WO2000026672A1

WO2000026672A1 - Diagnosis of breast cancer using antibody against cadherin-11

Info

Publication number: WO2000026672A1
Application number: PCT/US1999/025302
Authority: WO
Inventors: Stephen W. Byers; Carolyn M. Feltes; Michael Pishvaian
Original assignee: Georgetown University
Priority date: 1998-10-30
Filing date: 1999-10-27
Publication date: 2000-05-11

Abstract

Cadherin-11 is described as a marker for invasive breast cancer cells. Methods are provided that utilize anti-cadherin-11 monoclonal antibodies for the diagnosis, imaging, and treatment of breast cancer.

Description

DIAGNOSIS OF BREAST CANCER USING ANTIBODY AGAINST CADHERIN-1 1

Field of the Invention

The present invention relates to the diagnosis, imaging, and treatment of breast cancer. In particular, this invention relates to the diagnosis, imaging and treatment of breast cancer using antibody products specific for cadherin-11.

Background

Metastasis is a key event in the course of tumor progression in breast cancer. During the transition to invasive carcinoma, tumor cells detach from the primary tumor, invade the epithelial membrane, penetrate the underlying stroma, mix with the stromal cells, and further disseminate through the blood and lymphatic system to distant organs. The process of metastasis involves perturbations in both cell-cell and cell- matrix interactions with associated changes in expression of cell adhesion molecules such as cadherins.

Cadherins are transmembrane molecules that mediate calcium dependent cell- cell adhesion which is essential for the maintenance of tissue integrity and cell polarity. In breast cancer cell lines loss of expression of E-cadherin, one of the classical cadherins, is observed as cells become more invasive and less differentiated and is also associated with poor clinical prognosis. While the loss of E-cadherin expression appears to be one of the early events associated with the initiation of metastasis, it is not the sole factor.

Recent work has demonstrated the presence of other cadherins in pre-cancerous or fibroblast cells. For example, in spite of the lack of E-cadherin expression, most invasive cells still exhibit some calcium dependent adhesion which suggests expression of at least one additional member of the cadherin family.

Doctors making decisions on how to treat cancer patients require techniques to detect invasive cells, both at the time of initial diagnosis and during the course of the disease. The presence of such cells affects the prognosis and directs the extent and type of therapy. The grading of cells in the clinical prognosis of cancer, includes observing cells from tissue samples obtained by needle biopsy for irregularity in nuclear size and shape, the number of hyperchromatic nuclei and mitotic frequency, and the extent of tubule formation. However, the error rate in identifying or failing to identify invasive cancer cells is high because visual observation of cells is subjective.

One focus of research in the cancer field has been to develop radiolabeled monoclonal antibodies capable of binding to cell surface markers specifically expressed by invasive cancer cells. Imaging cancer cells using such antibodies would be a more objective measure than visual observation of invasive cells. There thus exists a need in the art for identification of cell surface markers expressed by invasive cancer cells.

Brief Summary of the Invention

The present application demonstrates that cadherin- 11 is expressed by invasive and poorly differentiated breast cancer cell lines. Cadherin-11 was shown to be localized to the cell membrane and in such a location may facilitate tumor cell invasion and metastasis. These findings indicate that cadherin- 11 can function as a marker for invasive cancer cells.

The present invention therefore relates to the diagnosis, imaging, and treatment of breast cancer. In particular, the invention relates to the diagnosis, imaging and treatment of breast cancer using antibodies specific for cadherin-11.

In one aspect, an anti-cadherin-11 antibody product conjugated to a detectable moiety is provided. The antibody product may be a monoclonal antibody, a cadherin-

11 binding antibody fragment, or a single chain antibody. The antibody products may be humanized by procedures commonly known to those skilled in the art. The term "detectable moiety" as used herein refers to chemicals, enzymes, stains or proteins that provide a means for visualizing the antibody product conjugated thereto. Standard methods for conjugating antibody products to detectable moieties exist in the art.

Examples of detectable moieties are fluorescent chemicals, radionuclides, dyes, enzymes, or chromophoric chemicals. A preferred radionuclide is technetium-99m.

Antibody products specific for cadherin- 11 may be generated by standard methods in the art. Cadherin-11 itself is described in U.S. Patent No. 5,597,725 issued September 28, 1997 to Shintaro Suzuki, which is hereby incorporated by reference in its entirety. Antibodies to cadherin-11 are also sold by various companies. According to another aspect of the invention, a method of detecting invasive breast cancer cells is provided. The term "detecting" as used herein refers to visualization as well as the diagnosis. In the method, cells to be evaluated are contacted with an antibody product of the invention conjugated to a detectable moiety and reactions between the cells and the antibody product are observed. Binding of the detectable antibody product to the cells indicates that the cells are invasive. The method may be practiced in vitro on cells samples such as those obtained by needle biopsy or may be practiced in vivo using, for example, gamma cameras.

In yet another aspect of the present invention, a kit comprising the antibody product and a detectable moiety is provided. In the kit, the antibody may or may not be already conjugated to the detectable moiety.

