AU2006202023B2 - Treatment of metastatic disease - Google Patents

Description

12-MAY-2006 17:37 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 S-1- To TREATMTNT OF METASTATIC DISEASE Field of the invention SThe present invention relates to diagnosis and treatment of metastatic Ci 5 disease. More particularly, this invention relates to the use of an epithelial cell tyrosine kuiasc that is overexpressed in movtutatiu tumor cells as the target for the diagnosis and treatment of mtastatic disease. Most particularly, this invention Cc~ Ci relates to the use of compounds that interact with and alter expression of the epithelial C< cell tyrosine kioase.

C SBackgr.ound and Summary of the Invention o Cancer is a disease of aberrant signal transduction. The most dangerous forms of cancer are malignant cells that metastasize to distant sites in a body. Metastatic cells have acquired the ability to break away from the primary tumor, translocate to distant sites, and colonize distant and foreign microcnvironments- Cancer cell metastasis requires cellular capacity to I) detach from a primary tumor, 2) migrate and invade utough 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 are linked to cell adhesions. Cell adhesions control the physical interactions of cells with their microenvironment. Cell adhesions also initiate signals that dictate tumor cell growth, death, and differentiation. At the cellular level, metastatic cells have overcome restraints upon cell growth and migration that result from physical linkages and signals conveyed by cell-cell contacts. Malignant cells often have increased interactions with surrounding extracellular matrix (ECM) proteins that provide linkages and signals that promote several aspects of metastasis.

Levels of protein tyrosine phosphorylation regulate a balance between cell-cell and cell-ECM adhesions in epithelial cells. Elevated tyrosine kinase activity weakens cell-cell contacts and promotes ECM adhesions. Alteration in levels of tyrosine phosphorylation is believed to be important for tumor cell invasiveness.

Tyronine phosphorylation is controlled by cell membrane tynrsinc kinases, and increased expression of tyrusine kinases is known to occur in metastatic cancer cells.

COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:38 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.6

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0 -2- EphA2 is a 130 kDa receptor tyrosine kinase that is exprcssed on adult C epithelia. A member of the Eph family of tyrosine kinases known as Ephrins, EphA2 is a transmembranc receptor tyrosine kinase with a cell-bound ligand. EphA2 expression has been found to be altered in many metastatic cells, including lung, C 5 breast, colon, and prostate tumors. Additionally, the distribution and/or 0 phosphorylation of EphA2 is altered in metastatic cells. Moreover, cells that have o been transformed to overexpress EphA2 demonstrate malignant growth, and sO stimulation of EphA2 is sufficient to reverse malignant growth and invasiveness.

0 EphA2 is a powerful oncoprotein. The present invention is directed to compounds and methods that target EphA2 for the diagnosis and treatment ofnmetastatic cancers.

One approach to cancer therapy is the administration of proformed antibodies to predetermined tumor antigens. This process is known as passive antibody treatment. An example of passive antibody treatment is the use of Herceptin® for the treatment of breast cancer. Herceptin is a humanized form of a murinT monoclonal antibody specific for the extracellular domain ofHer2/Neu. The basis for trealment with Herceptine is that 25-30% of melastatic breast cancers overexpress the Her2/Neu receptor tyrosine kinase. Herceptinr has been well tolerated in clinical trials and shows much promise for the maintenance and regression of metastatic breast cancer.

Effective passive immunotherapy for treatment of tumors requires isolation and preparation of an antibody that: 1) targets an antigen that is overexpressed in metastatic tumors; 2) targets an extracellular upitope of said antigen; 3) is not cross-reactive with any other antigen in a patient's circulation; and 4) exhibits tumoricidal or tumoristatic activity.

In a preferred embodiment, this invention relates to the selection and use of antibodies that are specific to an extracellular epitope of EphA2. The methods of this invention include the preparation, selection, and use of EphA2 specific antibodies for cancer therapy.

Another approach to cancer treatment is to use agonists to stimulate expression. For example, EphrinAl-F,, the extracellular domain of ephrinAl linked to immunoglobulin heavy chain (see Mino, et al., EphA2 kinase associates with focal adhesion kinase and upon activation, inhibits integrin-mediated cell adhesion COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:38 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.7/28

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-3and migration. Nature Cell Biol 2. 62-69 (2000)), can be used to increase the C phosphotyrosine content of EphA2. Thus, in another preferred rnbodiment, this invention relates to the use ofagonists or antagonists to alter the expression of EphA2 in mclastatic cells.

5 Thus, this invention is directed to the use of agonists and antagonists to C' alter the expression ofEphA2. EphA2 may he targeted by use of artificial or hybrid Cl forms of the protein, protein inhibitors, antisense oligonucleotidcs, or small molecule Is0 o inhibitors. Also, while a preferred embodiment is directed to use of monoclonal C antibodies, polyclonal, artificial, and hybrid antibodies are known in the art. It should be understood that use oftechniques known in the art to target EphA2 are within the scope of this invention.

One aspect of this invention is a pharmaceutical composition for the trlatment of mammalian metastatic tumors which overexpress EphA2, comprising a compound that specifically interacts with an extracellular epitope of EphA2 in an amount effective to reduce metastatic disease and a pharmaceutically acceptable carier. In the preferred embodiment, the pharnaceutical composition comprises the antibody 12D6, an antibody that specifically binds to an extracellular epitope of EphA2 Another aspect of this invention is a method of treating a patient having a metastatic tumor which overexpresses EphA2. The method comprises administering to the patient a therapeutic amount of a compound that binds to an exrraccllular epitope of EphA2. In a preferred embodiment, the compound is an antibody.

