WO2005037194A2

WO2005037194A2 - Collagen interactions with prostate cancer cells

Info

Publication number: WO2005037194A2
Application number: PCT/US2004/032516
Authority: WO
Inventors: Christopher L. Hall; Michael W. Long
Original assignee: The Regents Of The University Of Michigan
Priority date: 2003-10-02
Filing date: 2004-10-04
Publication date: 2005-04-28
Also published as: WO2005037194A3

Abstract

Osteoblastic lesions of the pelvis and vertebral column are the most frequent distant metastases formed in human prostate cancer (CaP). Since Type I collagen comprises 98% of the total protein in bone, the role of collagen binding in to metastatic prostate cancer cells was examined. While bone metastatic bound collagen, non-metastatic prostate cance cells were incapable of collagen binding. Antibodies to both α2β1 integrin and the GRGDTP peptide inhibited this binding, confirming the integrin-mediated attachment. In addition, metastatic prostate cancer cells exhibited an increased ability to migrate towards collagen I, as compared to non-metastatic cells, and α2β1-blocking antibodies prevented this migration.

Description

DESCRIPTION

COLLAGEN INTERACTIONS WITH PROSTATE CANCER CELLS BACKGROUND OF THE INVENTION

This application claims benefit of priority to U.S. Provisional Serial No. 60/508,230, filed October 2, 2003, the entire contents of which are hereby incorporated by reference. The government owns rights in the present invention pursuant to NLH grant RO1 DK 061456.

1. Field of th e Invention The present invention relates generally to the fields of biology and medicine. More particularly, it concerns methods for inhibiting prostate bone cancer metastasis.

2. Description of Related Art Prostate cancer (CaP) is the second leading cause of cancer death in men, with an estimated 189,000 men/year diagnosed as having prostate cancer. It is notable that 83% of all prostate cancers are discovered in the local and regional stages. In 2002, an estimated 30,200 men are expected to die of prostate cancer. Spine metastasis represents 90% of prostate cancer metastasis, and recurrence is common (45% risk within 2 years). Current therapies consist of surgical intervention, radiotherapy, hormone therapy, and chemotherapy. All of these have extensive side effects, and are directed at eradication of the primary tumor, often missing metastatic lesions, or not preventing this process. Thus, improved methods for dealing with metastatic bone cancer are needed.

Moreover, certain cancers, such as prostate and breast are characterized by metastases that induce localized bone proliferation. In the case of CaP, these cells clearly produce a signal that induces cells to produce bone tissue, whereas the others often form bone-eroding (osteolytic) lesions. Thus, the isolation of these factors not only provides a tool for inducing bone proliferation in damaged bone tissues, for halting erosion in osteolytic lesions, and their identification would permit intervention in osteogenic metastatic cancers. SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided a method of inhibiting metastasis of a cancer cell to bone comprising administering to a patient suffering from cancer a composition that interferes with binding of a cancer cell to Type 1 collagen. The patient may suffer from a cancer that is an osteoblastic cancer such as prostate cancer. The patient may suffer from a cancer that is osteolytic, such as breast cancer, multiple myeloma, prostate adenocarcinomas, sarcomatoid renal cell carcinoma, anal gland carcinoma, metastatic follicular thyroid carcinoma, leptomeningeal gliomatosis, metastatic cervical carcinoma, melanoma, medullary thyroid carcinoma, astrocytoma, ghoblastoma multiform, osteosarcoma, gastrinoma, bladder carcinoma, esophageal cancer, lung cancer, intercranial glioma, parotid carcinoma, bronchial carcinoid, renal cell carcinoma, and gastrointestinal carcinoid tumors.

The composition may be a peptide, a polypeptide, a peptidomimetic, a nucleic acid or an organopharmaceutical. The nucleic acid may be an expression construct that expresses a polypeptide product, an expression construct that expresses an antisense molecule, or a small interfering RNA. The antisense molecule or small interfering RNA may downregulate expression of Type I collagen in bone or 2/β 1 integrin in the cancer cell. The nucleic acid may comprise a sequence derived from the promoter, exons and/or introns of Type I collagen or α2/βl integrin genes. The polypeptide may be a monoclonal antibody, is part of a polyclonal antisera, is a F(ab), is a F(ab')₂ or a single chain antibody. The monoclonal antibody, polyclonal antisera, F(ab), F(ab')₂ or single chain antibody may bind to Type I collagen on bone or α2/βl integrin on the cancer cell. The polypeptide or peptide may comprise a sequence derived from Type I collagen or α2/βl integrin. The composition may either bind to, mimic or reduce the expression of Type 1 collagen of bone. The composition may either bind to, mimic or reduce the expression of α2/β 1 integrin of the cancer cells.

The composition may be is administered local to a tumor, regional to a tumor or systemically. The regional administration may comprise administration into a lymphatic tissue. The composition may be administered more than once, such as on a recurring basis. The method may further comprise administering to the patient a second cancer therapy, such as surgery, radiation, chemotherapy, hormonal therapy, immunotherapy or gene therapy. The patient may be a human, may appear to be in remission, or may be in relapse.

In another embodiment, there is provided a method of identifying an agent useful in preventing cancer cell metastasis to bone comprising (a) providing isolated α2/βl integrin; (b) providing isolated Type I collagen; (c) providing a candidate substance; and (d) contacting the α2/βl integrin in the presence of the candidate substance with the Type I collagen under conditions supporting the binding of α2/βl integrin to Type I collagen, wherein a difference in the binding seen in step (d), as compared to the binding in the absence of the candidate substance, identifies the candidate substance as bone metastasis inhibitor. The Type I collagen and/or the α2/βl integrin may be bound to a support, and the candidate substance may be peptide, a polypeptide, or peptidomimetic.

In yet another embodiment, there is provided a method of identifying an agent useful in preventing cancer cell metastasis to bone comprising (a) providing cell expressing 2/βl integrin; (b) providing a source of Type I collagen; (c) providing a candidate substance; and (d) contacting the cell in the presence of the candidate substance with Type I collagen under conditions supporting the binding of α2/βl integrin to the cell, wherein a difference in the binding seen in step (d), as compared to the binding in the absence of the candidate substance, identifies the candidate substance as bone metastasis inhibitor. The cell may be a prostate cancer cell line, such as LNCaP_coι, the binding may be measured by FACS, and the candidate substance may be a peptide, a polypeptide, a peptidomimetic, a nucleic acid or an organopharmaceutical.

