Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-10
2001-09-18
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C536S023100, C536S023500
Reexamination Certificate
active
06291235
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to human colon cell and colon cancer cell associated antigens, nucleic acid molecules, proteins and peptides. Specifically, the proteins and peptides of the invention, which are encoded by the nucleic acid molecules of the invention, are found both in and on the surface of human colon cells and human colon cancer cells, and bind to colon cancer antibodies. The protein, in a monomeric form, has a molecular weight of about 40-45 kD as determined by SDS gel electrophoresis under non-reducing conditions and about 49-55 kD when subjected to SDS-PAGE under reducing conditions. This protein, peptide fragments thereof and multimeric thereof can be used to develop reagents and methods useful in the diagnosis and treatment of cancer.
BACKGROUND OF THE INVENTION
Colorectal carcinoma is a malignant neoplastic disease. There is a high incidence of colorectal carcinoma in the Western world, particularly in the United States. Tumors of this type often metastasize through lymphatic and vascular channels. Many patients with colorectal carcinoma eventually die from this disease. In fact, it is estimated that 62,000 persons in the United States and 8000 persons in Australia die of colorectal carcinoma annually.
To date, systemic therapies and chemotherapies have been developed for the treatment of colorectal cancer. However, no therapies have exhibited sufficient anti-tumor activity to prolong the survival of colorectal carcinoma patients with metastatic disease, with any degree of reliability. As a result, a need still exists to develop methods and products for the successful treatment of colorectal carcinoma.
Monoclonal antibody A33 is a murine immunoglobulin that has undergone extensive preclinical analysis and localization studies in patients (see Welt et al.,
J. Clin. Oncol.,
8:1894-1906 (1990), Welt et al.,
J. Clin. Oncol.,
12:1561-1571 (1994), and Welt et al.
J. Clin. Oncol.
14: 1787-1797 (1996). This antibody binds to an antigen found in and on the surface of normal colon cells and colon cancer cells. This antigen is known as the A33 antigen.
In carcinomas originating from the colonic mucosa, the A33 antigen is expressed homogeneously in more than 95% of cases. The A33 antigen has not been detected in a wide range of other normal tissues studied. Its restricted expression defines this system as essentially “organ-specific” (colon, rectum and small bowel).
Immunofluorescence experiments have revealed that mAb A33 is internalized into the macropinosomes of A33 antigen-positive cells in vitro (Daghighian et al;.
J. Nuc. Med.,
37: 1052-1057 (1996). In a mouse model, mAb'A33 has been found to localize to xenografts of human colon cancer in substantial amounts, and it can be identified in the cytoplasm of transplanted colon cancer cells within the first hour after administration. Rapid tumor localization and high level of antibody uptake by tumors are thought to be related to the following factors: (1) A33 antigen is not secreted, and targeting of mAb A33 to tumor cells is therefore not impeded by shed A33 antigen diffusing from tumor cells to the vascular system; (2) mAb A33 is rapidly internalized into the cell once it binds to A33 antigen on the cell membrane, thereby increasing the amount of cell associated antibody; and (3) some colon cancer cell lines express large amounts of A33 antigen, binding up to 800,000 mAb A33 molecules per cell. Due to these properties, a need exists to isolate, characterize and sequence the A33 antigen, as well as related proteins with similar characteristics.
Many purification protocols typically utilize reduction steps in order to analyze proteins of interest by SDS-gel electrophoresis. In this way, proteins can be identified and monitored more easily. The inventors of the instant application found that surprisingly, by utilizing reducing conditions, they were unable to identify the target A33 protein by Western blotting. Standard techniques had to be changed so as to completely remove reducing steps in order to identify, monitor and characterize the A33 antigen of the invention. Once the antigen was isolated, studies on its behavior under reducing conditions could be carried out.
Purification of the A33 antigen has been further complicated by co-migration of other proteins, including actin, to about the same position on one and two dimensional gel electrophoresis. In addition, mAb A33 binds non-specifically to actin. The inventors of the instant application identified the Fc. regipon of the antibody as being responsible for the non-specific binding to actin. Removal of the Fc region has allowed the inventors to prevent actin binding. As actin is not a cell surface antigen on colon carcinoma cells, and is not sensitive to reduction, it became clear to the inventors that actin could not be the target for monoclonal antibody A33.
The difficulty in identifying, isolating and characterizing this antigen is evidenced by the fact that although the existence of the A33 antigen has been known for more than a decade, this is the first successful purification, isolation and sequencing of the antigen.
As described herein, the inventors of the instant application have identified, isolated and characterized the A33 antigen. The inventors have also isolated cDNA encoding the A33 antigen, determined the nucleotide sequence of the cDNA, and deduced the amino acid sequence for the A33 antigen. The A33 antigen, also referred to herein as the A33 protein, can be utilized to develop clinical reagents and methods useful in the prognosis, diagnosis and treatment of cancer and other diseases, in particular, cancers such as colon, rectum, gastric and small bowel mucosa cancer.
SUMMARY OF THE INVENTION
This invention is directed to an isolated protein which is found inside and on the surface of normal human colon cells and human colon cancer cells, as well as to peptide fragments of said protein. The protein and peptides are bound by the A33 colon cancer antibody or by polyclonal antibodies raised against regions of the protein sequence. When analyzed by SDS gel electrophoresis, the isolated glycoprotein of the invention has a molecular weight of about 40-45 kD, when non-reducing conditions are utilized and about 49-55 kilodaltons under reducing conditions. This invention further relates to nucleic acid molecules encoding said protein, and to the use of said glycoprotein, peptides and nucleic acid molecules in the diagnosis and treatment of cancer.
REFERENCES:
Welt et al., J. Clin. Oncol. vol. 12, pp 1561-1571, 1994.*
King et al., B. J. Cancer, vol. 72, pp 1364-1372, 1995.*
Daghighian et al, J. Nucl. Med. vol. 37, pp 1052-1057, 1996.
Burgess Antony
Catimel Bruno
Heath Joan
Ji Hong
Johnstone Cameron
Fulbright & Jaworski LLP
Jones W. Gary
Ludwig Institute for Cancer Research
Souaya Jehanne
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