A third aspect of this invention is a method for detecting the presence of lmtastatic cells. The method includes use of a labeled antibody specific to an extracellular cpitope of EphA2. A ccll sample is incubated with the antibody, unbound antibody is removed, and the bound labeled antibody is detected.

An additional aspect of this invention is a method for producing antibodies which inhibit metastatic tumor proliferation by specifically bioding to an extracellular epitope of EphA2. This method includes injecting tyrosine phosphorylated proteins into the lymph nodes of a mammal, harvesting the lymph COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:38 From:WYNNES PAT&TM RTT 0733421292 To:0262837999 P.81128

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0 0 -4nodes, fusing the lymph node cells with mycloma cells to form hybridomas, and Ci selecting hybridomas which produce antibodies specific for EphA2.

In still another aspect of this invention, a pharmaccutical composition for treatment of a mammalian metastatic tumor is provided, the composition Ci 5 comprising a compound that alters expression of EphA2 in an amount effective to C reduce metastatic proliferation of said tumor, and a pharmaceutically acceptable Scarrier Lherefor. in a preferred embodiment, the composition comprises an ephrin.

IO Additional features of the present invention will become apparent to o those skilled in the art upon consideration of the following detailed description of preferred cmbodiments exemplifying the best mode of carrying out the invention as presently perceived.

Brief Description of the Drawings Fig. 1 is an overview of the RMMS procedure, through which the antibodies of this invention are generated; Fig. 2A-C show a scries of western blots showing EphA2 expression in human cell lines; Fig. 2A is a western blot showing EphA2 expression in various human prostate cancer cell lines; Fig. 2B is similar to Fig. 2A, except showing EphA2 expression in a human prostatic opithelial cell line and expression in the cell line after transformation by oncogenic K-Ras or X-i.rradiation; Fig. 2C is similar to Fig. 2B, except showing EphA2 exprcssion in another human prostatic epithelial cell line and expression in the cell line after transformation by oncogenic K-Ras or X-irradiation; Fig. 3 is similar to Fig. 2, except showing EphA2 expression in canine prostatic cancer cells; and Fig. 4 shows predicted antibody binding plotted against cell density in a screening procedure for antibodies which are specific for an extracellular epitope of EphA2.

COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-20B6 17:39 From:WYNNES PRT&TM ATT 0733421292 To:0262837999 P.9/28 SDetailed Description of the Tnvention SEphA2 is expressed differently in normal and metastatic cells. In normal breast and prostate epithelial cells, EphA2 is enriched in within sites of cell adhesion. Conversely, in metastatic prostate cells EphA2 is diffusely distributed, and C 5 in metastatic breast cancer cells EphA2 is redistributed into the membrane ruffls.

F.phA2 expression is also known to he altered in lung and colon malignancies, and it is believed that EphA2 altered expression occurs in other types of metastasis, C particularly epithelial malignancies. Thus, techniques designed to alter EphA2 expression can be exploited to diagnose and treat metastatic disease.

In a preferred embodiment, antibodies specific for tyrosine 0 phosphorylated proteins in cancer cells have been isolated and used to target cancer o cells in passive immunotherapy. This approach is based upon the fact that many tyrosine kinases, Her2/Neu, are expressed by oncogenes and are therefore overexpressod in cancer cells. The present invention is directed to the production and use of antibodies capable of recognition of and specific binding to extracellular epitopes of the tyrosine kinase EphA2. The antibodies are produced by selected hybridomas, themselves the product of fusion of mycloma cells with lymph node cells harvested from animals subjected to a specific inoculation protocol designed for increased sensitivity and diversity of responding hybridomas.

To produce these hybridomas, tyrosine phosphorylated proteins from Ras-transformed human epithelial cells were isolated by aflinity chromatography using existing phosphotyrosine specilic antibodies. The tyrosine phosphorylated proteins are then used as an immunogen for producing monoclonal antibodies according to the procedure illustrated in Fig. 1. Low-dose amounts of tyrosine phosphorylated proteins are injected proximal to lymph nodes of a mammal, every other day, over a ten day period (the RIMMS strategy). B cells from engorged lymph nodes arc then isolated and fused with Bcl-2-overexpressing myeloma cells, to minimize apoptosis after fusion. This method results in increased diversity, specilicity, and cost-effectiveness of hybridoma production. The hybridomas are screened to identify those hybridomas producing antibodies that distinguish malignant from normal cancer cells.

COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:39 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.10/28 -6- Ilybridomas producing antibodies specific to EphA2 have been C selected. Use of the RIMMS technique has resulted in the production of a multiplicity ofhybridomas producing monoclonal antibodies that specifically bind EphAZ. To date, at least 450 hybridomas have been identified which produce Cl 5 antibodies capable of distinguishing malignant from normal cancer cells. Of the first four such hybridomas to be chracterized, two recognize independent opitopes on EphA2. The first, D7, produces an antibody recognizing an intracellular cpitope. The C second, B2D6, produces an antibody that specifically binds an extracellular epitope of C" EphA2, a characteristic that enables its effective use for the diagnosis and treatment C' 10 of selected metastatic tumors.

