereof of the present invention and detecting binding of the antibody or antigenically-reactive fragment thereof to an H2A histone protein in the sample. The detection of the binding of the antibody or antigenically-reactive fragment thereof to an H2A histone protein indicates the presence of a double-stranded break in DNA.
The invention may best be understood with reference to the following detailed description of the preferred embodiments.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated on the discovery that mammalian cells and mice respond to agents that introduce DNA double-stranded breaks with the immediate and substantial phosphorylation of histone H2AX. For many years, scientific paradigms envisioned the role of histones in chromatin and chromosomes as limited to the compaction of DNA. However, it has been discovered that H2AX is intimately involved in the recognition of regions of chromatin containing a DNA double-stranded break. The present invention seeks to capitalize on the formation of the phosphorylated H2A protein, termed &ggr;-H2A, in order to provide a means of sensitive detection of DNA double-stranded breaks. Therefore, the present invention provides an isolated or purified antibody or antigenically-reactive fragment thereof which specifically binds to a C-terminal phosphorylated serine in an H2A histone protein and methods of use.
As used herein, an “antibody” is a protein comprising one or more polypeptides selected from immunoglobulin light chains, immunoglobulin heavy chains, and antigen-binding fragments thereof, which are capable of binding to at least one antigen. An antibody of the present invention includes intact monoclonal and polyclonal immunoglobulins. An antibody of the present invention also includes immunoglobulin types IgA, IgD, IgE, IgG, IgM and subtypes of any of the foregoing, wherein the light chains of the immunoglobulin may be kappa or lambda type. Antibodies of the present invention additionally include bifunctional antibodies in which one arm of the antibody is specific to &ggr;-H2A and the other arm is specific for another antigen. By “isolated” is meant the removal of an antibody or antigenically-reactive fragment thereof from its natural environment. By “purified” is meant that a given antibody or fragment thereof, whether one that has been removed from nature (isolated from blood serum) or synthesized (produced by recombinant means), has been increased in purity, wherein “purity” is a relative term, not “absolute purity.”
An “antigenically-reactive fragment” of an antibody includes segments of immunoglobulins that retain antigen-binding specificity, for example, Fab, Fab′, F(ab′)
2
and F(v) fragments. Preferably, the antigenically-reactive fragment retains the ability to bind selectively &ggr;-H2A. The antibody or fragment thereof may be a single-chain antibody. The antibody or fragment thereof may be a heavy chain monomer, dimer or trimer, a light chain monomer, dimer or trimer, a dimer consisting of one heavy and one light chain, and the like.
One of ordinary skill in the art will appreciate that the isolated or purified antibody or antigenically-reactive fragment thereof of the present invention may include various deletions, additions or substitutions which either do not affect the binding affinity of the antibody or, preferably, enhances the affinit
Bonner William M.
Rogakou Emmy
Brusca John S.
Leydig , Voit & Mayer, Ltd.
The United States of America as represented by the Department of
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