WO1998056917A1

WO1998056917A1 - Methods and compositions for the expression of a bone and prostate derived growth factor

Info

Publication number: WO1998056917A1
Application number: PCT/US1998/011925
Authority: WO
Inventors: Leland W. K. Chung; Chuan Gao
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1997-06-10
Filing date: 1998-06-10
Publication date: 1998-12-17

Abstract

The role of tumor cell-host stromal interaction and stromal-specific growth factors in prostate cancer growth, progression and metastasis to the axial skeleton were investigated. A bone and prostate-derived growth factor (BPGF-1) was identified and cloned from an expression cDNA library prepared from human bone stromal cells. The cloned BPGF-1 cDNA comprised 3171 nucleotides with a single open reading frame of 1620 nucleotides. The BPGF-1 encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity. Polyclonal antibodies generated from the synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a single putative BPGF-1 protein with an apparent molecular weight of 70 kDa. Southern blot analysis of human genomic DNA revealed that there is a single copy of BPGF-1 gene of 15 kb in size within BamHI sites. The cDNA for BPGF-1 encodes a protein that stimulates the proliferation of prostatic epithelial cells. The BPGF-1 gene is expressed predominantly in bone, prostate and seminal vesicles, with substantially higher expression in the bone than that of the prostate and seminal vesicles.

Description

DESCRIPTION

METHODS AND COMPOSITIONS FOR THE EXPRESSION OF A BONE AND PROSTATE DERIVED GROWTH FACTOR

BACKGROUND OF THE INVENTION The present application is a continuation-in-part of co-pending U.S. Patent Application

Serial No. 08/283,701, filed August 1, 1994, which is a continuation-in-part of U.S. Patent Application Serial No. 07/859,228, filed March 30, 1992, now abandoned. The entire text of the above-referenced disclosure is specifically incorporated by reference herein without disclaimer. The government may own rights in the present invention pursuant to grants from the

National Cancer Institute.

1. Field of the Invention

The present invention relates generally to the field of cancer and also to polypeptides with growth-promoting activities. The invention is particularly directed to the identification of a growth factor, primarily from bone and prostate tissues, that has the capability to stimulate the growth of prostate cells and which promotes the metastasis of prostate cancer to bone tissues. The invention is further directed to novel in vivo and in vitro assay methods, both to detect and quantitate such growth factor activity, and to screen for potential anti-cancer therapeutic substances. The preparation and use of monoclonal antibodies against such growth factors is also disclosed.

2. Description of the Related Art

Cellular interactions between mesenchymal and epithelial cells are believed to be an integral part of embryonic development (Kratochwil, 1972), which continues through adulthood by maintaining differentiated organ growth and function (Frank et al., 1970). These interactions have also been proposed to be involved in the regulation of hormonal responsiveness (Cunha & Chung, 1981) and may play an important inductive and/or permissive role in the pathogenesis of tumor growth (Pitot et al., 1985; DeCosse et al., 1973; Hodges et al., 1977) and metastases (Chackel-Roy et al., 1989; Horak et al., 1985). The growth of a number of epithelial malignancies are influenced by their surrounding stroma, including the urinary bladder (Camps et al., 1990; Hodges et al., 1977), prostate (Camps et al., 1990; Kabalin et al., 1989), colon (Picard et al, 1986), and breast (Miller et al., 1989).

The increased incidence of prostate cancer during the last decade has established prostate cancer as both the most prevalent cancer and the second leading cause of cancer deaths in men (Carter & Coffey, 1990). Most patients dying of prostate cancer experience painful and sometimes crippling osseous metastases with up to 84% having bony metastases at autopsy (Franks, 1956). Prostate cancer is known to selectively spread to the cancellous bones of the axial skeleton, where it is the only malignancy to consistently produce osteoblastic lesions (Cook & Watson, 1968). Metastatic growth of prostate cancer in bone marrow is rapid and virulent; in contrast, growth of primary prostate cancer is generally slower, suggesting that interaction between prostate and bone cells may lead to enhance prostate cancer cell seeding and rapid growth (Rossi et al., 1992, Chung, et al., 1992 ).