\O While the RIMMS strategy has proven to be valuable in the production O of EphA2 specilic antibodies, other techniques are known in the art for producing antibodies to a specific antigen, and these techniques are within the scope of this invention.

It is known in the art to use antibodies to detect the presence or overexpression of a specific protein. Because EpbA2 is ovcrexpressed in metastatic cells, EphA2-specific antibodies of this invention may be used to detect this overexpression and, thus, to detect metastatic disease. Such techniques include but are not limited to western blotting, precipitation, agglutination, and ELISA assays.

These techniques are well known in the art. Also, the extracellular epitope specificity of EphA2-specific antibodies of this invention can be exploited to detect changes in EphA2 localization which are associated with metaslasis. In normal breast and prostate epithelial cells, EphA2 is enriched in within sites of cell adhesion, whereas in metastatic cells, EphA2 distribution is altered. In metastatic prostate cells EphA2 is diffusely distributed, and in metastatic breast cancer cells EpbA2 is redistributed into the membrane ruffles. EphA2 expression is also known to be altered in lung and colon malignancies, and it is believed that EphA2 altered expression occurs in other types of metastasis, particularly epithelial malignancies Techniques such as immunuhistological staining or immunofluorescent microscopy are well known in the art and may be used to visualize EphA2 distribution. See, for example, U.S. Patent No. 5,514,554. In order to detect overexression or altered distribution of EphA2, the EphA2-specific antibodies may be labeled covalently or non-covalently with any of a COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:39 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.11/2e -7-

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O number of known detectable labels, such fluorescent or radioactive substances, as is CA known in the art. Alternatively, a secondary antibody specific tor the antibodies of this invention is labeled with a known detectable label and used to detect the EphA2bfspeci antibodies in the above techniques. Thus, the antibodies of this invention Cl 5 provide methods to detect metastatic transformation.

The present invention also employs antibodies specfic for an extracellular epitopc of EphA2 in therapeutic compositions and methods for use.

C, When used for in vive therapy, a pharmaceutical composition administered to a 0 patient comprises EphA2-specific antibodies in therapeutically effective amounts in a 0 10 pharmaceutically acceptable carrier. In a preferred embodiment, the EphA2-specifie NO antibodies have been "humanized." Humanized antibodies include "chimeric o antibodies" made by splicing genes from a mouse (or other mammal) antibody of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. Such techniques are known in the art. See, for example, U.S. Patent No. 5,811,098. Tn addition to antibodies, natural or artificial I.igands, peptides, anti-sense, ATP analogues, or other small molecules capable of specifically targeting EphA2 may be employed.

An example of another way to target EphA2 is the use of ephrins to activate or inhibit EphA2. For example, EphrinA the extracellular domain of ephrinAl linked to immunoglobulin heavy chain, increases the phosphotyrosine content of EphA2. EphrinAl-F, reverses the malignant behavior of EphA2 transformed cells. Thus, another preferred embodiment of this invention is a pharmaceutical composition comprising an cphrin or a hybrid form ofephrin administered in a therapeutic amount.

Therapeutic amounts are amounts which eliminate or reduce the patient's tumor burden, or which prevent or reduce the proliferation of metastatic cells. The dosage will depend on many parameters, including the nature of the tumor, patient history, patient condition, the possible co-use of other oncolytic agents, and methods of administration. Methods of admiistration include injection parenteral, subcutaneous, intravenous, intraperitoneal, etc.) for which the antibodies are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:40 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.12/28 -8-

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O oleate, or liposomes. Typical dosages may range from about 0.01 to about 20 mg/kg, C and more particularly from about 0.1 to about 10 mg/kg. Other methods of administration include oral and transdermal. Acceptable carriers for oral ingestion in accordance with the present invention can be formulated using art-recognized C( 5 techniques into pharmaceutically acceptable liquid carriers or in combination with pharmaceutically acceptable solid carriers in the form of tablets, capsules, caplets, or gel-seals. Other effective methods of administration and dosages may be determined (C by routine experimentation and arc within the scope of this invention.

Therapeutic methods employing EphA2-specific antibodies may be combined with chemotherapy, surgery, and radiation therapy, depending on type of IN the tumor, patient condition, other health issues, and a variety of factors. The o methods may also include immunoconjugates for targeted immunotoxin-mediated therapy, wherein antibodies of this invention ae covalently or non-covalently conjugated to various cytotoxic agents, further enhancing toxicity to targeted cells.

See, for example, U.S. Patent No. 5,872,223. Such agents, including various bacterial toxins Pseudomunas exotoxin), ricin A-chain, daunorubicin, methotrexate, and ribosome inhibitors trichosantin). Also, the antibodies of this invention may be labeled with alpha, beta, or Auger electron emitters, resulting in immunoconjugates for targeted radiotherapy.

Thus, EphA2-specfic antibodies may be used in a variety of methods and compositions for detecting and treating metastatic disease.

