WO2006068822A1 - Notch receptor agonists and uses - Google Patents

Abstract

The present invention is directed to methods of alleviating prostate disorders by activation of the Notch 1 receptor. Also provided herein are methods for detecting and diagnosing prostate disorders, wherein the specific disorder is prostate cancer.

Description

NOTCH RECEPTOR AGONISTS AND USES

FIELD OF THE INVENTION

The present invention relates to the activation of Notch receptor signaling to decrease cancer cell proliferation

BACKGROUND OF THE INVENTION

Notch ) encodes a large protein with a single transmembrane domain a large extracellular domain that has many tandem EGF- like repeats Notch 1 receptor is a signaling molecule that functions in cell development and differentiation The Notch family includes several members for the receptor as well as hgand Binding of hgand to a Notch receptor triggers the cleavage of the receptor at a site in the intracellular domain (ICD), ieleasing the activated form of the receptor which then migrates to the nucleus

For men, prostate cancer is the second most fatal cancer after lung cancer In advanced stages, prostate cancer metastasizes to the bone Depending on the stage of the cancer, prostate cancer treatment involves one or a combination of the following therapies surgery to remove the cancerous tissue, radiation therapy, chemotherapy, androgen deprivation (e g hormonal therapy) in the case of prostate cancer The majoπtv of patients who undergo hormone theiapy progress to develop androgen-independent disease Currently, there is no effective tieatment for the 20-40% of prostate cancer patients who develop recurrent disease after surgery or radiation therapy, or for those in whom the cancer has metastasized at the time of diagnosis Chemotherapy has its toxic side effects, especially in elderly patients There is a need for new forms of prostate cancer therapy The present invention provides alternative methods of treating cancer that overcome the limitations of conventional therapeutic methods as well as offer additional advantages that will be apparent from the detailed description below

SUMMARY OF THE INVENTION In one embodiment, the invention concerns a method of increasing or decreasing the proliferation of prostate cancer cells by modulating the activation of the Notch 1 receptor

In other embodiments, the invention provides for methods of detecting, diagnosing and alleviating prostate cancer by contacting biological samples suspected of prostate cancer Detection and diagnosis of prostate cancer in the biological sample may include determining level of Notch 1 expression, effects of Notch 1 ligands on Notch 1 receptor, or probing the biological sample with Notch 1 receptor Treatment may include contacting the biological sample with agonists to Notchl receptor or administration of a soluble Notchl ligand

In another embodiment, the invention provides an antibody which binds, preferably specifically, to Notchl Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single- chain antibody Another embodiment of the invention concerns agonists and antagonists of Notchl receptor as defined herein In a particular embodiment, the agonist is an anti-Notch 1 antibody, a soluble Notch l ligand, or a small molecule In a still further embodiment, the invention concerns a method of identifying agonists to Notch 1 receptor which comprise contacting the Notch 1 receptor with a candidate molecule and monitoring a biological activity mediated by said Notch 1 receptor. Preferably the Notch 1 receptor is a native Notch 1 receptor.

Another embodiment of the present invention is directed to the use of constitutively active Notch 1 receptor, or an agonist thereof, for the preparation of a medicament useful in the treatment of a condition which is responsive to the constitutively active Notch 1 receptor or an agonist thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A-C. Gamma-secretase inhibitors down-reguiated Notch 1 signaling and affected prostatic branching morphogenesis in culture.

Figure 2Al -E. Gamma-secretase inhibitors induced phenotypes that resembled those of pre-differentiation stages.

Figure 3A1-C4. Epithelial cells in inhibitor-treated prostate co-expressed CK8 and CK14.

Figure 4Al -C3. Strong proliferation of epithelial cells in prostate tissue treated with γ-secretase inhibitors.

Figure 5A-E2. Induced Notchl gene deletion caused prostatic epithelial dysplasia.

Figure 6A1-C. Co-expression of CK8-CK14 in epithelial clusters of iNotchϊ'^" prostate

Figure 7A-C. Elevated proliferation of epithelial cells in Notch! knockout prostate tissue.

Figure 8A1-B2. Down-regulation of Notchl and Hey-1 expression in a large number of human prostate adenocarcinoma samples.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention encompasses several aspects. One aspect.of the invention is a method of inhibiting or decreasing the proliferation of cancer cells by administering an agonist of Notch receptor which results in activation of Notch signaling. In a specific embodiment, the Notch receptor is Notchl and the cancer is prostate cancer. Another aspect of the invention is a method of destroying cancer and tumor cells which express a Notchl receptor by administering to a patient in need thereof, a therapeutically effective amount of a composition comprising a Notchl receptor binding partner effective for that purpose. A further aspect of the invention is a method of alleviating cancer , in particular, prostate cancer, by administering an agonist of Notchl receptor. For therapeutic applications, the activators of Notchl signaling can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabeled compounds, or with surgery, cryotherapy, and/or radiotherapy.

The Notchl receptor binding partner useful in destroying cancer cells, in particular prostate cancer cells, includes soluble ligands of the receptor, antibodies and fragments thereof, and small molecules that bind the Notchl receptor. The binding partners can be conjugated to a cytotoxic agent. The antibodies are preferably internalizing and/or growth inhibitory antibodies. The cytotoxic agent can be a toxin, antibiotic, radioactive isotope or nucleolytic enzyme. A preferred cytotoxic agent is a toxin, preferably a small molecule toxin such as calicheamicin or a maytansinoid.

In all the above embodiments, the cancer includes are as described below. In a preferred embodiment, the cancer is prostate cancer. In all the above embodiments, the soluble ligand includes truncated forms of the native or natural ligand lacking the transmembrane domain, and immunoglobulin fusions of these soluble ligands where, e.g., the soluble ligand is fused to the Fc portion of an IgG. Other than the truncation, the soluble ligand can have further amino acid variation from the native sequence. These amino acid variants of the native ligand can, in the portion of the sequence that corresponds to the native sequence, have one or more amino acid changes. These amino acid changes can, e.g., confer upon the ligand, ability to constitutively activate the Notchl receptor, greater binding, longer half-life and greater stability in vivo.

The agonists and binding partners of Notchl receptor can be synthetically or recombinantly produced or otherwise isolated.

The invention also provides agonists of Notch 1 receptor that inhibit or decrease cancer cell growth or proliferation. Also provided are compositions comprising one or more agonists of Notch l receptor effective for use in the aforementioned methods, and a carrier. A Notchl receptor agonist includes any molecule that can produce an activated signaling form of the receptor, including soluble ligands of the receptor, antibodies and fragments thereof, small molecules that bind the Notchl receptor extracellularly or that can enter the cell and act intracellularly, and molecules that have enzymatic activity in cleaving the receptor to release the activated form. In a preferred embodiment, the antibody in the composition is a humanized antibody. In a preferred embodiment, the carrier is a pharmaceutically-acceptable carrier. The compositions can be provided in an article of manufacture or a kit.