In another aspect of the invention, anti-cadherin-11 antibody products may be used as targeting molecules for agents that cause cancer cell death. Agents contemplated by the invention include, for example, radioactive isotopes such as In¹¹¹, I¹²⁵, or I¹³¹; agents which bind DNA such as alkylating chemicals or various intercalating antibiotics; antimetabolites such as methotrexate; agents which act on cells surfaces such as venoms and toxins; and protein synthesis inhibitors such as diptheria toxin. Compositions comprising anti-cadherin-11 antibody products and such agents in a pharmaceutically acceptable diluent, adjuvant, or carrier are provided. The amount of the anti-cadherin-11 antibody may be in the range of about 1 mg/ml to about

10 mg/ml. The compositions may be used in a method of treating breast cancer in which the composition is administered to a patient in need of such treatment.

Additional features and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.

Detailed Description of the Invention

Example 1 reports the expression of cadherin-11 in invasive fibroblastoid cell lines. Example 2 describes methods of detecting invasive breast cancer cells using labeled anti-cadherin-11 antibody products of the invention. Example 3 describes the treatment of human breast cancer using combinations of anti-cadherin-11 antibody products and other agents. Example 1 The expression of cadherin-11 in breast cancer cell lines was investigated. Cadherin-11 had been previously found to be expressed only in embryonic mesenchymal tissues, osteoblasts, and invasive tumors of the stomach and the kidney. In seven breast cancer cell lines that were examined in this invention, five of the cell lines expressed cadherin-11 and also a splice variant of cadherin-11. This variant which is described in Okazaki et al, J. Biol. Chem., 269: 12092-19098 (1994) and Kools et al, Clin. Exp. Metas., 14: 52 (1996) arose from a 179 bp insertion that results in a protein that lacks the majority of the wild-type (WT) cytoplasmic domain, including the catenin-binding regions. The expression of this variant has been associated with invasive tumors.

The expression of cadherin-11 in breast cancer cell lines was examined by RT- PCR. Two sets of primers were used: the first amplified a region that encodes the extracellular domain of cadherin-11 and thus would recognize both the wild type and variant cadherin-11; the second set amplified only a portion of the variant cadherin-11. β-actin primers were used as a experimental control. MRC-5, a human embryonic lung fϊbroblast cell line which has been shown to express cadherin-11 was used as a positive control. Cadherin-11 and variant mRNA were expressed in five invasive cell lines, MDA-MB-157, MDA-MB-231, BT549, HS578T, and MCF-7^. In MCF-7_ADR cells, which are considerably less invasive than the others, the variant is expressed at low levels. Two invasive cell lines, MDA-MB-436 and MDA-MB-435 did not express cadherin-11 nor did any of the non-invasive cell lines.

These results were confirmed by Northern blot using a 1.6 kb fragment of cadherin-11 cDNA as a probe. The probe identified a 4.4 kb band from the same five cell lines. MDA-MB-157 and MCF-7_ADR cells express much lower levels of cadherin-

11 mRNA than MDA-MB-231, BT549 or HS578T cells, despite the presence of similar levels of total RNA and GAPDH expression.

In order to elucidate cadherin-11 protein expression, 10 μg of protein from a detergent lysate was run on a polyacrylamide gel, and the subsequent blot was probed with an anti-cadherin-11 monoclonal antibody (mAb) (ICOS Corporation ). This mAb recognizes the extracellular portion of cadherin-11 and should recognize both WT and variant protein. In Western blots, cadherin-11 runs as an approximately 120 kDa band. The cell lines which made cadherin-11 mRNA also expressed cadherin-11 protein. MDA-MB-157 and MCF-7_ADR cells do express cadherin-11 protein, but at very low levels. The putative variant protein would be approximately 7 kDa smaller than the wild type, however a smaller band could not be definitively identified on this Western blot. The Western blot was reprobed several times to examine the expression of other adherens junction proteins. Interestingly, an anti-E-cadherin Western blot demonstrated that E-cadherin and cadherin-11 are never co-expressed. Finally, most cell lines expressed -catenin and β-catenin. To determine whether cadherin-11 interacts with α-catenin and β-catenin in vivo, confluent cells were lysed in an NP40 buffer, precleared lysate was immunoprecipitated with polyclonal antibodies to either α-catenin or β-catenin. Western blots were performed first with the anti-cadherin-11 mAb, then with a pancadherin polyclonal, then with a mAb to E-cadherin, and finally to antibodies to α- catenin (polyclonal) or β-catenin (monoclonal). Immunoprecipitation with rabbit IgG as a non-immune control reveals no immunoreactive bands in any of the Western blots. The Westerns confirmed that the same five cells lines express cadherin-11 and that in all five cases cadherin-11 is associated with both α-catenin and β-catenin (except in MDA-MB-157 cells with lack α-catenin). Additional experiments were performed to determine that cadherin-11 protein was primarily found in an NP-40 insoluble pool indicating it is membrane-bound and non-cytoskeletally associated and that it localized in the membrane to sites of cell-cell contact (which is typical of cadherin family members) but was also found in lamellapodia-like extensions and regions of contact with substrate. These results suggest that cadherin-11 has several functions. One function is a form of cell-cell adhesion that involves the cytoplasmic catenins, but is not associated with the cytoskeleton. Another is contacting the cell-matrix, particularly in leading extensions of the cell. Such a complex would be much more transient and could facilitate the ability of a motile cell to interact with its surroundings, which could include both matrix proteins, as well as other mesenchymal cells. Significantly, the expression of cadherin-11 in the tumor cells appears to allow interactions with the underlying stroma and facihtates invasion. In fact, cadherin-11 may act to specifically target metastatic tumor cells to sites which express cadherin-11 such as facilitating association of metastatic cells with osteoblasts in the bone allowing establishment of a secondary tumor in the bone. Thus, unlike other cadherins, such as E-cadherin, which have invasion suppressor function cadherin-11 expression apparently enhances tumor cell invasiveness.