In still yet another embodiment, there is provided a method of identifying an agent useful in preventing cancer cell metastasis to bone comprising (a) providing cell expressing oc2/βl integrin; (b) providing a source of Type I collagen; (c) contacting the cell with a candidate substance; and (d) measuring migration of the cell in the presence of the candidate substance towards Type I collagen, wherein a difference in the migration seen in step (d), as compared to the migration in the absence of the candidate substance, identifies the candidate substance as bone metastasis inhibitor.

In yet a further embodiment, there is provided a method of identifying an agent useful in preventing cancer cell metastasis to bone comprising (a) providing cell expressing α2/βl integrin; (b) providing a source of Type I collagen; (c) contacting said cell with a candidate substance; and (d) measuring the level of a transcript or polypeptide for a gene involved in bone cell metastasis, wherein a decrease in the level seen in step (d), as compared to the level in the absence of said candidate substance, identifies said candidate substance as bone metastasis inhibitor. Also provided is an engineered cancer cell designated as LNCaP_coι.

In yet another embodiment, there is provided a method of identifying an agent useful in preventing cancer cell metastasis to bone comprising (a) providing an cancer cell; (b) providing a candidate substance; (c) contacting the cell with the candidate substance, (d) measuring the growth of the cancer cell, and (e) determining cellular growth or proliferation, wherein a reduction in the growth or proliferation seen in step (e), as compared to the growth or proliferation in the absence of the candidate substance, identifies the candidate substance as bone metastasis inhibitor. Growth is defined as to increases or decreases in cell size, protein content, or maturational characteristics, without affecting cell number, whereas proliferation goes to cell number. The cell may be a prostate cancer cell line, such as LNCaP_coι, the proliferation or growth may be measured by MTT assay or other proliferation assays or by growth assays such as change in cell size by FACS analysis or changes in cell protein content by biochemical methods or by combinations of both categories of measurement. The assays may be performed on in vitro, for example, onplastic or on culture dishes coated with proteins such as collagen, and the candidate substance may be a peptide, a polypeptide, a peptidomimetic, a nucleic acid or an organopharmaceutical.

In yet another embodiment, there is provided a method of identifying an agent useful in preventing cancer cell metastasis to bone comprising (a) providing an cancer cell; (b) providing a candidate substance; (c) contacting the cell with the candidate substance; (d) injecting the cell into an animal, and (e) determining cellular invasion/metastasis, wherein a reduction in cellular invasion/metastasis seen in step (e), as compared to cellular invasion/metastasis in absence of the candidate substance, identifies the candidate substance as bone metastasis inhibitor. The cell may be a prostate cancer cell line, such as LNCaP_coι, the invasion and/or metastasis may be measured by Faxitron or other radiographically based methods, or histological examination using antibodies to cancer-specific markers such as prostate specific antigen (PAS), and the candidate substance may be a peptide, a polypeptide, a peptidomimetic, a nucleic acid or an organopharmaceutical. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Adhesion of Collagen-Selected LNCap Cells to ECM Molecules. Parental LNCaP cells were passaged on Type I collagen. Selected cells (LNCaP_coι) were then allowed to adhere to collagen or fibronectin (control). FIG. 2. α2/βl Integrin Expression on LNCaP_coι Cells. FIG. 3. Anti-Integrin Antibodies and RGD-Containing Peptide Sequences Inhibit LNCaP_coi Cell Adhesion to Collagen. FIG. 4. LNCCaP_coi Cells Show Increased Migration Towards Type I Collagen. Co = type 1 collagen; ab = anti-α2βl integrin; control = media only. FIG. 5. Attachment-Dependent Proliferation of Prostate Cancer Cells on Plastic or Protein-Coated Culture Dishes. Co = collagen, LNCaP, C4-2B, and LNCaPcoi = prostate cancer cells.

FIG 6. In Vivo Invasion of Cancer Cells in bone. Shown are representative plane x- ray/faxitron (top) and histological sections stained for PSA (bottom) of normal, uninjected tibia or those injected with 5xl0⁵ LNCaP or LNCaPcoi cells. Left-hand panels = non-injected tibia, center Panels = LNCaP cells injected; right-hand Panels = LNCaPCol cells injected. Faxitron = X-ray analysis of bone density, PAS LHC = immunohistochemistry for prostate specific antigen (PAS; dark intratibial deposits in middle and right hand panel).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Binding to organ specific factors is believed to facilitate the selective metastasis of cancer cells, notably prostate cancer in men. Therefore, the inventors tested whether bone metastasis of human prostate cancer cells is mediated by binding to Type I collagen, a factor that comprises 98% of the total protein in the bone. The data presented below show that metastatic human prostate cancer cells use the collagen receptor, α2/βl integrin, to mediate both the attachment to, and chemotactic migration towards, Type I collagen. Thus, compounds or molecules that interfere with this interaction (such as those than bind to, compete with or inhibit the expression of these molecules) serve as effective therapies against bone metastasis in human prostate and other cancers.

I. Type I Collagen

Collagens are insoluble, extracellular glycoproteins found in all animals, and are the most abundant proteins in the human body. They are essential structural components of all connective tissues, such as cartilage bone tendons ligaments fascia skin. Nineteen types of collagen have been found (so far) in humans. The major ones are: (a) Type I, the chief component of tendons, ligaments, and bones; (b) Type II, representing more than 50% of the protein in cartilage (also used to build the notochord of vertebrate embryos); (c) Type III, which strengthens the walls of hollow structures like arteries, the intestine, and the uterus; (d) and Type IV, which forms the basal lamina of epithelia. The basal lamina is often called the basement membrane, but is not related to lipid bilayer membranes. A meshwork of Type IV collagens provides the filter for the blood capillaries and the glomeruli of the kidneys. The other 15 types are probably equally important but they are much less abundant.