Another aspect of the invention is an isolated nucleic acid encoding an internalizing or cytotoxic anti- Notch 1 receptor antibody of the invention, as well as a vector comprising the nucleic acid. The human Notchl DNA sequence can be found using GenBank Accession Number: HUMTANl . Also provided by the invention are cells including E.coli and hybridomas that produce the above-described antibodies.

"Notch" encompasses all members of the Notch receptor family and in particular, Notch 1. An "agonist" of Notch receptor in addition to binding Notch receptor, has a direct effect on a Notch receptor bearing cell. The Notch receptor agonist will bind Notch receptor, and as well, initiate or mediate the signaling event associated with the Notch receptor, such as, for example, to cause the intracellular domain of Notch to be cleaved and translocated to the nucleus. Here it induces the synthesis of transcriptional repressors known as HES-I. When ECD truncated forms of Notch are tested , the HES-I promoter is strongly stimulated. Activation of the HES-I promoter can be assayed in vitro. The ability to induce Notch receptor activation can be quantified using techniques known in the art such as reporter constructs such as Beta-galactosidase, chloroamphenicol acetyl transferase (CAT) or luciferase.

The proliferation of cancer cells is reduced or arrested when the Notch receptor is activated by contact with an added or administered Notch receptor agonist or activator, compared to situation in the absence of agonist. Proliferation assays are described below. The Notch ligands include Jagged 1 , Jagged2, Delta and Delta4.

Prostate cancer specifically includes prostate adenocarcinoma and related metastases. The term "amino acid sequence variant" refers to a polypeptide that has amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants of Notch 1 receptor will possess at least about 70% homology with the native sequence Notch 1 receptor, preferably, at least about 80%, more preferably at least about 85%, even more preferably at least about 90% homology, and most preferably at least 95%. The amino acid sequence variants can possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.

"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably. silver stain. Isolated polypeptide includes polypeptide HI situ within recombinant cells, since at least one component of the Notch 1 polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step

The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecifϊc antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non- reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.

An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et ai, Proc. Natl, Acad. ScL USA, 81 :6851 - 6855 ( 1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')_2> and Fv fragments; diabodies; linear antibodies (see US patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

"Single-chain Fv" also abbreviated as "sFv" or "scFv"' are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal

Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V_H and V_L domains such that inter- chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V_H and V_L domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149. The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-I, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-I and IgA-2), IgE, IgD or IgM. An anti-Notchl receptor antibody that "internalizes" is one that is taken up by (i.e., enters) the cell upon binding to cell surface Notchl on a mammalian cell. The internalizing antibody will of course include antibody fragments, human or humanized antibody and antibody conjugate. For therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill a Notchl -expressing cancer cell. Depending on the potency of the antibody or antibody conjugate, in some instances, the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugate to the antibody is sufficient to kill the tumor cell. Whether an antibody internalizes upon binding Notchl on a mammalian cell can be determined by various assays e.g., by confocal or electron microscopy using fluorescent or radiolabeled antibody. An "antibody that inhibits the growth of cancer cells expressing Notchl receptor or a "growth inhibitory" antibody is one which binds to and results in measurable growth inhibition of cancer cells expressing or overexpressing Notchl receptor. Preferred growth inhibitory anti-Notchl receptor antibodies inhibit growth of Notchl receptor expressing tumor cells (e.g., prostate cancer cells) by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g. from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1- 10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if, e.g., administration of the anti-Notch 1 receptor antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

A "Notch receptor-expressing cancer" is a cancer comprising cells that have Notch receptor protein present on the cell surface. A "Notch receptor-expressing cancer" produces sufficient levels of Notch receptor on the surface of cells thereof, such that a Notch receptor agonist or antibody can bind thereto and have a therapeutic effect with respect to the cancer. A cancer which "overexpresses" Notch receptor is one which has significantly higher levels of Notch receptor at the cell surface thereof, compared to a noncancerous cell of the same tissue type. Notch 1 receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the Notch 1 receptor protein present on the surface of a cell (e.g. via an immunohistochemistry assay; FACS analysis). Alternatively, or additionally, one may measure levels of

Notchl receptor-encoding nucleic acid or mRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study Notchl receptor overexpression by measuring shed antigen in a biological fluid such as serum, e.g, using antibody-based assays (see also, e.g., U.S. Patent No. 4,933,294 issued June 12, 1990; WO91/05264 published April 18, 1991; U.S. Patent 5,401,638 issued March 28, 1995; and Sias et al. J. Immunol Methods 132: 73-80 (1990)). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g. a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g. by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. "PIN" is prostatic intraepithelial neoplasia, a hyperproliferation of prostatic epithelial cells. "Alleviation of cancer" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of alleviation include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is "alleviated" for a Notch 1 receptor-expressing cancer if, after receiving a therapeutic amount of a Notchl receptor agonist according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer, and reduced morbidity and mortality. To the extent the Notchl receptor agonist or antibody may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.

"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR). For prostate cancer, the progress of therapy can be assessed by routine methods, usually by measuring serum PSA (prostate specific antigen) levels; the higher the level of PSA in the blood, the more extensive the cancer. Commercial assays for detecting PSA are available, e.g, Hybritech Tandem-E and Tandem-R PSA assay kits, the Yang ProsCheck polyclonal assay (Yang Labs, Bellevue, WA), Abbott Imx (Abbott Labs, Abbott Park, IL), etc. Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).

The term "therapeutically effective amount" refers to an amount of an agonist, antibody or a drug effective to "alleviate" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See preceding definition of "alleviating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides. and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits etc. Preferably, the mammal is human. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At²", I¹³', I¹²⁵, Y⁹⁰, Re'⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²'², P³² and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various anti-tumor or anticancer agents disclosed below. Other cytotoxic agents are described below.

A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially a Notch 1 receptor expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of Notch 1 receptor expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and anti-neoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.

A "small molecule" is defined herein to have a molecular weight below about 500 Daltons. An "isolated nucleic acid" is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule.

