Antibodies as a cancer diagnostic

a cancer and antibody technology, applied in the field of metastatic disease diagnosis, can solve the problems of little known about epha2 function, and achieve the effects of reducing cell-cell contact, increasing interaction, and reducing cell adhesion

Inactive Publication Date: 2007-07-12
PURDUE RES FOUND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004] Various cancer cells, including breast cancer cells, are known to exhibit altered cell adhesion. As compared to normal breast epithelia, transformed human breast epithelial cells have decreased cell-cell contacts and increased interactions with the surrounding extracellular matrix. These changes facilitate increased detachment and migration of cancer cells away from cell colonies and are directly linked with alteration in tyrosine phosphorylation of cell membrane proteins. Tyrosine phosphorylation is a potent form of cell signal transduction, and alteration in levels of tyrosine phosphorylation is believed to be important for tumor cell invasiveness. Thus, regulation of tyrosine phosphorylation represents a promising target for therapeutic intervention against metastatic cancer. Tyrosine phosphorylation is controlled by cell membrane tyrosine kinases, and increased expression of tyrosine kinases is known to occur in metastatic cancer cells.

Problems solved by technology

Although cloned a decade ago, see Lindberg, R. A. and Hunter, T., “cDNA Cloning and Characterization of Eck, an Epithelial Cell Receptor Protein-tyrosine Kinase in the Eph / elk Family of Protein Kinases,” Mo. Cell. Biol. 10 (12), 6316-6324 (1990), rather little is known about EphA2 function, largely because EphA2-specific antibodies previously have been difficult to generate.

Method used

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Examples

Experimental program
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example 1

[0028] The antibodies produced by the D7 hybridoma are used to detect differential expression of EphA2 between normal prostate epithelial cells and metastatic cells. FIG. 1 shows EphA2 expression in various human prostate cell lines. Referring first to FIG. 1A, three metastatic cell lines, LNCAP, DU145, and PC3, are tested for levels of EphA2 expression. It is known that, of these three cell lines, LNCAP is the least invasive, DU145 is somewhat more invasive, and PC3 is the most invasive. EphA2 expression is determined by western blotting with D7 antibodies. As can be seen in FIG. 1A, EphA2 expression positively correlates with invasiveness.

[0029] In FIG. 1B, D7 antibodies are used to test EphA2 expression in normal MLC cells as compared to expression in transformed cells. Normal MLC cells, MLC cells which have been transformed by K-Ras, and MLC cells with have been transformed by X-irradiation are studied. As can be seen in FIG. 1B, EphA2 is overexpressed in both of the transforme...

example 2

[0030] EphA2 antibodies are used to detect altered EphA2 expression in metastatic mammary cells. EphA2 is expressed in normal mammary epithelial cells. FIG. 2 illustrates altered EphA2 expression in mammary tumor cell lines. As can be seen in FIG. 2, western blots from whole cell lysates using D7 antibodies reveal that EphA2 expression is completely absent in cells derived from non-metastatic breast tumors (ZR75-1, BT474, SKBR3, MDA-MB-435). By contrast, EphA2 is overexpressed in metastatic breast cancer cell lines (MDA-MB-435, MDA-MB-231). Thus, EphA2 antibodies detect altered EphA2 expression in breast cancer cells, which can be used to diagnose metastasis. Moreover, in non-metastatic breast epithelial cells, loss of EphA2 occurs early in the disease, and testing with EphA2-specific antibodies provide information relevant to invasiveness even when other known markers remain normal. Thus, D7 antibodies are useful as a diagnostic, even in early stages of disease.

example 3

[0031] EphA2 antibodies in combination wvitlh other antibodies are used to detect further alterations in EphA2 expression. As discussed above in Example 2, wvesterni blots using D7 can distinguish between non-metastatic and metastatic tumors, with non-metastatic tumors failing to express EphA2, and metastatic cells overexpressing EphA2. However, different results are found when tyrosine phosphorylation is studied. Using a phosphotyrosine-specific antibody, it has been found that EphA2 is phosphorylated in normal cells, but it is not phosphorylated in metastatic cells. Thus, while EphA2 specific antibodies can qualitatively detect a difference between metastatic and non-metastatic mammary tumor cells, diagnostics incorporating both an EphA2-specific antibody and a phosphotyrosine-specific antibody provides a sensitive test for distinguishing between normal, non-metastatic, and metastatic mammary cells.

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Abstract

Method and kits are provided for the detection and diagnosis of metastatic disease. More particularly, the methods and kits employ compounds that can detect EphA2, a specific epithelial cell tyrosine kinase that is overexpressed in metastatic tumor cells. In one embodiment the compound is an antibody capable of binding to an epitope of EphA2.

Description

RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 60 / 149,259, filed Aug. 17, 1999, which is expressly incorporated by reference herein.FIELD OF THE INVENTION [0002] The present invention relates to diagnosis of metastatic disease. More particularly, this invention relates to reagents that can detect a specific epithelial cell tyrosine kinase that is overexpressed in metastatic tumor cells. Most particularly, this invention relates to reagents which bond to an intracellular epitope of the epithelial cell tyrosine kinase, and the use of these reagents for cancer diagnosis. BACKGROUND AND SUMMARY OF THE INVENTION [0003] Cancer cell metastasis requires cellular capacity to 1) detach from a primary tumor, 2) migrate and invade through local tissues, 3) translocate to distant sites in the body (via lymph or blood), 4) colonize a foreign site, and 5) grow and survive in this foreign environment. All of these behaviors ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/574
CPCC12Q1/6886C12Q2600/112C12Q2600/158G01N33/57407G01N33/57415Y10S436/813G01N33/57423G01N33/57434G01N33/57438G01N33/57496G01N33/57419
Inventor KINCH, MICHAEL S.ZANTEK, NICOLE D.
Owner PURDUE RES FOUND INC
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