Type I collagen is the single most abundant protein in mammals. The strength and integrity of all connective tissues, including bone and vasculature, is dependent upon the presence of collagen fibrils. The basic structure of Type I collagen follows the formula

II. α2/βl Integrin Integrins are receptor proteins which are of crucial importance, and are involved in a variety of cellular functions such as wound healing, cell differentiation, homing of tumor cells and apoptosis. They are part of a large family of cell adhesion receptors involved in cell- extracellular matrix and cell-cell interactions. Functional integrins consist of two transmembrane glycoprotein subunits, called α and β, that are non-covalently bound. The α subunits all share some homology to each other, as do the β subunits. The receptors always contain one chain and one β chain and are thus called heterodimeric. Both of the subunits contribute to the binding of ligand. From 17 α and 8 β subunits, some 22 integrins are formed in nature, which implicates that not all possible combinations exist. The β4 subunit for instance can only form a heterodimer with the α6 subunit. On the other hand the βl subunit can form heterodimers with ten different α subunits. Because not all the βl/α heterodimers have the same ligand specificities, it is believed that the α chain is at least partly involved in the ligand specificity. The diversity of the integrins is increased by the alternative splicing of some integrin messenger RNA's.

Integrins are composed of long extracellular domains which adhere to their ligands, and short cytoplasmic domains that link the receptors to the cytoskeleton of the cell. There is however one exception to this rule, which is the β4 subunit. β4 contains a very large cytoplasmic domain of some 1000 amino acids, while the other integrins only have cytoplasmic domains of up to 60 amino acids.

The structure between the α subunits is very similar. All contain 7 homologous repeats of 30-40 amino acids in their extracellular domain, spaced by stretches of 20-30 amino acids. The three or four repeats that are most extracellular, contain sequences with cation-binding properties. These sequences are thought to be involved in the binding of ligands because the interaction of integrins with their ligand is cation-dependent. All the α subunits share the 5 amino acid motif GFFKR, which is located directly under the transmembrane region.

Integrins differ from other cell-surface receptors in that they bind their ligands with a low affinity and that they are usually present at 10-100 fold higher concentration on the cell surface. The integrins, however, can only bind their ligands when they exceed a certain mimmal number of integrins at certain places, called focal contacts and hemidesmosomes. So, when the integrins are diffusely distributed over the cell surface, no adhesion will be present, but when after a certain stimuli these integrins cluster for example in focal contacts their combined weak affinities give rise to a spot on the cell surface which has enough adhesive (sticking) capacity to adhere to the extracellular matrix. This is a very useful situation because in this way cells can bind simultaneously, but weakly, to large numbers of matrix molecules and still have the opportunity to explore their environment without losing all attachment to it by building or breaking down focal contacts.

Integrin-ligand interactions are accompanied by clustering and activation of the integrins on the cell surface, which is also accompanied by the transduction of signals into intracellular signal transduction pathways that mediate a number of intracellular events. Signaling through integrins depands on the formation of focal adhesions, dynamic sites in which cytoskeletal and other proteins are concentrated and which regulate migration and the shape of a cell (Scwartz, 1992).

Integrins can bind to an array of ligands. Common ligands are for example fibronectin and laminin, which are both part of the extracellular matrix or basal lamina's. Both of the ligands mentioned above are recognized by multiple integrins. For adhesion to ligands, both integin subunits are needed, as is the presence of cations. The α chain is the part that has cation binding sites.

Collagen is a particularly important ligand for both the αl/βl and α2/βl. l/βl and α2/βl integrin belong to a class of integrins that contain an inserted or "I" domain in their α subunits. It is this domain that is primarily responsible for binding collagen. The integrin "I" domains are also sometimes referred to as "A" domains and are connected to the body of the integrin by their N and C termini (Xiong et al, 2000). α2/βl integrin also serves as the receptor for the human pathogen echovirus-1 (Emsley et al, 1997). α2/βl integrin binds preferentially to type I collagen (Dickeson et al, 1999). α2/βl integrin is also capable of binding type IV collagen but at a lower affinity than type I (Xu et al, 2000). The I domain of 2/β 1 integrin is a 200 amino acid stretch that contain the residues necessary for the cation dependant adhesion to collagen. Integrins that contain an I domain typically adopt a dinucleotide-binding fold. α2/βl integrin features a metal ion dependant adhesion motif, more commonly referred to as a MIDAS site. This MIDAS site is at the center of a trench shaped section of the I domains and contains the residues necessary to bind to collagen. The MIDAS site is made up of six residues D151, S153, S155, T221, D254, and E256 (Emsley et al., 2000). α2/βl integrin has been shown to bind collagen in the presence of magnesium and manganese ions, but not in the presence of calcium. In the presence of the appropriate divalent cation, α2/βl integrin binds to the collagen triple helix. The collagen triple helix is composed of repeating sequences GXY (X is usually proline and Y is usually hydroxyproline) (Xu et al, 2000). The specific sequence that binds to the A2 I domain has been identified as GFOGER (O= hydroxyproline) (Emsley et al, 2000).

III. Cancers Involving Bone The present invention provides methods for inhibiting the involvement of bone metastasis in certain cancers. In one aspect, the cancer is an osteoblastic cancer, where metastasis results in abnormal bone growth. Such cancers are represented by prostate cancer. In another aspect, the cancer may be an osteolytic cancer, such as multiple myeloma, breast cancer, sarcomatoid renal cell carcinoma, anal gland carcinoma, metastatic follicular thyroid carcinoma, leptomeningeal gliomatosis, metastatic cervical carcinoma, melanoma, medullary thyroid carcinoma, astrocytoma, ghoblastoma multiform, osteosarcoma, gastrinoma, bladder carcinoma, esophageal cancer, lung cancer, intercranial glioma, parotid carcinoma, bronchial carcinoid, renal cell carcinoma, and gastrointestinal carcinoid tumors.

IV. Peptide and Polypeptides hi one aspect of the invention, a putative inhibitor is a peptide or polypeptides that is derived from or mimics the structure of Type I collagen or α2/βl integrin. Peptides are generally of about 10 to no more than about 50 amino acids in length comprising a segment of 10 or more residues from the polypeptide discussed above. These peptides (or fragments) of the polypeptides that may not retain all of the functions of Type I collagen or α2/βl integrin, but will retain their binding function.