The treatment strategies available for patients with metastatic prostate cancer have, in the past, focused primarily on androgen deprivation and/or radiation therapy. Such therapeutic modalities have palliative value, but have not resulted in a cure or in significant increases in patient survival rate. Recently, suramin, a drug known to disrupt the interaction of growth factors and their receptors, was shown to inhibit prostate tumor cell growth both in vitro and in vivo (LaRocca et al., 1990). However, the extreme toxicity of suramin in vivo prevents its clinical use in human treatment. The "seed and soil" hypothesis initially described in 1889 (Paget, 1989) proposes that tumor cells may selectively grow in certain organs due to their particular properties. More recently, some such properties have been proposed to be relevant to prostate cancer development, including enhanced adhesion (Nicolson & Winkelhake, 1975; Sherman et al., 1980), chemotaxis (Varani, 1982; Hujanen & Terranova, 1985), and preferential growth at certain sites (Manishen et al., 1985; Hart, 1985).

Several factors have been hypothesized to be responsible for the metastasis of prostate cancer to bone tissues. For example, it has been proposed that prostate cancer cells selectively seed the lumbar spine and pelvis via a paravertebral venous plexus through which retrograde flow from the prostate to the spine may occur at times of increased intraabdominal pressure (Batson, 1940; Shevrin et al., 1988). However, this theory falls short, since most tumor cells in the venous circulation also pass through the lungs (Nicolson, 1979), and yet the incidence of clinically apparent lung metastases in patients dying of prostate cancer is low (Elkin & Mueller, 1979; Johnson, 1982). Furthermore, kinetic distribution studies using radio-labeled tumor cells have not shown a correlation between organ seeding and subsequent metastatic formation (Fidler & Nicolson, 1976; Potter et al., 1983), suggesting that factors other than the simple mechanical arrest of tumor cells are responsible for the development of prostate cancer bony metastasis.

Recent work has provided some evidence that prostate cancer cell growth may be under autocrine influences involving androgen-mediated regulation of TGF-α, EGF receptor, or bFGF (Wilding et al., 1989; Nonomura et al., 1988; Lu et al., 1989). It has also been suggested that paracrine-mediated pathways involving the stromal compartment play a role in prostate cancer progression (Camps et al., 1990; Chung et al., 1989; Chackel-Roy et al., 1989; Kabalin et al., 1989). Clinically, the interaction between prostate cancer cells and osteoblasts is apparent from the enhanced growth rate of bony metastatic lesions and accompanying osteoblastic reaction. Primary benign and malignant prostatic neoplasms have been shown to express bFGF (Mydlo et al., 1988; Story et al., 1987). Prostatic osteoblastic factor, a soluble substance found in benign hyperplastic and malignant prostatic tissue that stimulates osteoblasts, may well be an FGF-like substance (Jacobs et al., 1979; Nishi et al., 1988), although it may be a distinct and as yet undefined growth factor (Perkel et al., 1990).

Despite many studies, including those described above, it is evident that the factors involved in prostatic carcinogenesis, progression, and nonrandom metastasis remain poorly defined. Moreover, the actions of those few growth factors which have been shown to stimulate prostate cell growth in vitro have not been examined in vivo. The identification of growth factor(s) which exhibit prostate cell growth-promoting activity in vivo would be an important development, creating new avenues of clinical investigation and treatment.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to the identification and characterization of novel growth factors, primarily present in bone tissues, but also present in prostate tissues, that have the capability to promote normal prostate cell growth and prostate cancer cell growth and metastases. The invention is further directed to novel in vivo assay methods, both for the WO 98756917 , PCT/US98/11925 _ -

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identification of factors which promote prostate cancer cell growth and to the identification of potential therapeutic compounds for use in treatment strategies.