"Vector" includes shuttle and expression vectors. Typically, the plasmid construct will also include an origin of replication (e.g., the CoIEl origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively, of the plasmids in bacteria. An "expression vector" refers to a vector that contains the necessary regulatory elements for expression of the antibodies including antibody fragment of the invention, in bacterial or eukaryotic cells

A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a Notch 1 polypeptide or antibody thereto) to a mammal The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes

The Notch 1 receptor agonists and prostate cancer cell growth inhibitory antibodies of the invention also have various non-therapeutic applications In view of the fact that the use of PSA as a tool for screening or diagnosing prostate cancer is controversial, the agonists and antibodies of the present invention can be useful for diagnosis and staging of Notch 1 -expressing cancers (e g , in radioimaging) The antibodies are also useful for puiification or immunoprecipitation of Notch 1 from cells, for detection and quantitation of Notch 1 in vitro, e g in an ELISA or a Western blot to kill and eliminate Notch 1 -expressing cells from a population of mixed cells as a step in the purification of other cells

The Notch 1 receptor agonists and antibodies can be fused to a heterologous polypeptide sequence The antibody can be modified in the Fc region to provide desired effector functions As discussed in more detail in the sections below, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e g , via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism

The present Notch 1 receptor agonists and antibodies are useful for treating a Notch 1 receptor expressing cancer or alleviating one or more symptoms of the cancer in a mammal Such a cancer includes prostate cancer and leukemias The antibody is able to bind to at least a portion of the cancer cells that express Notch 1 receptor in the mammal and preferably is one that does not induce or that minimizes HAMA response (human anti-mouse antibody) In a preferred embodiment, the antibody is effective to destroy or kill Notch 1 receptor-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to Notch 1 receptor on the cell Such an antibody includes a naked anti-Notch 1 receptor antibody (not conjugated to any agent) Naked antibodies that have cytotoxic or cell growth inhibition properties can be further harnessed with a cytotoxic agent to render them even moie potent in tumor cell destruction Cytotoxic properties can be conferred to an anti-Notchl receptor antibody by, e g , conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described below The cytotoxic agent or a growth inhibitory agent is preferably a small molecule Toxins such as calichearrucin or a maytansinoid and analogs or derivatives thereof, are preferable I. Production of anti-Notch-1 Antibodies

(ι) Polyclonal antibodies

Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobuhn, or soybean trypsin inhibitor, using a bifunctional or denvatizing agent, e g , maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N=C=NR, where R and R¹ are different alkyl groups Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

(H) Monoclonal antibodies _v Monoclonal antibodies may be made using the hybridoma method first described by Kohler et at,

Nature, 256:495 ( 1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol.,

133:3001 (1984); and Brodeur et ai, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980). Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g, by i.p. injection of the cells into mice. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g.. using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures {e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et ai, GOT. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et ai, Nature, 348:552-554 (1990). Clackson et ai, Nature, 352:624-628 (1991) and Marks et ai, J. MoI. Bio!., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et ai, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et ai, Niic. Acids. Res., 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified, for example, by substituting human heavy chain and light chain constant domain (C_H and C_L) sequences for the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et ai, Proc. Natl Acad. ScL USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide. The non- immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen- combining site having specificity for a different antigen.

(Hi) Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an 'import" variable domain Humanization can be essentially performed following the method of Winter and co workers (Jones et al , Natuie, 321 522-525 ( 1986) Reichmann et al , Natui e, 332 323-327 ( 1988), Verhoeyen et al Science, 239 1534-153t> ( 1°>88)), by substituting hypervaπable region sequences for the corresponding sequences of a human antibody Accordingly, such ' humanized ' antibodies are chimeric antibodies (U S Patent No 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some hypervaπable region residues and possibly some FR residues are substituted by iesidues from analogous sites in rodent antibodies

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use According to the so-called ' best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al , J Immunol , 151 2296 ( 1993), Chothm et al J MoI Biol , 196 901 (1987)) Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains The same framework may be used for several different humanized antibodies (Carter et al , P we Natl Acad Sa USA, 89 4285 (1992), Presta et al , J Immunol , 151 2623 (1993))

It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences Three- dimensional immunoglobulin models are commonly available and aie familiar to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, / e , the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen In this way, FR residues can be selected and combined from the recipient and import sequences so that the desned antibody characteristic, such as increased affinity for the target antigen(s), is achieved In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding Vaπous forms of a humanized anti-Notchl receptor antibody are contemplated For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an lmmunoconjugate Alternatively, the humanized antibody may be an intact antibody, such as an intact IgGl antibody

(ιv) Human antibodies

As an alternative to humanization, human antibodies can be generated For example, it is now possible to produce transgenic animals (e g , mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J_H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits er a/., Proc. Natl. Acad. Sci. USA, 90:2551 ( 1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggemann et al, Year in lmmuno., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in- frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 ( 1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624- 628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. MoI. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Patent Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275).

(v) Antibody fragments In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al. , Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)).

However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above.

Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')₂ fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')₂ fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046.

Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641 ,870 for example. Such linear antibody fragments may be monospecific or bispecific.

(vi) Bispecific antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Notch 1 receptor protein. Other such antibodies may combine a Notchl receptor binding site with a binding site for another protein. Alternatively, an anti-Notch 1 receptor arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), Fc γRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to the Notch l receptor- expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express

Notchl receptor. These antibodies possess a Notchl receptor-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-γ, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')₂ bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcγ antibody is shown in WO98/02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et a!., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et ai, EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_H2, and C_H3 regions. It is preferred to have the first heavy-chain constant region (CH I) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination

According to another appioach described in U S Patent No 5,731 , 168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from iecombinant cell culture The preferred interface comprises at least a part of the C_H3 domain In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g tyrosine or tryptophan) Compensatory "cavities" of identical or similar size to the large side chain(s) aie created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e g alanine or threonine) This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin Such antibodies have for example, been proposed to target immune system cells to unwanted cells ((J S Patent No 4,670,980), and tor treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089) Heteroconjugate antibodies may be made using any convenient cross-linking methods Suitable cross-linking agents are well known in the art, and are disclosed in U S Patent No 4,676,980, along with a number of cross-linking techniques

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature For example, bispecific antibodies can be prepared using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies aie proteolytically cleaved to generate F(ab')₂ fragments These fragments are reduced in the presence of the dithiol complexing agent, sodium arsemte, to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) deπvatives One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E coli, which can be chemically coupled to form bispecific antibodies Shalaby et al , J Exp Med , 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule Each Fab' fragment was separately secreted from E coli and subjected to directed chemical coupling in vitio to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human

T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol , 148(5) 1547-1553 (1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. ScL USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et ah, J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 ( 1991).

To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG^, IgGβ, or IgG^ that is responsible for increasing the in vivo serum half-life of the IgG molecule.

(vii) Screening for antibodies with the desired properties Techniques for generating antibodies have been described above. One may further select antibodies with certain biological characteristics, as desired. The growth inhibitory effects of an anti-Notch 1 receptor antibody of the invention may be assessed by methods known in the art, e.g., using cells which express Notch 1 receptor either endogenously or following transfection with the Notchl receptor gene. For example, tumor cell lines and Notchl receptor-transfected cells may treated with an anti-Notch 1 receptor monoclonal antibody of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing ³H-thymidine uptake by the cells treated in the presence or absence an anti-Notch 1 receptor antibody of the invention. After antibody treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Preferably, the Notchl receptor agonist will inhibit cell proliferation of a Notchl receptor-expressing tumor cell in vitro or in vivo by about 25- 100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, at an antibody concentration of about 0.5 to 30 μg/ml. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of the anti-Notchl receptor antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days. Maytansine and mavtansinoids In one preferred embodiment, an anti-Notchl receptor antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent Nos. 4, 137,230: 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821 ; 4,322,348; 4,331 ,598; 4,361 ,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371 ,533, the disclosures of which are hereby expressly incorporated by reference.