Peptides may be produced de novo using chemical synthesis. Fragments, including the N- terminus of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below). Alternatively, treatment of the polypeptide with proteolytic enzymes, known as proteases, can produce a variety of N-terminal, C-terminal and internal fragments. Examples of fragments may include contiguous residues of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300, 400 or more amino acids in length. These fragments may be purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration). 1. Variants of Polypeptides Amino acid sequence variants of Type I collagen or α2/βl integrin can also be substiturional, insertional or deletion variants, where binding functions are retained. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, without the entire loss of other functions or properties. Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure (Johnson et al, 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule.

2. Purification of Proteins It will be desirable to purify the novel polypeptide sequences of the present invention or variants thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC. Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally- obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur. Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "- fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide. There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample. Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight. Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.). A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins; other lectins that have been include lentil lectin and wheat germ agglutinin, which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N- acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus. The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.

3. Synthetic Peptides As discussed above, the present invention encompasses peptides of the larger polypeptide sequences. Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.

Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young (1984); Tarn et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

V. Nucleic Acids Nucleic acids are a particular composition that may be used in conjunction with the present invention. These molecules fall into two different categories - nucleic acid vectors that express proteins of interest, and inhibitory nucleic acids. Each of these are discussed below. With regard to vectors, the expression of fragments of either Type I collagen or α2/βl integrin are contemplated as inhibitors of bone metastasis. With regard to inhibitory nucleic acids, antisense, ribozymes and siRNA directed against α2/βl integrin sequences are contemplated. 1. Expression Vectors DNA vectors form important further aspects of the present invention. The term

"expression vector or construct" means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed into mRNA. The transcript may be translated into a protein, but it need not be.

Thus, in certain embodiments, expression includes both transcription of a gene and the translation of its RNA into a gene product (protein). In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense constructs. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The promoter may be in the form of the promoter that is naturally associated with a gene encoding a bone cell spheroid enhancing protein, as may be obtained by isolating the 5' non- coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Of particular use are promoters and enhancers that direct transcription of genes that are specific for or highly expressed in bone tissue, osteoblasts and bone precursor cells. For instance, the promoter and enhancer elements of type I collagen, alkaline phosphatase, other bone matrix proteins such as osteopontin, osteonectin and osteocalcin, as well as c-Fos, which is expressed in large amounts in bone and cartilaginous tissues in the generation process, would all be useful for the expression of bone cell spheroid enhancing constructs of the present invention. In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of nucleic acids. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression are contemplated as well, provided that the levels of expression are sufficient for a given purpose. Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will generally process the genomic transcripts to yield functional mRNA for translation into protein. Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude or more larger than the cDNA gene. However, it is contemplated that a genomic version of a particular gene may be employed where desired.

2. Inhibitory Nucleic Acids The second category of nucleic acids are generally termed inhibitory nucleic acids. While these may be used directly, they may be provided using DNA vector, as discussed above. The term "antisense nucleic acid" is intended to refer to the oligonucleotides complementary to the base sequences of DNA and RNA. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target nucleic acid and interfere with transcription, RNA processing, transport and/or translation. Targeting double-stranded (ds) DNA with oligonucleotide leads to triple-helix formation; targeting RNA will lead to double- helix formation. Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. Antisense RNA constructs, or DNA encoding such antisense RNAs, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject. Nucleic acid sequences comprising "complementary nucleotides" are those which are capable of base-pairing according to the standard Watson-Crick complementary rules. That is, that the larger purines will base pair with the smaller pyrimidines to form only combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T), in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. As an alternative to targeted antisense delivery, targeted ribozymes may be used. The term "ribozyme" refers to an RNA-based enzyme capable of targeting and cleaving particular base sequences in oncogene DNA and RNA. Ribozymes either can be targeted directly to cells, in the form of RNA oligo-nucleotides incorporating ribozyme sequences, or introduced into the cell as an expression construct encoding the desired ribozymal RNA. Ribozymes may be used and applied in much the same way as described for antisense nucleic acids. In certain embodiments, the nucleic acid is an RNA molecule. For example, the RNA molecule can be a messenger RNA (mRNA) molecule, hi other embodiments, the RNA molecule is an interfering RNA. RNA interference (RNAi) is a form of gene silencing triggered by double-stranded RNA (dsRNA). DsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity. Fire et al. (1998); Grishok et al. (2000); Ketting et al. (1999); Lin and Avery (1999); Montgomery et al. (1998); Sharp (1999); Sharp and Zamore (2000); Tabara et al. (1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction. RNAj offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene. Fire et al. (1998); Grishok et al. (2000); Ketting et al (1999); Lin and Avery (1999); Montgomery et al. (1998); Sharp (1999); Sharp and Zamore (2000); Tabara et al. (1999). RNAj also is incredibly potent. It has been estimated that only a few copies of dsRNA are required to knock down >95% of targeted gene expression in a cell. Fire et al. (1998). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, C. elegans and Drosophila. Grishok et al. (2000); Sharp (1999); Sharp and Zamore (1999). In order to mediate the effect transgene expression in a cell, it will be necessary to transfer the expression vectors of the present invention into a cell. Such transfer may employ viral or non- viral methods of gene transfer. This section provides a discussion of methods and compositions of gene transfer.