This invention further concerns bone and prostate derived growth factor (BPGF) genes and nucleic acid segments, proteins, peptides, and related compositions, and methods of making and using such genes and proteins, for example, in various diagnostic and treatment embodiments. Also provided are nucleic probes and primers, vectors, and recombinant host cells. The present invention further encompasses the generation of monoclonal and polyclonal antibodies directed against these growth factor polypeptides and their use in cancer diagnosis and treatment. In important embodiments, the present invention concerns the substantial purification of such prostate cell growth-promoting factor(s) from human bone tissues. The term "substantially purified human growth factor" as used herein refers to a growth factor composition isolatable from human bone fibroblasts from which has been removed various non- growth-promoting components, and which composition substantially retains its prostate cell growth promoting activity.

Further embodiments of the present invention relate to methods of purifying one or more of the foregoing growth factors. A particularly preferred source for isolating such growth factors is the cell-conditioned media obtained from human bone or prostate fibroblasts. Such conditioned media were chosen by the inventors as potential sources of prostate cell growth factors because of the frequent metastasis of prostate cancer to the axial skeleton. Since the human bone fibroblast conditioned media was found to be a particularly rich source of growth factors, it is contemplated to be the preferred starting material for the purification of such growth factors. However, other starting materials may also be employed, such as, for example, human prostate cancers, human osteogenic sarcomas, or bone marrow aspirates, preferably obtained from prostate cancer patients.

The preferred approach used to isolate such growth factors involves first culturing human bone fibroblasts to produce the human growth factor polypeptides. After obtaining the growth factor polypeptides, for example, by removing conditioned media from the cells, the resultant cell-free polypeptides can then be assayed, characterized and used as a starting material for further purification of the growth factors. During the purification process, it is contemplated that assays will be conducted at various intervals using any one of, or a combination of, the assay methods disclosed herein.

The method preferred by the present inventors to obtain a substantially purified human growth factor in accordance herewith is affinity chromatography, and in particular, affinity chromatography employing a heparin sepharose column. To perform heparin sepharose chromatography in this manner, one would first pass a sample of the cell-free growth factor polypeptides, for example, as contained within conditioned media, over the column in a low salt containing buffer, such as lOmM Tris/Hcl, lmM PMSF, pH 7.4, to allow binding to the column, and then wash the column with the same buffer to remove any non-binding species. The components that bind to the column can be eluted using the above buffer with an increased salt concentration, such as 1 M or 2 M NaCl, or by employing a buffered salt gradient, for example, of 0-3 M NaCl. Following assays of the eluted material, the active fractions can be identified, and such fractions selected and pooled.

The growth factors of the present invention are proposed to have utility in a variety of embodiments. Importantly, they are contemplated to be of use in vivo in stimulating the growth of prostate grafts. Also, since the tumor formed under the stimulation of these growth factors was found to be extremely angiogenic, the growth factors of the present invention are also reasoned to be powerful angiogens, and as such are contemplated to have utility in further clinical embodiments. These include, for example, the promotion of wound healing, organ growth and/or regeneration, and the promotion of epithelial sprouting.

Furthermore, the growth factors can be used either alone or in conjunction with other components in novel tissue culture media. Although preferred, there is no general requirement that the growth factors be provided in their most purified state for use in such embodiments; indeed, it is contemplated that conditioned media containing the growth factors could be suitably directly employed in tissue culture protocols.

Assays for Prostate Cell Growth

Various methods are contemplated to be of use in determining prostate cell growth, i.e., for use in assaying the activity of prostate growth-promoting factors. In preferred embodiments, it is contemplated that such assays may be directed to analyzing the growth of prostate cancer cells, rather than normal prostate cells, simply as a matter of convenience. Such assays include, but are not limited to: in vitro assays, such as the uptake and elution of crystal violet dye; the MTT assay for the staining and quantitation of live cells in a culture dish; and the incorporation of radioactive or non-radioactive labels, such as H-thymidine or bromodeoxy uridine, respectively, into TCA-precipitable cellular DNA. A preferred in vitro assay for use in accordance with the present invention is contemplated to be the soft agar colony-forming assay. The soft agar colony-forming assay is an indication of transformation, as only transformed cell types can grow in soft agar. Methods of conducting an assay of this kind will be known to those skilled in the art in light of the present disclosure. For example, one could first place placing agar, such as 0.6% (w/v) agar, into the bottom of each well on a plate, and seed the wells with an appropriate number of NbE-1 cells, such as 2,000 cells. A feeder layer of less concentrated agar, such as 0.3 to 0.4% (w/v) agar, containing the potential growth factor substances to be analyzed, would then be placed on top of the cells, from which the candidate substances can diffuse and come into contact with the cells. The number of soft agar colonies subsequently formed would be recorded after an appropriate time interval, for example, on the order of 3 to 4 weeks after seeding. Both the cells and the agar could then be prepared and resuspended in media such as T-medium containing approximately between 5 and 10% foetal calf serum, if desired.