EXAMPLE 1 Characterization of EphA2 Expression in Metastatic Cells Following the RIMMS strategy using tyrosine phosphorylated proteins from Ras-transtbrmed human epithelial cells, hybridomas were screened, and an antibody specific for EphA2 has been isolated. This antibody was used to assess the levels of EphA2 expression in nontrafnormed prostatic epithelial cells and prostatic ttmor cells. Low levels of EphA2 expression were found in non-transformed prostatic epithelial cells, but this EphA2 expression was enriched within sites ofcellccUl contact and interacted with cell-bound ligand. Compared to non-transformed cells, two features distinguish EphA2 in metastatic prostate cancer cells: I) EphA2 is COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MRY-2006 17:40 From:WYNNES PAT&TM ATT 0733421292 To:062837999 P.13/28

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-9overexpressed; 2) EphA2 is diffusely distributed and does not appear to interact with Cl ligand. To confirm these data, western blots were performed using the EphA2 specific antibodies. EphA2 overexpression in human prostate cancer cells (LNCAP, DU145, PC3) directly correlates with their invasiveness in vitro and in vivo, Of the l 5 three lines tested, LNCAP is the least aggressive, DU145 is more aggressive, and PC3

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Cl is the most aggressive. As seen in Fig. 2, DU145 cells exhibit higher levels of EphA2 0 C expression than LNCAP, and PC3 cells exhibit even higher levels of EphA2 o expression. Similarly, as shown in Figs. 2B and 2C, EphA2 expression is elevated in o variants of human prostatic epithelial cells transformed by oncogenic K-Ras or Xirradialion. The three lanes in Fig. 2B show "normal" MCL prostatic epithelial cells.

and K-Ras and X-ray transformed cell lines derived therefrom. Similarly, the three lanes of Fig. 2C show "normal" 267B1 prostatic cpitheial cells, and K-Ras and X-ray transformed cell lines derived therefrom, As seen in Figs, 2B and 2C, the transformed cells all exhibited elevated BphA2 levels. Fig. 3 shows similar western blots, except using prostatc cancer cell lines from dogs. As shown in Fig. 3, consistent with the results from human cells, EphA2 is overexpressed in metastatic prostatic carcinoma cells derived from dogs with spontaneous prostate cancer.

rhe metastatic prostate cell lines can be subdivided into three categories: I) cells derived from primary prostate tumors; 2) cells derived from metastases that are poorly metastatic in vivo; 3) cells derived from metastases that are highly metastatic in vivo. The western blots using EphA2-specific antibodies have revealed that EphA2 expression is elevated in all cells derived from metastases, with highest EphA2 expression in cells that retain metastatic potential in vive (as assessed using athymic mouse models). Interestingly, B2D6 studies have shown that EphA2 is overexpressed in cells from prostate cancer metastases coimpared to lines established from the primary tumor of same patient. Taken together, these results all reveal EpbA2 overexpression in metastatic prostate tumor cells.

Similar EphA2 expression patterns have been found with breast cancer cells. In normal mammary epithelial cells, EphA2 is enriched within the cell-cell junctions. By contrast, non-metastatic breast cancer cells do not express EphA2, while metastatic breast cancer cells overexpress EphA2. In metastatic breast cancer COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:40 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.14/28

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cells, .EphA2 is redistributed into the membranc rmffls and, thus, is available for Santibody binding.

EXAMPLE 2 C 5 In Vitro Targeting of Metastatic Cells 0 EphA2 overexpression renders metastatic cells susceptible to antibodyo mediated selected killing with the present antibodies specific for an extracellular O epitopc of EphA2. While normal cells express EphA2, it is believed that ligand 0 binding or clustering within sites of cell-cell contact occludes extracellular epitopes in normal cells and renders them inaccessible to antibodies specific for an extracellular epitope of EphA2. The tumor selectivity of the antibodies of the present invention is believed to rival or exceed that ofHercoptinS for targeting metastatic cancer.

EphA2 overexpression provides a basis for targeting metastatic cancer cells with EphA2-specific antibodies. Antibodies specific for an extracellular epitope of EphA2, such as those produced by hybridoma B2D6, may be used to alter selectively (versus normal cells) the proliferative or invasive behaviors ofmetastadi cancer cells. In both metastasis-derived and laboratory-induced transformation, EphA2 overexpression correlates with invasivencss, whereas non-invasive cells have lower levels of EphA2 expression.

To measure the effect of 132D6 on cell growth, cells are incubated with purified B2D6, and cell proliferation is measured by counting cells microscopically (using a hemacytometer) and by measuring DNA synthesis. For example, normal growth media is supplemented with B2D6 and BrdU, and BrdU incorporation is measured over the following four hours. To measure the effects of B2D6 over longer times, samples are counted at 24 hour intervals, with BrdU added to the culture media for the final four hours of incubation. As a third measure of cell growth, the effect of B2D6 on the growth of metastatic cells in soft agar is determined. Soft agar plating assays are used, wherein 2x104 metastalic cells are plated atop agar, in the presence or absence of B2D6 (0-10 nM), and colony growth is evaluated at three-day intervals thereafter.

It is believed that B2D6 decreases the growth of metastatic cells.

Preliminary results reveal that B2D6 aggregates EphA2 and blocks about 50% of COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:41 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.15/28 0 0

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-11growth of metastatic breast cancer cells (which also overexpress EphA2) over the first Cl four hours of incubation. Although EphA2 is not tyrosine phosphorylated in metastatic breast cancer cells, tyrosine phosphorytation is restored these B2D6 treated cells. Thus, B2D6 is believed to restore normal EphA2 function.

Cl 5 Additional studies with prostate cancer cells are being performed to

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Cl determine if longer incubations with B2D6 further inhibit metastatic cell growth.