3. Viral Vector-Mediated Transfer The expression constructs discussed above may be incorporated into an infectious particle to mediate gene transfer to a cell. Additional expression constructs as described herein may also be transferred via viral transduction using infectious viral particles, for example, by transformation with an adenovirus vector of the present invention as described herein below. Alternatively, lentiviral, retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a gene(s) of interest. Thus, in one example, viral infection of cells is used in order to deliver therapeutically significant genes to a cell. Typically, the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus. Though adenovirus is exemplified, the present methods may be advantageously employed with other viral vectors, as discussed below. Adenovirus. Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. The roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained -acting elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990). The products of the late genes (Ll, L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 map units) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5' tripartite leader (TL) sequence which makes them preferred mRNAs for translation. In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products. The two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease. The large displacement of DNA is possible because the cis elements required for viral « DNA replication all are localized in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non- defective adenovirus (Hay et al, 1984). Therefore, inclusion of these elements in an adenoviral vector should permit replication. In addition, the packaging signal for viral encapsidation is localized between 194-385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al, 1987). This signal mimics the protein recognition site in bacteriophage λ DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure. El substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al, 1991). Previously, it has been shown that certain regions of the adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants. Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element, as provided for in the present invention, derives from the packaging function of adenovirus. It has been shown that a packaging signal for adenovirus exists in the left end of the conventional adenovirus map (Tibbetts, 1977). Later studies showed that a mutant with a deletion in the El A (194-358 bp) region of the genome grew poorly even in a cell line that complemented the early (E1A) function (Hearing and Shenk, 1983). When a compensating adenoviral DNA (0-353 bp) was recombined into the right end of the mutant, the virus was packaged normally. Further mutational analysis identified a short, repeated, position-dependent element in the left end of the Ad5 genome. One copy of the repeat was found to be sufficient for efficient packaging if present at either end of the genome, but not when moved towards the interior of the Ad5 DNA molecule (Hearing et al, 1987). By using mutated versions of the packaging signal, it is possible to create helper viruses that are packaged with varying efficiencies. Typically, the mutations are point mutations or deletions. When helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper. When these helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions. Given a limiting amount of packaging factor, the virus containing the wild-type signals are packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity should be achieved. Retrovirus. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed Ψ, functions as a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990). In order to construct a retroviral vector, a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol and env genes but without the LTR and Ψ components is constructed (Mann et al, 1983). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and Ψ sequences is introduced into this cell line (by calcium phosphate precipitation for example), the Ψ sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al, 1975). An approach designed to allow specific targeting of retrovirus vectors recently was developed based on the chemical modification of a retrovirus by the chemical addition of galactose residues to the viral envelope. This modification could permit the specific infection of cells such as hepatocytes via asialoglycoprotein receptors, should this be desired. A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, the infection of a variety of human cells that bore those surface antigens was demonstrated with an ecotropic virus in vitro (Roux et al, 1989). Adeno-associated Virus. AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription. The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript. AAV is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication. Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block. The terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al. 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. The ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration. AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, 1995 ; Chatterjee et al, 1995; Ferrari et al, 1996; Fisher et al, 1996; Flotte et al, 1993; Goodman et al, 1994; Kaplitt et al, 1994; 1996, Kessler et al, 1996; Koeberl et al, 1997; Mizukami et al, 1996; Xiao et al, 1996). AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1995; Flotte et al, 1993). Similarly, the prospects for treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart, appear promising since AAV- mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al, 1996; Flotte et al, 1993; Kaplitt et al, 199 A; 1996; Koeberl et al, 1997; McCown et al, 1996; Ping et al, 1996; Xiao et al, 1996). Lentivims. Lentivirus vectors based on human immunodeficiency virus (HIV) type 1 (HIV-1) constitute a recent development in the field of gene therapy. A key property of HIV-1- derived vectors is their ability to infect nondividing cells. High-titer HIV-1 -derived vectors have been produced. Examples of lentiviral vectors include White et al. (1999), describing a lentivirus vector which is based on HIV, simian immunodeficiency virus (SIV), and vesicular stomatitis virus (VSV) and which we refer to as HIV/SrVpack/G. The potential for pathogenicity with this vector system is minimal. The transduction ability of HIV/SIVpack/G was demonstrated with immortalized human lymphocytes, human primary macrophages, human bone marrow-derived CD34(+) cells, and primary mouse neurons. Gasmi et al. (1999) describe a system to transiently produce HΓV-1 -based vectors by using expression plasmids encoding gag, pol, and tat of HIV-1 under the control of the cytomegalovirus immediate-early promoter. Other Viral Vectors. Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) canary pox virus, and herpes viruses may be employed. These viruses offer several features for use in gene transfer into various mammalian cells. 4. Non-viral Transfer DNA constructs of the present invention are generally delivered to a cell, or inhibitory nucleic acids themselves in certain situations, and these may be transferred using non-viral methods. Several non-viral methods for the transfer of expression constructs into cultured mammalian cells are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harlan and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979), cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Once the construct has been delivered into the cell the nucleic acid encoding the bone cell spheroid-enhancing construct may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. hi a particular embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et αl., 1997). These DNA-lipid complexes are potential non- viral vectors for use in gene therapy. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Using the β-lactamase gene, Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection. Also included are various commercial approaches involving "lipofection" technology. Hong et al. (1997) describe stable complexes of cationic liposomes with plasmid DNA were prepared by (1) including a small amount of polyethyleneglycol-phospholipid conjugate or (2) condensing the DNA with polyamines prior to the formation of liposome-plasmid complexes. These preparations were stable for months at 4°C and gave reproducible high transfection activity for in vivo gene delivery. In these formulations cholesterol, not dioleoylphosphatidylethanolamine, was the helper lipid effective for sustaining high transfection activity in vivo. Commercially available lipofection reagents include CellPhect Transfection Kit (Amersham-Pharmacia Biotech), CytoFectene Reagent (Bio-Rad), CLONrectin Reagent (Clontech), Cytofectin (Glen Research), Perfect Lipid™ Transfection Kit (rnvitrogen), EuFectin (JBL Scientific), Lipofectamine™ 2000, Lipofectamine Plus™, Lipofectamine™, DMRLE-C Reagent (Life Technologies), ExGen 500 (MBI Fermentas), TransT LT-1 and LT-2 (PanVera), Transfast™ and Tr_x™ Reagents (Promega), SuperFect™ Transfection Reagent (Qiagen), LipoTAXI™ (Stratagene) and Genetransfer HMG- 1 ,-2 Mixture (Wako Chemicals USA). In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al, 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Other vector delivery systems which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor- mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferring (Wagner et al, 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al, 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0 273 085). hi other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, the human prostate-specific antigen (Watt et al, 1986) may be used as the receptor for mediated delivery of a nucleic acid in prostate tissue. In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of CaPO₄ precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of CaPO precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a CAM may also be transferred in a similar manner in vivo and express CAM. Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. VI. Antibodies In one embodiment of the present invention, one will desire to prepare antibodies against α2/βl integrin and/or Type I collagen. An antibody can be a polyclonal or a monoclonal antibody. In one embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isofoπns of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) Multiple Antigenic Peptide (MAP) or bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs. MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, and frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 107 to 2 x 108 lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, around 1 x 10-6 to 1 x 10-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methofrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purities and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells. This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

VII. Combination Therapies In order to increase the effectiveness of a cancer therapy that targets α2/βl integrin or Type I collagen, it may be desirable to combine such therapies with other agents effective in the treatment of hyperproliferative disease, such as anti-cancer agents. An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s). Thus, in the context of the present invention, it is contemplated that α2/βl integrin or Type I collagen therapy could be used in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention. The α2/βl integrin or Type I collagen therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and the α2/βl integrin or Type I collagen therapy are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other, hi some situations, it may be desirable to extend the time period for treatment significantly, however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Various combinations may be employed, α2/βl integrin or Type I collagen therapy is "A" and the secondary agent, such as radio- or chemotherapy, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A A/A/B B/A/A/A A/B/A A A/A/B/A

Administration of the agents of the present invention to a patient are believed to follow general protocols for the administration of chemotherapeutics or gene therapeutics (depending on the agent), taking into account the toxicity, if any, of the agent. It is expected that the treatment cycles would be repeated as necessary.