The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-Notch 1 receptor antibodies.

Examples include At²", I¹³', I¹²⁵, Y⁹⁰, Re'⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc^99m or I¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123 again, iodine- 131 , indium- 1 1 1 , fluorine- 19, carbon- 13. nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc^99m or I¹²³, .Re^ιsδ, Re'⁸⁸ and In^{11 1} can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,CRC Press 1989) describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyIdithio) propionate (SPDP), succinimidyl-4-(N- maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-Notch 1 receptor antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" {e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide).

(viii) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Patent No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5- fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide- containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D- amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β- lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-Notchl receptor antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984).

(be) Other antibody modifications

Other modifications of the antibody are contemplated herein. For example, the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington 's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., ( 1980). (JC) Purification of anti-Notch I receptor antibody When using recombinant techniques, the antibody can be produced intracellular^, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et ai, Bio/Technology 10: 163- 167 ( 1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γl, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a C_H3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation,

Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

II. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preserv atives (such as octadecyldimethylbenzyl ammonium chloride, hexamethomum chloride, benzalkonium chloride, benzethonium chloride phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextπns, chelating agents such as EDTA, tonicifiers such as trehalose and sodium chloride, sugars such as sucrose, mannitol trehalose or sorbitol, surfactant such as polysorbate, salt-forming counter-ions such as sodium, metal complexes (e g Zn-protein complexes), and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) The antibody preferably comprises the antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other For example, in addition to the anti-Notch 1 receptor antibody which internalizes, it may be desirable to include in the one formulation, an additional antibody, e g a second anti-Notch 1 receptor antibody which binds a different epitope on Notch 1 receptor, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant Such molecules are suitably present in combination in amounts that are effective for the purpose intended

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980)

Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyπc acid

The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes

In one particular embodiment, an lmmunoconjugate comprising the anti-Notch 1 receptor antibody conjugated with a cytotoxic agent is administered to the patient Preferably, the lmmunoconjugate bound to the Notchl receptor protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell Examples of such cytotoxic agents are described above and include maytansinoids cahcheamicins, πbonucleases and DNA endonucleases

The anti-Notch 1 receptor antibodies or immunoconjugates are administered to a human patient, in accord with known methods, such as intravenous administration, e g , as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, lntra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes Intravenous or subcutaneous administration of the antibody is preferred

Other therapeutic regimens may be combined with the administration of the anti-Notch 1 receptor antibody The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) actne agents simultaneously exert their biological activities Preferably such combined therapy results in a synergistic therapeutic effect

It may also be desirable to combine administration of the anti-Notch 1 receptor antibody or antibodies, with administration of an antibody directed against another tumor antigen associated with the particular cancer

III. Administration of Anti-Notchl receptor Agonists and Antibodies

In another embodiment, the antibody therapeutic treatment method of the present invention involves the combined administration of an anti-Notch 1 receptor antibody (or antibodies) and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5- fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed , M C Perry, Williams & Wilkins, Baltimore, MD ( 1992)

The antibody may be combined with an anti-hormonal compound, e g , an anti-estrogen compound such as tamoxifen, an anti-progesterone such as onapπstone (see, EP 616 812), or an anti-androgen such as flutamide, in dosages known for such molecules Where the cancer to be treated is androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen independent, the anti-Notch 1 receptor antibody (and optionally other agents as described herein) may be administered to the patient

For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria The appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the seventy and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician The antibody is suitably administered to the patient at one time or over a series of treatments Preferably, the antibody is administered by intravenous infusion or by subcutaneous injections Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg body weight {e g about 0 l- 15mg/kg/dose) of antibody can be an initial candidate dosage for administration to the patient, whether, for example by one or more separate administrations, or by continuous infusion A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg ot the anti-Notch 1 receptor antibody However, other dosage regimens may be useful A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art

IV. Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacture containing materials useful for the alleviation of anti Notch 1 receptor expressing cancer, in particular prostate cancer The article of manufacture compπses a container and a label or package insert on or associated with the container Suitable containers include, for example bottles, vials, syringes, etc The containers may be formed from a variety of materials such as glass or plastic The container holds a composition which is effective for alleviating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) At least one active agent in the composition is an anti-Notch 1 receptor antibody of the invention The label or package insert indicates that the composition is used for treating prostate cancer, androgen independent prostate cancer, or androgen dependent prostate cancer, or bladder cancer The label or package insert will further comprise instructions for administering the agonist or antibody composition to the cancer patient Additionally, the aiticle of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution It may further include other materials desiiable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes

Kits are also provided that are useful for various purposes , e g , for Notch 1 receptor cell killing assays, for purification or immunoprecipitation of Notch 1 receptor from cells For isolation and purification of Notch 1 receptor, the kit can contain an anti-Notch 1 receptor antibody coupled to beads (e g , sepharose beads) Kits can be provided which contain the antibodies for detection and quantitation of Notch 1 receptor ;/; vitio, e g in an ELISA or a Western blot As with the article of manufacture, the kit compπses a container and a label or package insert on or associated with the container The container holds a composition comprising at least one anti-Notchl receptor antibody of the invention Additional containers may be included that contain, e g , diluents and buffers, control antibodies The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use V. Examples

Example 1 Expression of Notch 1 during prostatic development To determine the role of Notch 1 in regulating the development of prostate stem/progenitor cells, two inhibitors were used to inactivate Notch 1 signaling. The inhibitors used were WPE-III-31C (Esler et al., PNAS 99: 2720-2725 (2002)) and L-685,458 (Li et al., Nature 405:689-694 (2000); Shearman et al. Biochem. 39:8698- 8704 (2000)). These molecules were previously shown to specifically inhibit the γ-secretase activity of presenilin and Notch ] activation (Doerfler et al., PNAS 98:9312-9317 (2001); (Esler et al., PNAS 99: 2720- 2725 (2002)). The mouse myoblast cell line C2C12, which has intact Notch 1 signaling and has been extensively used for studying myogenesis (Yaffe and Saxel, Nature 398: 525-529 (1977); Nofziger et al., Development 126: 1689- 1702 ( 1999)). was used here to confirm that the γ-secretase inhibitors inhibited Notchl activity. As shown in Fig. IA-B, treatment of C2C12 cells with either WPE-III-31C or L-685,458 markedly reduced the level of Hes- 1 and Hey- 1, which are transcription factors belonging to the HES and HERP families of downstream Notchl effectors (Iso et al., J. Cell. Phys. 194:237-255 (2003)). The efficacy of these inhibitors is shown in Fig. IA and I B, where γ-secretase inhibitors were able to down-regulate Hes-1 and Hey-1 mRNA expression in C2C12 myoblasts. The C2C12 myoblast cells were grown in differentiation-preventing high serum medium, and either WPE-III-31C (2 μM) or L-685,458 ( 1 μM) inhibitor was added to -60% confluent cells the night before RNA extraction. Taqman analysis of the RNA demonstrates that the inhibitors significantly reduced Hes-1 and Hey- 1 mRNA levels, thus showing that these two inhibitors are effective in reducing Notchl signaling.

