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Cspcna isoform antibodies and uses thereof

Cspcna isoform antibodies and uses thereof description/claims

This application claims priority to U.S. Ser. No. 60/675,275, filed Apr. 27, 2005 and U.S. Ser. No. 60/689,614, filed Jun. 9, 2005.

The present disclosure relates to detection and treatment of malignant cells involving the use of antibodies that bind specifically to a cancer specific protein.

One of the least understood and most complex disease processes is the transformation that occurs as a cell becomes malignant. This process involves both genetic mutations and proteomic transformations, the result of which allows the cell to escape normal controls; preventing inappropriate cell division. All cancers are unique and distinct from other cells, as well as other cancers. Despite this uniqueness, cancer cells share some common attributes. Most cancer cells proliferate outside of the normal cell cycle controls, exhibit morphological changes and exhibit various biochemical disruptions to cellular processes.

Cancer is usually diagnosed when a tumor becomes visible well after the first on-set of cellular changes. Many cancers are diagnosed after a biopsy sample is examined by histology for morphologic abnormalities, evidence of cell proliferation and genetic irregularities. Effective treatment for malignancy often depends on the ability to detect reliably the presence of malignant cells at early stages of a disease so that an effective treatment can begin at a stage when the disease is most susceptible to such treatment. Thus, there is a need to be able to reliably detect a potentially malignant cell that has not progressed to the histological stage recognized as malignant, but which can progress to a malignant state. There is also a need for a rapid, minimally invasive technique that can reliably detect or treat malignant cells or potentially malignant cells.

Proliferating cell nuclear antigen (PCNA) is a 29 kDa nuclear protein and its expression in cells during the S and G2 phases of the cell cycle, makes the protein a good cell proliferation marker. It has also been shown to partner in many of the molecular pathways responsible for the life and death of the cell. Its periodic appearance in S phase nuclei suggested an involvement in DNA replication. PCNA was later identified as a DNA polymerase accessory factor in mammalian cells and an essential factor for SV40 DNA replication in vitro. In addition to functioning as a DNA sliding clamp protein and a DNA polymerase accessory factor in mammalian cells, PCNA interacts with a number of other proteins involved in transcription, cell cycle checkpoints, recombination, apoptosis, and other forms of DNA repair. Besides being diverse in action, PCNA's many binding partners are linked by their contributions to the precise inheritance of cellular functions by each new generation of cells. PCNA may act as a master molecule that coordinates chromosome processing.

Malignant cancer cells express an isoform of PCNA termed cancer specific PCNA (csPCNA) and non-malignant cells express an isoform termed non-malignant PCNA (nmPCNA). Effective compositions and methods to distinguish the two isoforms are needed for diagnosis and treatment of cancers.

Antibodies to cancer specific isoform of proliferating cell nuclear antigen (csPCNA) and uses thereof are disclosed. Antibodies specifically bind to the cancer specific isoform of PCNA but do not bind to the non-malignant isoform of PCNA (nmPCNA). The antibodies are produced from an immunogen that includes a peptide comprising an amino acid sequence found on the region of csPCNA protein that interacts with the Xeroderma pigmentosum group G (XPG).

A peptide region that corresponds to amino acid residues 126-133 of the human PCNA protein, SEQ ID NO.: 1 (LeuGlyIleProGluGlnGluTyr) is a suitable antigenic peptide for generating csPCNA antibodies. The antigenic peptides disclosed herein may include additional amino acid residues that improve immunogenicity of the peptide without substantially interfering with the specificity of the resulting antibodies to csPCNA. For example, the peptide may have the amino acid sequence of SEQ ID NO: 2 (CysGlyGlyGlyLeuGlyIleProGluGlnGluTyr). The resulting antibodies may be polyclonal or monoclonal antibodies or fragments thereof.

An isolated antibody disclosed herein specifically binds cancer specific proliferating cell nuclear antigen (csPCNA) isoform. The csPCNA isoform includes an amino acid sequence of SEQ ID NO: 3 and any variations, mutations including substitutions, insertions and deletions that do not affect the specificity of csPCNA specific antibodies. csPCNA specific antibodies do not bind to nmPCNA isoform.

In an embodiment, the antibody binds to an epitope that includes an amino acid sequence within the csPCNA protein that binds to Xeroderma pigmentosum group G (XPG) protein.

In an embodiment, the antibody binds to an epitope of csPCNA that includes an amino acid sequence selected from LGIPEQEY (SEQ ID NO: 1), VEQLGIPEQEY (SEQ ID NO: 5), LGIPEQEYSCVVK (SEQ ID NO: 6), LGIPEQEYSCVVKMPSG (SEQ ID NO: 7), EQLGIPEQEY (SEQ ID NO: 8), QLGIPEQEY (SEQ ID NO: 9), LGIPEQEYSCVVKMPS (SEQ ID NO: 10), LGIPEQEYSCVVKMP (SEQ ID NO: 11), LGIPEQEYSCVVKM (SEQ ID NO: 12), LGIPEQEYSCVV (SEQ ID NO: 13), LGIPEQEYSCV (SEQ ID NO: 14), and LGIPEQEYSC (SEQ ID NO: 15).

In an embodiment, the antibody includes a monoclonal antibody or a chimeric antibody or a recombinant antibody or a single chain antibody.

In an embodiment, the antibody is an antibody fragment selected from Fab, Fab′, or F(ab′)2.

The antibodies may be associated with a detectable agent and the detectable agent is selected from a fluorescent label, radio label, chromatogenic label, and an enzymatic label.

A composition includes an isolated and substantially purified antibody that is specifically bound to an epitope of cancer specific proliferating cell nuclear antigen (csPCNA), wherein the epitope includes an amino acid sequence of LeuGlyIleProGluGlnGluTyr (SEQ ID NO: 1).

A method for detecting a cancer specific proliferating cell nuclear antigen (csPCNA) isoform in a biological sample includes the steps of:

contacting the biological sample with an antibody that specifically binds cancer specific proliferating cell nuclear antigen (csPCNA) isoform;

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