1. Chemotherapy Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing. 2. Radiotherapy Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV- irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

3. Immunotherapy hnmunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pl55. 4. Genes In yet another embodiment, the secondary treatment is a gene therapy. Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues. i. Inducers of Cellular Proliferation The proteins that induce cellular proliferation further fall into various categories dependent on function. The commonality of all of these proteins is their ability to regulate cellular proliferation. For example, a form of PDGF, the sis oncogene, is a secreted growth factor. Oncogenes rarely arise from genes encoding growth factors, and at the present, sis is the only known naturally-occurring oncogenic growth factor. In one embodiment of the present invention, it is contemplated that anti-sense mRNA directed to a particular inducer of cellular proliferation is used to prevent expression of the inducer of cellular proliferation. The proteins FMS, ErbA, ErbB and neu are growth factor receptors. Mutations to these receptors result in loss of regulatable function. For example, a point mutation affecting the transmembrane domain of the Neu receptor protein results in the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. The modified oncogenic ErbA receptor is believed to compete with the endogenous thyroid hormone receptor, causing uncontrolled growth. The largest class of oncogenes includes the signal transducing proteins (e.g., Src, Abl and

Ras). The protein Src is a cytoplasmic protein-tyrosine kinase, and its transformation from proto-oncogene to oncogene in some cases, results via mutations at tyrosine residue 527. In contrast, transformation of GTPase protein ras from proto-oncogene to oncogene, in one example, results from a valine to glycine mutation at amino acid 12 in the sequence, reducing ras GTPase activity. The proteins Jun, Fos and Myc are proteins that directly exert their effects on nuclear functions as transcription factors. ii. Inhibitors of Cellular Proliferation The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors p53, pl6 and C-CAM are described below. High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al, 1991) and in a wide spectrum of other tumors. The p53 gene encodes a 393-amino acid phosphoprotein that can form complexes with host proteins such as large-T antigen and E1B. The protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene. A single genetic change prompted by point mutations can create carcinogenic p53. Unlike other oncogenes, however, p53 point mutations are known to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity. Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line. Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991). Another inhibitor of cellular proliferation is p 16. The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the Gj . The activity of this enzyme may be to phosphorylate Rb at late Gi. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6^mKA has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al, 1995). Since the plό¹¹^⁴ protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein. pl6 also is known to regulate the function of CDK6. p!6^m belongs to a newly described class of CDK-inhibitory proteins that also includes pl6^B, pl9, p21^WAF1, and p27^Hpl. The p!6^{m A} gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6 ^κ4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6^mK4 gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency of the plό¹¹^⁴ gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al, 1994; Cheng et al, 1994; Hussussian et al, 1994; Kamb et al, 199 ; Kamb et al, 199 ; Mori et al, 199 A; Okamoto et al, 199 A; Nobori et al, 1995; Orlow et al, 199 A; Arap et al, 1995). Restoration of wild-type pl6 ^κ4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995). Other genes that may be employed according to the present invention include Rb, APC,

DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, ρ27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS,

Dp, E2F, ras, myc, neu, ra erb, fins, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDALF, or their receptors) and MCC. iii. Regulators of Programmed Cell Death Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al, 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists. Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BCI_XL, Bcl_w, Bcls, Mcl-1, Al, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

5. Surgery Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, or 12 months. These treatments maybe of varying dosages as well.

6. Other agents It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include irnmunoniodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperprohferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; LL-2 and other cytokines; F42K and other cytokine analogs; or MLP-1, MJP-lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abililties of the present invention by establishment of an autocrine or paracrine effect on hyperprohferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperprohferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy. Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

VIII. Modulators and Screening Assays In still further embodiments, the present invention provides methods for identifying new compounds that inhibit bone cancer metastasis. It may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assays will be structurally related to α2/β 1 integrin or Type I collagen. Such compounds, which may include peptidomimetics of peptide modulators, may be used in the same mam er as the initial modulators. Certain compounds that mimic elements of protein secondary structure are designed using the rationale that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule. Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteins, which are known to be highly antigenic. Likely β-turn structure within a polypeptide can be predicted by computer-based algorithms. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains. The generation of further structural equivalents or mimetics may be achieved by the techniques of modeling and chemical design known to those of skill in the art. The art of computer-based chemical modeling is now well known. Using such methods, a chemical that specifically modulates bone cell spheroid formation can be designed, and then synthesized, following the initial identification of a compound that modulates bone cell spheroid formation, but that is not specific or sufficiently specific to other human or animal bone formation properties. It will be understood that all such sterically similar constructs and second generation molecules fall within the scope of the present invention. It also is possible to use antibodies to ascertain the structure of a target compound activator or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen. Alternatively, candidate substances may be derived from natural samples, such as rain forest and marine samples. Candidate compounds may include fragments or parts of naturally- occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, and fungi. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators. On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds. As used herein the term "candidate substance" refers to any molecule that may potentially inhibit cancer metastasis. The candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. Specific inhibitors include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for a target molecule. Such compounds are described in greater detail elsewhere in this document. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors.

1. In vitro Assays A quick, inexpensive and easy assay to run is an in vitro assay. Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads, permitting parallel screening of multiple candidates. One example of a cell free assay is a binding assay. While not directly addressing function, the ability of an inhibitor to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of an inhibotor molecule to 2/β 1 integrin or Type I collagen may, in and of itself, be inhibitory, due to steric, allosteric or charge- charge interactions. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determining of binding. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding. A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods.