Cl 'Non-transformed epithelial cells express some EphA2, albeit much less than O metastatic cells, and some toxicity to non-transformed cells is possible. The Sminimum effective and maximun non-toxic dosage levels of antibodies in accordance with one aspect of this invention can be identified by routine experimentation, but preferably, typical doses will range from about 0.1 to about 20 mg/kg ofpatient body weight. The preferred dose will depend on many parameters, including the nature of the tumor, patient history, patient condition, the possible co-use of other oncolytic agents, and methods of administration. Antibody levels that best discriminate between normal and metastatic cells wil I be used in treatment of rlctastatic tumors overexpressing EphA2 proteins.

EXAMPLE 3 In Vitro Antibody Mediated Cytotoxicity Preliminary results demonstrate that EphA2 antibodies impede metastatic cell growth. To measure antibody-directed cytotoxicity, preferably, nonradioactive versions of Cr-rclcasc assays using target cells (normal or mctastatic prostalic pilbclial cells) labeled with europium chloride (EuClI) and diethylenetriamine-pentaacetic acid (DTPA) are performed. After washing away unincorporated Eu naphthoyltrifluoroacetone (NTA) and trioctylphosphine oxide are incubated with the cytolytic agents and assay supernatants. The luminescence of resultant ternary complex (Eu^/NTAltrioctylphosphinc oxide) is measured using a fluorescence microplate reader. The sensitivity of this Eu'-releasc assay for complement-mediated cytolysis has been reported to be fivefold better than "Crrelease assays. To determine specific lysis, parallel samples are hyponically lysed by adding distilled water. Untreated samples and isotype matched antibodies serve as negative controls.

COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MRY-2006 17:41 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.16/28

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0 -12- To model complement-mediated death in vitro, cells are labeled with B2D6 and exposed to sera that has not been heat-inactivated. For antibody dependent cellular cytotoxicity both control and B2D6-treated prostate cells are incubated with peripheral blood mononuclear cells (PBMC; 10:1 EIT ratio).

Cf 5 It is believed that complement and ADCC both promote specific 0 killing of the metaslases. Antibody dose can be varied in order to establish LDs, 0 measurements for metastatic and normal cells. Antibody concentrations that aO maximize specific killing ofmetastatic cells (PC3, 267-Ras, 267-X) while minimizing o the death to non-transformed prostatic epithclia (267, MLC) are determined by routine c l 10 experimentation. Also, ADCC can be combined with complement to further enhance tlumor killing by treating the samples with tumor necrosis factor (TNF). There is evidence that TNP potentiates the killing of tumor cells by EGFR and Hcr2-speific antibodies. It is expected that humanizing the antibodies of this invention would provide for better complement or ADCC results.

It is believed that B206 kills tumor cells through complement cascade or ADCC. However, covalent or non-covalent conjugation of the present antibodies to art-recognized cytntoxic agents can further enhance toxicity to targeted cells.

Examples of toxins appropriate for immunoconjugation include Pseudomonas exotoxin or ricin A-chain.

EXAMPLE 4 In Viw targeting of Metastatic Cells The present EphA2 antibodies, particularly those produced by hybridoma B2D6 are effective in blocking the growth and invasiveness of prostate cancer cells in vivo. The efficacy of B2D6 in blocking the growth of primary prostate tumors using subcutaneous implantation of PC3 tumor cells in mice is determined by use of subcutaneous models. The primary advantages of subcutaneous models are the ease of implantation and subsequent monitoring of tumor size. 5xl0' PC3 cells are inoculated subcutaneously into the right craniolateral thorax (axilla) using aseptic technique. Tumors are measured every 3-4 days using vernier calipers until they reach a volume of 0.2-0.3 cm'. At that time, the mice are divided into four groups (8animals each): Group 1 (vehicle control), Groups 2-4 are treated with 0.1, 1.0, or COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MRY-2006 17:41 From:WYNNES PRT&TM ATT 0733421292 To:0262837999 P.17/28 0 0

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-13mg/kg B2L)6, administered intraperitoneally, twice a week. The mice are then Cl monitored every 3 days to measure tumor volume (with vernier calipers), body weight, and life span. After no greater than 60 days past implantation, the animals are sacriliced and postmortem evaluations oftumorigencsis, including mcasurement and C 5 weight of implanted tumors and proximal lymph nodes, macroscopic evaluation of C soft tissues for tumors (lymph nodes and lung), and formalin fixation of the primary C tumor and tissues, are performed. The tissues are evaluated by o iinmiinohistochemistry using D7 (another EphA2 specific antibody that is amenahle 0 to immunohistochemistry) to determine the level of EphA2 expression in the tumors.

In particular, tumor cells that escape B2D6 treatment are studied to determine whether they have low levels of EphA2 expression. Also, EphA2 expression in the individual animals is correlated with tumor invasiveness.