Example 2: Notchl expression in branching morphogenesis.

To examine the role of Notch l signaling in prostate development with regard to branching morphogenesis, a convenient ex vivo organ culture system was used (Thomson and Cunha, Development 126: 3693-3701 (1999); Wang et al., J. Biol. Chem. 278: 18506-18513 (2003); Wang et al., J. Biol. Chem. 279: 24733-24744 (2004)). Early postnatal rat ventral prostates were cultured with the γ-secretase inhibitors and their effect on growth and branching morphogenesis was examined. Rat P3 ventral prostate was freshly dissected and incubated with serum-free medium containing an inhibitor. The concentration of WPE-III-31C or L-685,458 was 2 μM and 1 μM respectively. There was reduced branching and enlarged epithelial tubules in P3 prostate explants treated with inhibitors for 6 days. As shown in Fig. 1C, treatment of postnatal day 3 (P3) prostates with inhibitor WPE-III-31C or L-685,458 for 6 days (P3+d6) resulted in greatly reduced branching and markedly enlarged epithelial tubules in the prostate as compared to the untreated control, suggesting that Notchl signaling is crucial for branching morphogenesis in prostate development. Immunohistochemistry on untreated prostate tissue sections with an antibody against p63, an epithelial cell marker which stains all epithelial cells at early stage of development (such as P3) and stains only basal epithelial cells at later stages of development (after P5) revealed a dramatic difference in p63 expression pattern between the control prostate tissue and the prostate tissue treated with γ-secretase inhibitors. While control cultures exhibited lumen formation (Fig.2Al), prostate treated with either one of the inhibitors showed much less lumen formation (Fig.2B l and Fig.2Cl). Also shown is DAPI counterstaining (Fig.2 Al, B l, Cl) and p63 immunostaining (Fig.2 A2, B2, C2) on cryosections of untreated P3 prostate or prostate that had received either WPE-III-31C or L-685,458 treatment for 6 days in culture (P3+d6). The staining is (D 1 , DAPI; D2, p63) for a pre-differentiation stage at the start of culture, P3 pre-treated, are also shown for comparison. There is a drastic difference in lumen formation, luminal and basal cell layer segregation and spacing of p63-positive cells between WPE-III-31C (Fig 2 B 1 -2) or L-685,458 (Fig C 1-2) treated and untreated control tissues (Fig Al-2) Luminal and basal epithelial layer segregation (Fig 2A l -2) and well-spaced basal cells (p63-positive cells, Fig 2A2) were evident in control prostates whereas WPE-III-31C or L-685,458 treated prostates displayed clustering of p63-positive epithelial cells or thickening of basal layers (Fig 2B2 and 2C2) There is also a striking resemblance between treated (Fig 2 B,C) and pre-differentiation P3 tissues (Fig 2D) Overall, prostates treated with either inhibitor showed epitheha that lacked the well-organized columnar luminal epithelial cells juxtaposed to elongated basal cells that are seen in normal prostate tissue at this stage The phenotypes observed in prostates after 6 days of inhibitor treatment (P3+d6) had moiphology more similar to P3 prostate prior to treatment (P3 pre-treated) (Fig 2B2, 2C2 and 2D2), as they had un-segregated or moderately segregated epitheha consisting largely of p63-positive cells

The percentages of epithelial cells expressing p63 aie compared in (Fig 2E) The prostates treated with inhibitor showed significantly increased numbers of p63 positive cells, and were more similar to P3 stage control The percentage of epithelial cells expressing pt>3 in the piostate treated with the inhibitors (81 0% for WPE-III-31C and 75 9% for L-685,458 ) were similar to that of the P3 pre-treated prostate (90 5%), but differed significantly from the untreated control (38 7%) that had grown in culture for 6 days (Fig 2E) No apparent apoptosis was found by TUNEL assays in these tissues (data not shown), and these cells were actively proliferating (also see Fig 4), suggesting that these inhibitors did not cause general cytotoxic effects

Example 3 Expansion of progenitor-like prostate epithelial cells following Notch 1 inhibitor treatment In rodents, lobular divisions of prostate are formed just before birth and contain unbranched solid epithelial buds extended from urogenital sinus epithelium After birth, epithelial cells continue to proliferate and send out sub-branches that still consist of solid epithelial buds All epithelial cells at this stage are considered to be progenitor cells as they co-express the cytoskeletal proteins cytokeratin 8 (CK8) and cytokeratin 14 (CK14) and proliferate Around postnatal day 5, ductal canalization is initiated and some epithelial cells become post-mitotic and terminally differentiate into luminal cells, and other epithelial cells become basal cells (Hayward et al , Ann N Y Acad Sci 784 50-62 (1996)) After this stage, CK8 and CK14 expressions are largely mutually exclusive in that CK8 is mainly expressed in luminal cells and CK14 is mainly expressed in basal cells Prostate stem/progenitor cells are believed to reside in CK14-positive basal cell compartment and they can differentiate into luminal cells (B onkhoff, Eur Urol 30 201-205 (1996)) Since the p63 staining of prostate after inhibitor treatments resembled that of undifferentiated P3 prostate, we further determined whether epithelial cells in the inhibitor-treated prostate exhibited features of P3 prostate such as co- expression of both luminal (CK8) and basal (CK14) cell markers and high proliferating index (Hayward et al 1996)

As shown in Fig 3, the majority of epithelial cells in prostates treated with either inhibitors WPE-III- 31C or L-685,458 co-expressed CK8 and CK14, whereas control prostates grown from P3 for 6 days in culture showed segregation of CK8-positive luminal and CK14-positive basal layers In contrast to the confined localization of CK8 and CK14-positive cells in luminal or basal layers in control cultures, the CK8 or CK14 positive cells were seen intermingled and dispersed throughout the entire epithelial region in inhbitor treated prostates. In addition, epithelial cells in inhibitor-treated prostates were highly proliferative as demonstrated by BrdU incorporation (Fig.4B l and 4C l). The anti-CK8 antibody stained more strongly in the luminal layer and anti-CK14 antibody stained more strongly in the basal layer of the epithelia (Fig.3), and that some BrdU- positive cells had lost expression of CK 14 (Fig.4), indicating that differentiation still occurred but at a much lower level. The above data show that Notch 1 signaling exerts an inhibitory role on prostate stem/progenitor cell proliferation and promotes cell differentiation, and that disruption of this pathway results in an over- proliferation and incomplete differentiation of this cell population.

Example 4: Prostatic dysplasia in Notch 1 inducible knockout mice.