2. In cyto Assays The present invention also contemplates the screening of compounds for their ability to. inhibit cancer cell binding to and migration towards Type I collagen. Various cell lines can be utilized for such screening assays. These include but are not limited to the following cell lines: VCaP. DuCaP, LNCaP, LNCaPcoi, and B42B. Depending on the assay, culture may be required. The cell is examined using any of a number of different physiologic assays. Alternatively, molecular analysis may be performed, for example, looking at protein expression, mRNA expression (including differential display of whole cell or polyA RNA) and others.

3. In vivo Assays In vivo assays involve the use of various animal models, including transgenic animals that have been engineered to have specific defects, or carry markers that can be used to measure the ability of a candidate substance to reach and effect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred embodiment, especially for transgenics. However, other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons). Assays for modulators may be conducted using an animal model derived from any of these species. In such assays, one or more candidate substances are administered to an animal, and the ability of the candidate substance(s) to inhibit bone metastasis, as compared to a similar animal not treated with the candidate substance(s), identifies an inhibitor. As well, CaP cells or cell lines (e.g., C4 2B, or LNCaP_coι) that are known to be metastatic can be administered as targets for the compound. Treatment of these animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, or even topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.

Specifically contemplated routes are systemic intravenous injection, regional administration via blood or lymph supply, or directly to an affected site. Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.

IX. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Osteoblastic lesions of the pelvis and vertebral column are the most frequent distant metastases formed in human prostate cancer (CaP). Selective binding to organ specific factors likely facilitate specific metastasis of cancer cells. Therefore, the inventors evaluated whether bone metastasis of CaP cells is mediated by binding to Type I collagen, an extracellular matrix protein that comprises 98% of the total protein in the bone. To examine the role of collagen binding in the metastatic phenotype of CaP, human LNCaP cells, which routinely fail to bind to Type 1 collagen, were selected for collagen binding by serial passage on type 1 collagen. These collagen-selected cells (LNCaPcoi) were compared to parental LNCaP cells, and the highly metastatic, isogenic CaP variants LNCaP-LN3 (that metastasize to lymph nodes) and C4-2B (that metastasize to bone). The data show that equivalent numbers of LNCaPcoi cells adhere to collagen, as do the highly metastatic C4-2B cells (both cell types show an average of 34 ± 2% adhesion, n = 3; FIG. 1). In sharp contrast, the non- metastatic parental LNCaP and lymph node metastatic LN3 cells were incapable of collagen binding. LNCaPcoi attachment to fibronectin is equivalent to the parental LNCaP cells indicating the collagen-selection was specific for this, and not other, ECM proteins

Addressing the mechanism of this adhesive interaction, FACS analysis demonstrated that both C42B and LNCaPcoi express equivalent levels of the integrin caβi, a known collagen receptor (FIG. 2). Importantly, both antibodies to o2βl and the GRGDTP peptide, known to interfere with the integrin binding site, inhibit C42B and LNCaPcoi binding thus confirming the integrin-mediated attachment (FIG. 3). These observations also directly demonstrate the feasibility of using antibodies or peptides to inhibit metastatic CaP cell adhesion.

Type I collagen also directly effects CaP cells by acting as a chemoattractant. Cell migration assays show that both LNCaPcoi and C4-2B cells had an increased ability to migrate towards collagen 1 compared to parental LNCaP cells and Q2/31 blocking antibodies prevented this migration (FIG. 4). Thus, antibodies or peptides reduce the infiltration (i.e., migration and invasiveness) potential of CaP cells.

The LNCaPcoi cell line represents a novel entity and is the product of a unique approach, i.e., increasing the adhesive/metastatic phenotype of human LNCaP cells by serial passage on

Type 1 collagen. The inhibition of these cells from binding by antibody and peptide treatment clearly shows its utility as a screening tool for compounds and molecules that inhibit CaP collagen binding and hence metastasis.

EXAMPLE 2

The role of cell proliferation in the metastatic phenotype of prostate cancer (CaP) is shown in studies of human LNCaP cells, which routinely fail to bind to Type 1 collagen, that were selected for collagen binding by serial passage on type 1 collagen. These collagen-selected cells (LNCaPcoi) were compared to the proliferation of parental LNCaP cells, and the highly metastatic, isogenic CaP variants LNCaP-LN3 (that metastasize to lymph nodes) and C4-2B (that metastasize to bone). The data show that equivalent numbers of LNCaPcoi cells seeded in culture dishes (i.e., on plastic), or in culture dishes coated with collagen (to which they show increased adhesion or binding) proliferate more rapidly than do the other prostate cells (n = 3; FIG. 5). Thus, the non-metastatic parental LNCaP, the lymph node metastatic LN3 cells, and the bone-metastatic C4-2B all proliferated slower (decreased slope of proliferation curves in FIG. 5) and to a lesser degree than LNCaPcoi, on either surface. The inhibition of the proliferation of these cells from binding by antibody, peptide treatment, RNAs, etc., clearly shows utility as a screening tool for compounds and molecules that inhibit CaP collagen binding and hence metastasis. The in vivo invasiveness of CaP cells as a component of the metastatic phenotype was investigated using human LNCaP cells, selected for collagen binding by serial passage on type 1 collagen (i.e., LNCaPcoi cells as described above). Intra-tibial injections of equal numbers of LNCaP or LNCaPcoi cells were made into the marrow space and the animals followed for the development of CaP cells in vivo. Bone tumor growth was evaluated 9 weeks post injection by Faxitron radioscopic scan for changes in bone content, and by immunohistochemistry (LHC) for the presence of Prostate Specific Antigen (PAS-IHC) on the surface of the CaP cells (FIG. 6). Radiographic analysis indicates that LNCaPcoi cells stimulate the development of large bone tumors in injected animals (FIG. 6, top row). Compared to control, non-injected animals (top- row, left-hand panes), injected parental LNCaP cells (top row, center panel) cause little, if any, in crease in bone formation (i.e., there is little change in the radioopacity of the injected tibia). In sharp contrast, LNCaPcoi cells stimulate the formation of large bone tumors, as indicated by the bright white areas in the radiograph and a general increase in bone density, as indicated in the increased bone-opacity of the entire tibia (top row, right-hand panel). The presence of persisting LNCaP cells is seen in PAS-IHC studies of the injected tibia (bottom row center panel; non- injected is left-hand panel). While some residual LNCaP cells are apparent in this tibia (darker staining regions indicate P AS-positive cells), comparison of the radiographs immediately above show these residual LNCaP cells do not stimulate bone formation. Importantly, LNCaPcoi cells engraft and proliferate in injected animals, generating a large tumor within and surrounding the injected limb. Thus, this in vivo methodology provides a method of identifying agents useful in preventing cancer cell metastasis to bone in which injected cells are treated with antibody, peptide treatment, RNAs, etc., and the degree of cellular invasion/metastasis identifies the candidate substance as bone metastasis inhibitor. * * * * * * * * * * * * * * * * All of the compositions and or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

X. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. A method of inhibiting metastasis of a cancer cell to bone comprising admimstering to a patient suffering from cancer a composition that interferes with binding of a cancer cell to Type 1 collagen.