As target-negative controls for specificity, parallel studies are performed using DU145 cells, which express very low levels of EphA2. Whereas the growth of PC3 tumors are believed to be sensitive to B2D6, tumors caused by DU11145 are believed be insensitive. Statistical significance of B2D6 inhibition of turnorigenicity, overall metastatic frequency, and frequency of distant metastascs is tested using computerized statistical packages (PC/SAS Ver. 6.04), with differences considered significant if p<0.05, While subcutaneous implantation is a popular and valuable method for modeling tumor cell growth, differences between the microcnvironinceat of the skin and prostate can cause rather dramatic differences in cell behavior. For example, PC3 cells rarely metastasize when implanted subcutaneously whereas intraproslatic implantation (orthotopic) facilitates metastasis. Thus, PC3 cells are implanted in the prostate by exposing the prostate via laparotomy and inoculating tumor cells into prostate gland using a surgical microscope. After seven days, the mice begin receiving treatment with B2D6, as described above, and the animals are sacrificed no later than 60 days after implantation (or if the animals become moribund). The tumors are palpated at 3-5 day intervals, at which time data on tumor size, animal weight, and survival are collected. Post-mortem evaluations arc also performed as described above, with emphasis upon the effect of B2D6 upon metastatic potential (to COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:42 From:WYNNES PAT&TM ATT 0733421292 To:02621837999 P.18/28 0

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S-14lungs and regional lymph nodes). B2D6 is believed to block the primary tumor and <C metastatic potential of PC3 cells in a dose-dependent manner.

To minimize identification of strain or clonal-specific effects, identical analyses using other model systems can he employed. For example, the effects of C' 5 B2D6 on the growth or metastasis of tumors caused by implantation of K-Ras or X- 0 ray transformed 267B can he compared to the effects on MLC human prostatic o pithelial cell lines.

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SEXAMPLE Development of"Second Generation" EphA2-based Antibody Therapeutics Wlhile EphA2 ovcrcxpression in metastatic prostate cancer cells provides a degree of selectivity comparable to Herceptin in breast cancer, unique properties of EphA2 are believed to allow for even more selective targeting. In particular, EphA2 at the surface of non-transformed epithelia is tightly packed into cell-ccll contacts whereas EphA2 on metastatic cells is difflisely distributed. It is thus likely that some epitopes on EphA2 are accessible in metastases but protected by ligand in normal prostatic epithelia. The EphA2 antibodies that provide the optimal discrimination between normal and metastatic prostatic epithelia are selected.

The panel of antibodies generated previously are screened for epitopes on EphA2 that arc found at the cell surface. Using flow cytometry, EphA2 expression in a variety of "normal" 267B or MLC cells) and metastatic cells (PC3 cells) are compared. For example, confluent monolayers of normal and metastatic cells are labeled with B2D6. To insure that antibodies are selected that are specific for epitopes inaccessible in normal cells but accessible in metastatic cells, antibodies are selected whose binding decreases in normal cells with increasing cell density but whose binding remains constant in metastatic cells, as shown in Fig. 4. After labeling with flioruscein-secondary antibodies, EphA2 expression is evaluated by using flow cytometry. The antibodies that best distinguish between normal and metastatic cells are selected. Specificity for EphA2 is confirmed via westrn blotting and immunoprecipitation studies. Antibodies exhibiting the best selectivity are then humanized using art recognized techniques.

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EXAMPLE 6 Ci Altered EphA2 Expression Through Transfection To assess the consequences o EphA2 overoxpression, MCF-I0A vells were transfected with human EphA2 cDNA (EphA2) or a vector control (vector).

C 5 After establishing cultures ofMCF-1OA cells with stable overexpression of EphA2, CN microscopic evaluation revealed differences in the cell morphology as compared to c-i vector-transfected control cells (not shown). Non-transformed MCF-10A cells NO display an epithelial morphology and interact with one-another, even at low cell 0 density. In contrast, EphA2-overexpressing MCF-10A cells (MCF t adopt a fibroblast-like morphology and do not form cell-cell contacts, even at high cell density. To confirm that the mesenchymal morphology does not represent clonal variation, a separate sample of MCF-10A cells transfected with EphA2 cDNAs yielded identical results.

Ccll-ECM adhesions were evaluated by incubating cells on ECM at 37 "C for 30 minutes before vigorous washing to remove weakly adherent cells.

These assays revealed a 24-fold increase in ECM attachments in MCF hA2 cells relative to vector-transfected controls (P 4x Cell-cell adhesions were assayed by incubating cells in suspension and counting the average size of cell colonies.

Whereas vector-transfected MCF-10A cells interact with one-another in colonies with an average size of 4.1 cells, the average colony size of MCF '11N cells is reduced to 1.3 cells (P 3x10s).

Since stable cell-cell contacts cause EphA2 to become cnriched within sites of cell-cell contact, EphA2 subcellular localization was assessed by inmmniostaining with specific antibodies. The EphA2 on non-transformed cells was restricted to a narrow line where adjacent cells came into direct contact, with little staining of membrane that was not in contact with neighboring cells. In contrast, the pattern ofEphA2 stahiing on MCF 1 cells was diffuse, with little staining of cell-cell contacts.

The lack of EphA2 within cell-cell contacts in MCFE cells was intriguing since EphA2 is stimulated by ligands that are anchored to the cell membrane. To measure EphA2 stimulation, the phosphotyrosine content of immmuoprecipitated EphA2 was measured by Western blot analysis with COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MRY-2006 17:42 From:WYNNES PAT&TM ATT 0733421292 To:0262837999 P.20/28 0 0

(N

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phosphotyrosine specific antibodies. Whereas the EphA2 in vcctor-transfected MCF- Ci 10 cells was tyrosine phosphorylated, EphA2 was not tyrosine phosphorylated in MCF h

A

2 cells. The decreased phosphotyrosine content was confirmed using multiple EphA2 antibodies for immunoprecipitation (D7, B2D6) and different C" 5 phosphotyrosine-specific antibodies (4Gl 0, PY20) for Western blot analyses,

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0 EXAMPLE 7 O Malignancy and Metastasis Through EphA2 Transfcction 0 Malignant transformation was studied in vitro, and MCFPI M cells were found to colonize soft agar. Whereas vector-transfected MCF-10A cells formed 0.3 colonies per high-power field, while MCF p2 S cells displayed increased colony growth in soft agar, with an average of 3.0 colonies per high-power field (P 3x10").