To examine the effect of inactivation of Notch 1 pathway on prostate development in vivo, an interferon-inducible Notch 1 gene deletion mouse model was used (Radtke et al., Immunity 10: 547-558 (1999)). As Notch! ablation causes embryonic lethality (Swiatek et al., Genes Dev. 8: 707-719 ( 1994); Conlon et al., Development 121 : 1533- 1545 ( 1995 )), it was necessary to use an inducible system. Successful deletion of Notch 1 could be achieved in a number of tissues including thymus, bone marrow, liver, spleen, skin, lung, kidney and intestines by treatment of mice homologous for loxP-flanked allele and positive for Mx- C re transgene (hereafter iNotchl ^') with interferon-inducing agent polyl-polyC (Radtke et al., supra). To verify that gene deletion was induced, we examined Cre transgene expression, targeted gene deletion and Notch! downstream gene expression in mouse prostates collected 1 week after the last polyl-polyC injection. As shown in Fig.5 A, immunostaining of prostate cryosections for Cre recombinase revealed that Cre was induced in CVe- positive mice and its expression was mainly in the epithelium. Cre expression was not detected in Cre-negative mice (data not shown). The deletion of Notch! sequence was confirmed with PCR. As expected, mice genotyped positive for Cre produced a recombinant band of approximately 0.4 kb, which was absent in CVe- negative mice (arrow, Fig.5B). The corresponding wild type Notch! sequence, 3.9 kb in length, was not amplified under those conditions. The effect of Notch! deletion on downstream gene expression was evaluated using real-time RT-PCR. As shown in Fig.5C, Hey-1 mRNA was reduced by approximately 50% 1 week after induction. Hes-1 mRNA was also evaluated but no significant change was found, suggesting differential regulation of Notch 1 downstream gene expression in the prostate. The Cre expression. Notch! deletion and Hey-1 reduction data indicated that Notch! was deleted in the prostate of iNotchϊ'^' mice.

Histological analyses of prostates harvested 3 weeks after induction revealed a distinct phenotype, particularly in ventral lobes of prostate. Wild type prostates, at a stage corresponding to postnatal day 36-40, were relatively well developed (Fig.5Dl). The prostatic epithelium consists mostly of a single layer of cuboidal to columnar cells with ample cytoplasm and evenly distributed nuclear spacing (arrowhead, Fig.5Dl-D2) with infrequent tufts. The prostatic glands had open lumens that were frequently filled with abundant secretion and many desquamated cells, an indication of normal development and glandular maturity (Fig.5Dl-D2). In contrast, prostates from iNotch!^'A mice displayed reduced luminal secretion with rare or no desquamated cells (Fig.5El-E2). Instead, multiple tufting and occasional bridging were seen in multiple places. In many areas within the epithelium, clusters of epithelial cells were also detected (arrow, Fig.5El-E2). The cells in these focal areas did not maintain the normal columnar morphology of luminal epithelial cells or the laminar nature of basal cells, but instead displayed a spherical morphology. In addition, the nuclei of epithelial cells in iNotchϊ^1' tissue appeared more crowded with denser chromatin The phenotypes in the anterior lobes of iNotchl ' prostate were less evident than in ventral prostate These findings together demonstrate that the lack of Notch 1 slows down or prevents terminal differentiation and promotes proliferation of prostatic epithelial cells, thus, supporting the results from γ-secretase inhibitor studies The changes in organ morphology induced by Notch 1 deficiency are commonly seen in prostatic intraepithelial neoplasia (PIN), a precursor lesion to prostate cancer

Example 5 Increased immature cell population and enhanced proliferation in the prostatic epithelium of Notch 1 inducible knockout mice

The phenotypes of prostate epithelial cells, particularly those clustered focally in the epitheha of iNotchl ' prostate were examined further Overall, CK8 and CK14 staining was highly specific in control wild type tissue at this stage, with Ib 9% of epithelial cells that co-expressed CK8 and CK14 In contrast, the percentage of CK8/CK14-coexpressing epithelial cells increased to 70 0% in the iNotchl ^f prostate (Fig 6C) More importantly the focal clusters of cells in iNotchl ' prostate epitheha expressed both CK8 and CK14 (Fig 6A-B), indicative of a progenitor phenotype It was this cell population that was responsible for the increase in percentage of CK8/CK14-coexpressing epithelial cells To determine whether this alteration was due to enhanced cell proliferation, Ki-67 immunostaining was performed In contrast to the low proliferation detected in the epitheha of control mice, which was expected for 7-8 weeks old mice, proliferation in iNotch ¹ mice prostate was significantly enhanced (Fig 7) These results show that inactivation of Notch 1 signaling caused over-proliferation of progenitor cells in prostate which resulted in phenotypes that resemble neoplastic lesions

Example 6 Notch 1 and Hev-1 are dow n-regulated in human prostate adenocarcinomas

The suppressive role of Notch 1 signaling on prostate growth and neoplastic transformation identified in these models is consistent with the previous study with human prostate cancer cell lines where it was shown that exogenous expression of a constitutively active form of Notchl inhibits proliferation of prostate cancer cell lines DU145, LNCaP and PC3 (Shou et al , Cancer Res 61 7291-7297 (2001)) To further determine the clinical relevance of these findings, we examined the expression of Notchl pathway genes in a Gene Logic database, which includes data from large sets of microarray analyses on various diseased human tissues, including tumors, as well as respective normal control tissue samples Strikingly, we found that the Notchl gene and one of the downstream target genes, Hey-1, were down-regulated significantly in prostate adenocarcinomas As shown in Fig 8, the mean expression level of Notchl in 51 prostate adenocarcinoma samples was significantly lower than normal prostates from patients without prostate cancer (p<0 01) and normal prostate tissues adjacent to prostate tumors as well (p<0 0001) Similarly, the mean level of Hey-1 in 93 prostate adenocarcinomas was significantly lower than both normal and tumor-adjacent normal tissues (p<0 0001) These data indicate that Notchl pathway, in particular, Notchl and Hey-1, may play an important role during prostatic tumoπgenesis Other Notchl pathway genes including receptors Notch!, Notch3, Notch4, ligands DU-I, Jag-1, Jag-2, and downstream genes Hes-1, Hes-4, Hes-6 and Hey-2, were included in Affymetπx chips used for the database But no significant alteration in their expression levels was found, showing that these genes are not as important as Notchl and Hey-1 in prostate tumoπgenesis Example 7: Expression of Notch 1 in E. coli

This example illustrates preparation of an unglycosylated form of Notch 1 by recombinant expression in E. coli.

The DNA sequence encoding Notch 1 is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 ( 1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the Notch 1 coding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on. After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized Notch 1 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.