Cadherin-11 is therefore expressed in highly invasive and poorly differentiated breast cancer cell lines that lack E-cadherin expression. This indicates that cadherin-11 is a very specific marker for only the most invasive subset of tumors cells. As a molecular marker, it has the potential to identify highly malignant tumors that would require aggressive therapy.

Example 2 The antibody products of the invention are useful for detecting invasive breast cancer cells in vitro and in vivo. Anti-cadherin-11 antibody products are purified by techniques generally known in the art such as ammonium sulfate precipitation followed by dialysis then filtration sterilization or immunoaffinity chromatography.

For detection of invasive breast cancer cells in vitro, sections from a tissue biopsy are incubated with the antibody product and then washed. Bound antibody is visualized using, for example, horseradish peroxidase conjugated goat antimouse immunoglobulin (KPL, Gaithersburg, MD) and peroxidase activity is measured upon addition of diaminobenzidine hydrochloride (Sigma, St. Louis, MO). The art is replete with alternative visualization techniques (e.g. , immunofluorescence techniques).

For detection of invasive breast cancer cells in vivo, antibody products are conjugated to a detectable moiety. Presently preferred is the detectable moiety technetium-99m (99mTc). 99mTc pertechnetate is reduced with dithionite in the presence of the ligand to form 99mTc 4,5-bisthioacetamide pentanoate. The 99mTc complex is esterfied with 2,3,5,6-tetrafluorophenol using carbodiimide. The active ester of the complex is reacted with the antibody product to produce a 99mTc antibody product conjugate. The conjugate is then purified by gel filtration. The conjugate is administered to a patient by i.v. injection. Planar images of the patient are obtained immediately and at 3 and 8 hours postinfusion on a gamma camera and images are acquired and stored for processing. The images are then analyzed for changes in normal organ and tumor concentrations of activity. Both in vitro and in vivo, the amount of binding to cancer cells correlates with the invasive potential of the cells.

Example 3 The anti-cadherin-11 antibody products of the invention can be used in combination with a broad spectrum of agents to treat breast cancer by using the antibody products as targeting molecules. Combinations with agents such as radioactive isotopes In¹¹¹, I¹²⁵, or I¹³¹; agents which bind DNA such as alkylating chemicals or various intercalating antibiotics; antimetabolites such as methotrexate; agents which act on cells surfaces such as venoms and toxins; and protein synthesis inhibitors such as diptheria toxin are presently preferred. The method of linkage of the agent to the antibody product can be either covalent or non-covalent. Chemicals like carbodiimide can be used to covalently link the carboxy groups of a polypeptide agent to amino groups of the antibody product.

Bifiinctional agents such as dialdehydes or imidoesters can be used to link the amino group of a drug to amino groups of the antibody product. The Schiff base reaction can be used to oxidatively link the drug to the antibody product. Similarly, glycosidic enzymes can be conjugated to the antibody product to increase the binding effect of the enzymes.

Compositions comprising the antibody product and agent are then admimstered to patients in an amount cytotoxic to cancer cells. While the invention is described in terms of examples and preferred embodiments, it will be apparent to one of ordinary skill in the art that modifications and alternatives exist and can be practiced without departing from the spirit or scope of the following claims.

Claims

ClairoNWe claim:

1. A method of detecting invasive cancerous breast cells which comprises contacting cells with an anti-cadherin-11 antibody product conjugated to a detectable moiety and observing binding between the antibody product and cells.

2. The method of claim 2 wherein the detectable moiety is selected from the group consisting of fluorescent chemicals, radionuclides, dyes, enzymes, and chromophoric chemicals.

3. A kit for detecting invasive cancerous breast cells comprising an anti- cadherin-11 antibody product and a detectable moiety.

4. The kit of claim 5 wherein the detectable moiety is selected from the group consisting of fluorescent chemicals, radionuclides, dyes, enzymes, and chromophoric chemicals.