Vector and BphA2 overexpressing MCF-10A cells were allowed to interact with Matrigel (Collaborative, Bedford, MA). Non-transformed MCF-10A cells rapidly organized into spherical colonies when cultured on Matrigel whereas MCF 4 7 h1 cells adopted a stellate organization that was indistinguishable from the behavior of metastatic cells MDA-MB-231, MDA-MB,435), Since in vitro analyses of transformation do not always predict tumorigenic potential in vivo, control or EphA2-overexpressing MCF-10A cells were implanted into athymic (nuu) mice. Subcutaneous injection ofMCF E p t cells caused the formation of palpable tumors within four days in 19 of 19 mice. The median volume of resulting tumors related to the number of implanted cells and reached an average of 300 mnm' (for samples injected with 5x10' cells) within 10 days (Table Necropsy revealed that the tumors were firmly attached to the underlying axillary muscle and surrounded by fibrous tissue. Histologically, the neoplastic cells were invasive and associated with fibrous connective tissue. These neoplastic cells exhibited moderate cytoplasmic and nuclear pleiomorphism and formed dysplastic tubular and secreting structures, h control experiments, cells transfected with vector DNA failed to grow in athymic mice (0 of 13; Table I) and necropsy failed to identify any growth or invasion of these cells.

Since the highest levels of EphA2 were consistently found in breast cancer cells that are metastatic in vivo, IxlO 6 control or MCF4h1^A cells were injected COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MY-2006 17:43 From:WYNNES PAT&TM ATT 0733421292 To:062837999 P.21-128

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0 -17into the tail vein of athymic mice. Within seven days, necropsy revealed lung micrometasases within large vessels in 2 of 4 mice injected with MCF#A 2 cells (Table The mctastases were generally found to occlude large blood vessels but did C notl breach the vessel wall Inmmunohistachcmical staining with cytokeratin antibodies O 5 confirmed the epithelial nature of the thrombus and a lack ofanti-throxmbin staining revealed that the thrombus did not represent an abormal or atypical I outgrowth of endothelial cells. No lung colonization was observed in minice that had been injected with control MCF-10A cells (Table I).

Table I. Tumorigenic and Metastatic Potential of EphA2-Transformed MCF-1OA Cells Site of of Cells Incidence of Tumor Volume Cell Inoculation Injected Tumorigenicity (mm) CtrI Subcutaneous I x 10' 0/9 N/A EpA 2 1 x 10 9/9 66 1 Ctrl Subcutaneous 5 x 10' 0/4 N/A EphA2 5 x 10 10/10h 293 i Ctrl Tail Vein I x 10' 0/4 EphA 1 x 10' 2/4 EXAMPLE 8 Metastatic Targeting Using EphA2 Agonists To test if EphA2 could be stimulated by an agonist, MCF$A 2 cells were suspended in soft agar in the presence or absence of 0.5 mg/mL EphrinAl EphriuAl-F, increased the phosphotyrosine content of EpbA2, and EphrinAl-Ftreated rduced colony formation in soft agar by 49% relative to vehicle-treated controls (P 5x1 To test if EphA2 stimulation could alter cell behavior onil Matrigel, the MCF4hAZ cells were treated with 0.5 mrg/mL EphrinA I which COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:43 From:WYNNES PAT&TM RTT 0733421292 To:0262837999 P.22/28

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0 restored a spherical phenotype that was comparable to non-transformed MCF-1OA c- cells. Thus, EphA2 stimulation reverses the effects of EphA2 overexpression.

EphrinAl-F. Despitv its inability to interact with its endogenous ligands, the EphA2 in MCFn cells responded to cxogenous stimuli.

Although the invcntion has been described in detail with reference to o preferred embodimcnts, variations and modifications exist within the scope and spirit o of the invention as described and defined in the following claims.

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Va COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12

Claims (11)