Notch 1 may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding Notch 1 is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30⁰C with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate^«2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 niL water, as well as 110 itiM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSC^) and grown for approximately 20-30 hours at 30⁰C with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 I M and 0 02 M, respectively, and the solution is stirred overnight at 4⁰C This step results in a denatured protein with all cysteine residues blocked by sulfitohzation The solution is centπfuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min The supernatant is diluted with 3 5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tπs, pH 7 4) and filtered through 0 22 micron filters to clarify The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7 4 The protein is eluted with buffer containing 250 mM imidazole Fractions containing the desired protein are pooled and stored at 4°C Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence

The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of 20 mM Tπs, pH 8 6, 0 3 M NaCl, 2 5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA Refolding volumes are chosen so that the final protein concentiation is between 50 to 100 micrograms/ml The refolding solution is stirred gently at 4⁰C for 12-36 hours The refolding reaction is quenched by the addition of TFA to a final concentration of 0 4% (pH of approximately 3) Before further purification of the protein, the solution is, filtered through a 0 22 micron filter and acetonitπle is added to 2-10% final concentration The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0 1 % TFA with elution with a gradient of acetonitπle from 10 to 80% Ahquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled Generally the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitπle since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher acetonitπle concentrations In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples

Fractions containing the desired folded Notch 1 polypeptide are pooled and the acetonitrile removed using a gentle stream ot nitrogen directed at the solution Proteins are formulated into 20 mM Hepes, pH 6 8 with 0 14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered

Example 8 Expiession of Notchl in mammalian cells

This example illustrates preparation of a potentially glycosylated form of Notchl by recombinant expression in mammalian cells

The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector Optionally, the Notchl DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the Notchl DNA using ligation methods such as described in Sambrook et al , supra The resulting vector is called pRK5-Notchl In one embodiment, the selected host cells may be 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 μg pRK5-Notchl DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al , CeU, 31 543 (1982)] and dissolved in 500 μl of 1 mM Tπs-HCl, 0 1 mM EDTA, 0 227 M CaCl₂ To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl, 1 5 mM NaPO₄, and a precipitate is allowed to form for 10 minutes at 25⁰C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37⁰C The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds The 2^3 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml ^S-cysteine and 200 μCi/ml ³⁵S-methiomne After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel The processed gel may be dried and exposed to film for a selected period of time to re\ eal the presence of Notch 1 polypeptide The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays

In an alternative technique, Notch 1 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Natl Acad Sci 12 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-Notchl DNA is added The cells are first concentrated from the spinner flask by centπfugation and w ashed with PBS The DNA dextran piecipitate is incubated on the cell pellet for four hours The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0 1 μg/ml bovine transferrin After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expiessed Notch 1 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography In another embodiment, Notch 1 can be expressed in CHO cells The pRK5-Notchl can be transfected into CHO cells using known reagents such as CaPO₄ or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as ³⁵S-methionine After determining the presence of Notch 1 polypeptide, the culture medium may be replaced with serum free medium Preferably, the cultures are incubated for about b days, and then the conditioned medium is harvested The medium containing the expressed Notch 1 can then be concentrated and purified by any selected method

Epitope-tagged Notch 1 may also be expressed in host CHO cells The Notch 1 may be subcloned out of the pRK5 vector The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector The poly-his tagged Notchl insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones Finally, the

CHO cells can be transfected (as described above) with the SV40 driven vector Labeling may be performed, as described above, to verify expression The culture medium containing the expressed poly-His tagged Notchl can then be concentrated and purified by any selected method, such as by Ni²⁺-chelate affinity cliromatography Notchl may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure

Stable expression in CHO cells is performed using the following procedure The proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form

Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Current Piotocols of Molecular Biology Unit 3 16, John Wiley and Sons ( 1997) CHO expression vectors are constructed to have compatible restriction sites 5' and 3 of the DNA of interest to allow the convenient shuttling of cDNA's The vector used expression in CHO cells is as described in Lucas et al , Nucl Acids Res 24 °- (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dih>drofolate reductase (DHFR) DHFR expression permits selection for stable maintenance of the plasmid following transfection

Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect^® (Quiagen), Dosper^® or Fugene^® (Boehπnger Mannheim) The cells are grown as described in Lucas et al , supra Approximately 3 x 10⁷ cells are frozen in an ampule for further growth and pioduction as described below

The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0 2 μm filtered PS20 with 5% 0 2 μm diafiltered fetal bovine serum) The cells are then ahquoted into a 100 mL spinner containing ^QO mL of selective media After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37⁰C After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 x 10⁵ cells/mL The cell media is exchanged with fresh media by centπfugation and resuspension in production medium Although any suitable CHO media may be employed, a production medium described in U S Patent No 5,122,469, issued June 16, 1992 may actually be used A 3L production spinner is seeded at 1 2 x 10⁶ cells/mL On day 0, the cell number pH is determined On day 1, the spinner is sampled and sparging with filtered air is commenced On day 2, the spinner is sampled, the temperature shifted to 33⁰C, and 30 mL of 500 g/L glucose and 0 6 mL of 10% antifoam (e g , 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken Throughout the production, the pH is adjusted as necessary to keep it at around 7 2 After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0 22 μm filter The filtrate was either stored at 4⁰C or immediately loaded onto columns for purification

For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4⁰C After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -8O⁰C

Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 niM citric acid, pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tπs buffer, pH 9 The highly purified protein is subsequently desalted into stoiage buffer as described above for the poly-His tagged proteins The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation

Example 9 Expression of Notch 1 in Yeast

The following method describes recombinant expression of Notchl in yeast

First, yeast expression vectors aie constructed for intracellular production or secretion of Notchl from the ADH2/GAPDH promoter DNA encoding Notchl and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of Notchl For secretion, DNA encoding Notchl can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native Notchl signal peptide or other mammalian signal peptide, or, for example, a yeast alpha factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression ot Notch 1

Yeast cells such as yeast strain AB l 10 can then be transformed with the expression plasmids described above and cultured in selected fermentation media The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain

Recombinant Notchl can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters The concentrate containing Notchl may further be punfied using selected column chromatography resins

Example 10 Expression of Notchl in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of Notchl in Baculovirus-infected insect cells The sequence coding for Notchl is fused upstream of an epitope tag contained within a baculo virus expression vector Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding Notchl or the desired portion of the coding sequence of Notchl such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector

Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodoptetapugφeida ("Sf9") cells (ATCC CRL 1711) using hpofectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28⁰C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al , Baculovirus expression vectors A Laboratory Manual, Oxford Oxford University Press (1994) Expressed poly-his tagged Notchl can then be purified, for example, by Ni²⁺-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature, 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7 9, 12 5 mM MgCl₂, 0 1 mM EDTA, 10% glycerol, 0 1 % NP-40, 0 4 M KCl), and sonicated twice for 20 seconds on ice The sonicates are cleared by centπfugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 μm filter A Nr⁺ NTA agarose column (commercially available from Qiagen) ib prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer The filtered cell extract is loaded onto the column at 0 5 mL per minute The column is washed to baseline A_28O with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH b 0), which elutes nonspecifically bound protein After ieaching A₂₈₀ baseline again, the column is de\ eloped with a 0 to 500 mM Imidazole gradient in the secondary wash buffer One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni²⁺-NT A-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted Hisio-tagged Notch 1 are pooled and dialyzed against loading buffer