2. The method of claim 1, wherein said patient suffers from a cancer that is an osteoblastic cancer such as prostate cancer,.

3. The method of claim 1, wherein said patient suffers from a cancer that is osteolytic, such as multiple myeloma, breast cancer, sarcomatoid renal cell carcinoma, anal gland carcinoma, metastatic follicular thyroid carcinoma, leptomeningeal gliomatosis, metastatic cervical carcinoma, melanoma, medullary thyroid carcinoma, astrocytoma, ghoblastoma multiform, osteosarcoma, gastrinoma, bladder carcinoma, esophageal cancer, lung cancer, intercranial glioma, parotid carcinoma, bronchial carcinoid, renal cell carcinoma, and gastrointestinal carcinoid tumors.

4. The method of claim 1, wherein said composition is a peptide, a polypeptide, a peptidomimetic, a nucleic acid or an organopharmaceutical.

5. The method of claim 4, wherein said nucleic acid is an expression construct that expresses a polypeptide product, an expression construct that expresses an antisense molecule, or a small interfering RNA.

6. The method of claim 5, wherein said antisense molecule or small interfering RNA downregulates expression of Type I collagen in bone or α2/βl integrin in said cancer cell.

7. The method of claim 5, wherein said nucleic acid comprises a sequence derived from the promoter, exons and/or introns of Type I collagen or α2/βl integrin genes.

8. The method of claim 4, wherein said polypeptide is monoclonal antibody, is part of a polyclonal antisera, is a F(ab), is a F(ab')₂ or a single chain antibody.

9. The method of claim 8, wherein said monoclonal antibody, polyclonal antisera, F(ab), F(ab')₂ or single chain antibody binds to Type I collagen on bone or α2/βl integrin on said cancer cell.

10. The method of claim 4, wherein said polypeptide or peptide comprises a sequence derived from Type I collagen or α2/β 1 integrin.

11. The method of claim 1, wherein said composition either binds to, mimics or reduces the expression of Type 1 collagen of bone.

12. The method of claim 1, wherein said composition either binds to, mimics or reduces the expression of α2/βl integrin of said cancer cells.

13. The method of claim 1, wherein said composition is administered local to a tumor, regional to a tumor or systemically.

14. The method of claim 13, wherein regional administration comprises administration into a lymphatic tissue.

15. The method of claim 1, wherein said composition is administered more than once.

16. The method of claim 1, wherein said composition is administered on a recurring basis.

17. The method of claim 1, further comprising administering to said patient a second cancer therapy.

18. The method of claim 1, wherein said second cancer therapy is surgery, radiation, chemotherapy, hormonal therapy, immunotherapy or gene therapy.

19. The method of claim 1 , wherein said patient is a human.

20. The method of claim 1, wherein said patient appears to be in remission.

21. The method of claim 1, wherein said patient is in relapse.

22. A method of identifying an agent useful in preventing cancer cell metastasis to bone comprising:

(a) providing isolated α2/βl integrin;

(b) providing isolated Type I collagen;

(c) providing a candidate substance; and (d) contacting said α2/βl integrin in the presence of said candidate substance with said Type I collagen under conditions supporting the binding of α2/βl integrin to Type I collagen, wherein a difference in the binding seen in step (d), as compared to the binding in the absence of said candidate substance, identifies said candidate substance as bone metastasis inhibitor.

23. The method of claim 22, wherein Type I collagen is bound to a support.

24. The method of claim 22, wherein α2/β 1 integrin is bound to a support.

25. The method of claim 22, wherein said candidate substance is peptide, a polypeptide, or peptidomimetic.

26. A method of identifying an agent useful in preventing cancer cell metastasis to bone comprising:

(a) providing cell expressing α2/βl integrin;

(b) providing a source of Type I collagen;

(c) providing a candidate substance; and

(d) contacting the cell in the presence of said candidate substance with Type I collagen under conditions supporting the binding of α2/βl integrin to said cell, wherein a difference in the binding seen in step (d), as compared to the binding in the absence of said candidate substance, identifies said candidate substance as bone metastasis inhibitor.

27. The method of claim 26, wherein said cell is a prostate cancer cell line.

28. The method of claim 27, wherein said prostate cancer cell line is LNCaP_coι.

29. The method of claim 26, wherein binding is measured by F ACS .

30. The method of claim 26, wherein said candidate substance is a peptide, a polypeptide, a peptidomimetic, a nucleic acid or an organopharmaceutical.

31. A method of identifying an agent useful in preventing cancer cell metastasis to bone comprising:

(a) providing cell expressing α2/β 1 integrin;

(b) providing a source of Type I collagen;

(c) contacting said cell with a candidate substance; and

(d) measuring migration of said cell in the presence of said candidate substance towards Type I collagen, wherein a difference in the migration seen in step (d), as compared to the migration in the absence of said candidate substance, identifies said candidate substance as bone metastasis inhibitor.

32. A method of identifying an agent useful in preventing cancer cell metastasis to bone comprising:

(a) providing cell expressing α2/β 1 integrin;

(b) providing a source of Type I collagen;

(c) contacting said cell with a candidate substance; and

(d) measuring the level of a transcript or polypeptide for a gene involved in bone cell metastasis, wherein a decrease in the level seen in step (d), as compared to the level in the absence of said candidate substance, identifies said candidate substance as bone metastasis inhibitor.

33. An engineered cancer cell designated as LNCaP_coι.