12-MAY-2006 17:43 From:WYNNES PAT&TM ATT 0733421292 To:03262837999 P.23/28 VO -19- 0O o CLAIMS 1. A pharmaceutical composition comprising an EphA2 agonist selected from (i) San ephrin comprising an extracellular domain of EphrinA1 and (ii) an antibody that is specific to an extracellular epitope of EphA2; and a pharmaceutically acceptable ci S carrier. o 2. The composition of claim 1, wherein said EphA2 agonist is present in a o therapeutically effective amount for treating cancer. c 3. The composition of any one of the preceding claims, wherein said composition is suitable for injection. 4. The composition of any one of the preceding claims, wherein said pharmaceutically acceptable carrier is selected from the group consisting of water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, and liposomes. 5. Use of an EphA2 agonist selected from an ephrin comprising an extracellular domain of EphrinAl and (ii) an antibody that is specific to an extracellular epitope of EphA2 having at least one biological activity for reducing invasiveness, proliferation or volume of a tumor, for reducing metastasis of cancer cells, for impeding proliferation of cancer cells, for increasing the phosphotyrosine content of EphA2 in cancer cells, and/or for reducing the invasiveness of cancer cells compared to untreated cancer cells for the preparation of a pharmaceutical composition for treatment of a tumor or cancer including a population of cells that overexpress EphA2. 6. Use of an EphA2 agonist selected from an ephrin comprising an extracellular domain of EphrinAl and (ii) an antibody that is specific to an COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 03- SEP-2008 12:06 From:l"YNNHES F'HT&TM 1 T 07 -341292 Tu:02g283P 9 R/R 00 O extracellular epilope of EphA' for the manufacture of a medicament for the treatment Sof a tumor comprising a population of cells that overexpress EphA2. 0) 7. Use of an EphA2 agonist selected from an ephrin comprising an Sextracellular domarin ot EptrinAl and (ii) an antibody that is specific to an extracellular epitope of EphA2 for the manufacture of a medicament for the treatment C] Sof a patient having a cancer comprising a population of culls that overexpress o EphA2. 8. A method of treatment of a patient having a tumor or cancer comprising a 0 C population of cells that overexpress EphA2, said method comprising administering to the patient a therapeutically effective amount of an EphA2 agonist selected from (i) an ephrin comprising an extracellular domain of EphrinAl and (ii) an antibody that is specific to an extracellular epitope of EphA2. 9. The method of claim 8 wherein the EphA2 agonist has at least one biological activity for reducing invasiveness, proliferation or volume of a tumor, for reducing metastasis of cancer cells, for impeding proliferation of cancer cells, for increasing the phosphotyrosine content of EphA2 in cancer cells, and/or for reducing the invasiveness of cancer cells compared lo untreated cancer cells. The composition, use or method of any one of the preceding claims, wherein the EphA2 agonist comprises an antibody. 11. The composition, use or method of claim 10, wherein the antibody is a monoclonal antibody. 12. The composition, use or method of claim 11, wherein said monoclonal antibody is humanized.

13. The composition, use or method ol any one of the preceding claims, wherein the EphA2 agonist comprises an antibody that selectively binds to cancer cells. COMS ID No: ARCS-204687 Received by IP Australia: Time 12:17 Date 2008-09-03 12-MAY-2006 17:43 From:WYNNES PAT&TM ATT 0733421292 To:026837999 P.25z28 -21- 0O 0 14. The composition, use or method of any of the preceding claims, wherein the EphA2 agonist comprises an antibody conjugated to a cytotoxic agent. The composition, use or method of claim 14, wherein the cytotoxic agent is selected from the group consisting of a bacterial toxin, ricinA-chain, daunorubicin, methotrexate, a ribosome inhibitor, and a radioisotope.

16. The composition, use or method of claim 14, wherein the cytotoxic agent is a o radioisotope selected from the group consisting of an alpha emitter, a beta emitter, o and an Auger electron emitter. 0 c 17. The composition, use or method of any one of claims 1 to 9, wherein the EphA2 agonist comprises an ephrin that affects phosphorylatlon of EphA2.

18. The composition, use or method of any one of claims 1 to 9 or 17, wherein the EphA2 agonist comprises an ephrin comprising ephrinAl-Fc.

19. The composition, use or method of any one of the preceding claims, where the EphA2 agonist comprises a peptide.

20. The composition, use or method of any one of the preceding claims, wherein the EphA2 agonist comprises a peptide sequence defining an extracellular domain of EphrinAl.

21. The composition, use or method of claim 19, wherein the peptide sequence is linked to a second peptide sequence defining immunoglobulin heavy chain.

22. The composition, use or method of any one of the preceding claims, wherein the EphA2 agonist comprises an antisense oligonucleotide that affects EphA2 expression.

23. The use or method of any one of claims 5 to 22, wherein the population of cells forms at least a portion of a cancer tumor selected from the group consisting of breast, prostate, lung, and colon cancer tumors. COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12 12-MAY-2006 17:43 From:WYNNES PAT&TM ATT 0733421292 To:26283999 P.26/28 22- 0O O 24. The use or method of any one of claims 5 to 23, wherein the population of cells comprises cells selected from the group consisting of breast cancer cells, prostate cancer cells, lung cancer cells and colon cancer cells. The use or method of any one of claims 5 to 24, wherein the cells that overexpress EphA2 are epithelial cells.

26. The use or method of any one of claims 5 to 25 wherein the cancer cell or 0 population of cancer cells overexpress EphA2 as compared to normal cells. O 27. The use or method of any one of claims 5 to 26 wherein the cancer cell or 0 ci population of cancer cells is present in a mammalian patient.

28. A method for identifying an EphA2 antibody that is selective for EphA2 in cancer cells, the method comprising: comparing the binding of an EphA2 antibody as a function of cell density in noncancerous cells to the binding of the EphA2 antibody as a function of cell density in analogous cancer cells, wherein an observed decrease in binding of the antibody to noncancerous cells as cell density increases, coupled with an observed constant level of antibody binding to the cancer cells as cell density increases, is indicative of an EphA2 antibody selective for EphA2 in cancer cells. DATED THIS TWELFTH DAY OF MAY 2006 PURDUE RESEARCH FOUNDATION, GLAXO GROUP LIMITED AND THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL by its Patent Attorneys WYNNES PATENT AND TRADE MARK ATTORNEYS COMS ID No: SBMI-03580647 Received by IP Australia: Time 17:44 Date 2006-05-12