Alternatively pui ification of the IgG tagged (or Fc tagged) Notchl can be performed using know n chromatography techniques, including for instance, Protein A or protein G column chromatography

Example 11 Preparation of Antibodies that Bind Notchl

This example illustrates pieparation of monoclonal antibodies which can specifically bind Notchl Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra Immunogens that may be employed include purified Notchl, fusion proteins containing Notchl, and cells expressing recombinant Notch l on the cell surface Selection of the immunogen can be made by the skilled artisan w ithout undue experimentation

Mice, such as Balb/c, aie immunized with the Notchl immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro-oibital bleeding for testing in ELISA assays to detect anti-Notchl antibodies

After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of Notchl Three to four days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1, available from ATCC, No CRL 1597 The fusions generate hybπdoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopteπn, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids The hybπdoma cells will be screened in an ELISA for reactivity against Notchl Determination of

"positive" hybπdoma cells secreting the desired monoclonal antibodies against Notch l is within the skill in the art The positive hybπdoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-Notch 1 monoclonal antibodies Alternatively, the hybπdoma cells can be grown in tissue culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be emplo>ed

Example 12 Purification of Notch 1 Polypeptides Using Specific Antibodies

Native or recombinant Notchl polypeptides may be purified by a variety of standard techniques in the art of protein purification For example, pro-Notch 1 polypeptide, mature Notch 1 polypeptide, or pre-Notchl polypeptide is purified by immunoaffinity chromatography using antibodies specific for the Notchl polypeptide of interest In general, an immunoaffinity column is constructed by covalently coupling the anti-Notch 1 polypeptide antibody to an actn ated chromatographic resin

Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKJB Biotechnology, Piscataway N J ) Likew ise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology) The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufactuier's instructions

Such an immunoaffinity column is utilized in the purification of Notchl polypeptide by preparing a fraction from cells containing Notchl polypeptide in a soluble form This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively, soluble Notchl polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown A soluble Notch l polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of Notch l polypeptide (e g , high ionic strength buffers in the presence of detergent) Then, the column is eluted under conditions that disrupt antibody/Notch 1 polypeptide binding {e g , a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and Notchl polypeptide is collected

Example 13 Drug Screening

This invention is particularly useful for screening compounds by using Notchl polypeptides or binding fragment thereof in any of a variety of drug screening techniques The Notchl polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the Notchl polypeptide or fragment Drugs are screened against such transformed cells in competitive binding assays Such cells, either in viable or fixed form, can be used for standard binding assays One may measure, for example, the formation of complexes between Notchl polypeptide or a fragment and the agent being tested Alternatively, one can examine the diminution in complex formation between the Notch 1 polypeptide and its target cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs or any other agents which can affect a Notch 1 polypeptide-associated disease or disorder. These methods comprise contacting such an agent with a Notch 1 polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the Notch 1 polypeptide or fragment, or (ii) for the presence of a complex between the Notch 1 polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the Notch 1 polypeptide or fragment is typically labeled. After suitable incubation, free Notch 1 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to Notch 1 polypeptide or to interfere with the Notch 1 polypeptide/cell complex.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a Notch 1 polypeptide, the peptide test compounds are reacted with Notch 1 polypeptide and washed. Bound Notch 1 polypeptide is detected by methods well known in the art. Purified Notch 1 polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding Notch 1 polypeptide specifically compete with a test compound for binding to Notch 1 polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with Notch 1 polypeptide.

Example 14: Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e., a Notchl polypeptide) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the Notchl polypeptide or which enhance or interfere with the function of the Notchl polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)). In one approach, the three-dimensional structure of the Notchl polypeptide, or of a Notchl polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the Notchl polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the Notchl polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous Notchl polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry, 31 7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al , J Biochem , 1 13 742-746 ( 1993)

It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure This approach, in principle, yields a pharmacore upon which subsequent drug design can be based It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides The isolated peptides would then act as the pharmacore

By virtue of the present invention, sufficient amounts of the Notch 1 polypeptide may be made available to perform such analytical studies as X-ray crystallography In addition, knowledge of the Notch 1 polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography

Claims

What is Claimed is

1 A method of alleviating a prostate disorder in a mammal, comprising administering to said mammal, an effective amount of a Notch 1 polypeptide or an agonist thereof

2 A method of decreasing the proliferation of prostatic cells comprising contacting Notch 1 receptor with an agonist, thereby activating said Notch 1 receptor and decreasing the proliferation of said prostatic cells

3 The method of Claim2, wherein step of contacting occurs in \ itio

4 The method of Claim 1, wheiein said agonist is an anti-Notchl polypeptide antibody

5 The method of Claim 4 wherein said agonist is an antibody fragment

6 The method of Claim 4 wherein the antibody fragment is a Fab fragment

7 The method of Claim 1, wherein said agonist is a soluble Notch 1 polypeptide hgand

8 The method of Claim 7 wherein said soluble Notch 1 polypeptide hgand is selected from the group consisting of Jagged 1, Jagged2, Delta and Delta4

9 The method of Claim 1 , wherein the prostate disorder is prostate adenocarcinoma and metastatic prostate cancer

10 A method of diagnosing prostatic cancer in a mammal, comprising contacting a biological sample obtained from the prostate of said mammal with a Notch 1 probe, wherein increased expression of the

Notchl polypeptide is indicative of the presence of said prostatic cancer

11 The method of Claim 10 wheiein said Notchl probe is a nucleic acid probe

12 The method of Claim 11 wherein said Notchl probe is an antibody probe

13 A method of identifying a compound that activates the Notchl polypeptide, said method comprising contacting cells which express said Notchl polypeptide with a candidate compound, and determining the activation of said Notchl polypeptide

14 The method of Claim 13, wherein said candidate compound comprises an anti- Notch 1 antibody 15 The method of Claim 13, wherein said candidate compound comprises a soluble Notch 1 ligand selected from the group consisting of Jagged 1, Jagged2, Delta, and Delta4

16 An article of manufacture, comprising a container, a label on the container, and a composition comprising an active agent contained within the container, wherein the composition is effective for reducing prostate cancer in a mammal, the label on the container indicates that the composition can be used for treating prostate cancer, and the active agent in the composition is an agent activating the Notch 1 polypeptide

17 A method of detecting prostatic cancer in a sample suspected of containing a Notch 1 polypeptide, said method comprising contacting said sample with a Notch 1 probe, wheiein the binding of the probe is indicative of the presence of a Notchl polypeptide in said sample

18 The method according to Claim 17, wherein said sample comprises cells suspected of expressing said Notchl polypeptide

19 The method according to Claim 17, wherein said Notchl polypeptide is labeled with a detectable label or is attached to a solid support