A particularly important aspect of the present invention is the development of a novel in vivo assay for prostate cancer growth promoting activity. The development of such an assay is based on the inventors' observations that, although LNCaP human prostate cancer cells are nontumorigenic when administered at a dose of < 5 x 10 cells/site to athymic mice, cancer formation can be induced following co-administration of the non-tumorigenic prostate cells with other cells or compositions. This method, therefore, allows the inductive capabilities of any cell type, conditioned media, growth factor, hormone, carcinogen, or indeed any substance one desires to be examined following the co-administration of the substance and LNCaP cells, or other non-tumorigenic human cells, to mice.

The choice of LNCaP cells for use in such an assay is particularly preferred as such cells have certain advantageous features. For example, LNCaP cells produce prostate specific antigen (PSA), a human tissue-specific tumor marker, which can be used as one method to monitor in vivo prostate cancer cell growth. Moreover, LNCaP cells are the only androgen- responsive human prostate cancer cells that can be consistently grown in vitro. This is an important aspect of the invention that allows one to conduct parallel in vitro and in vivo assays of various compounds using the same prostate cancer cell types.

To conduct such an assay to investigate the capability of a given cell type to elicit

LNCaP growth in vivo, one would preferably co-inoculate suitable athymic mice, such as 6-8 week old BALB/c mice, with a number of LNCaP cells and an approximately equivalent number of cells of the cell type to be investigated (herein referred to as the "subject cell type").

Virtually any mode of co-inoculation is considered to be appropriate, such as subcutaneous, intravenous, or intraperitoneal injection. The administration of 1 x 10 to 5 x 10 cells per inoculant of each cell type is preferred, with the administration of 1 x 10 LNCaP cells and 1 x 10 of the subject cells being particularly preferred. One would suspend the cells in an appropriate medium, such as RPMI 1640 with 10% foetal bovine serum (FBS), prior to injection.

It is contemplated that one would also generally wish to perform parallel control studies to confirm the non-tumorigenic nature of the LNCaP and subject cells when administered independently. In such control studies one would administer the same number, or slightly more cells, such as in the order of 2 x 10⁶ to 5 x 10 , of each cell type.

Various methods are contemplated to be of use in assessing tumor development. The tumors can be measured at regular intervals and their volumes calculated according to the formula L x W x H x 0.5236 (Janek et al., 1975). After sacrifice, the tumors may be excised, weighed, and subjected to various morphological and biochemical analyses as desired.

Furthermore, the choice of LNCaP cells by the inventors also allows the serum levels PSA to be used as an indication of tumor progression.

In further important embodiments, the present invention provides modifications of this in vivo assay model which have been developed to allow the investigation of the effects of substances other than intact cells on prostate cancer growth. This modified method is based upon the adsoφtion of a concentrated substance onto a solid matrix and the co-administration of the matrix and LNCaP cells to the experimental animal. The adsorbed matrix serves as a reservoir for delivery of the particular substance to the live animal. It is contemplated that this method will be particularly useful for analyzing substances such as conditioned media from various cell types and the partially and fully purified growth factors. To conduct such an assay, one would modify the protocol described immediately above by substituting the co-administration of LNCaP cells with subject cells for the co-administration of LNCaP cells with the adsorbed matrix. A particularly preferred matrix for use in such embodiments is Gelfoam, which is commercially available from Upjohn (Kalamazoo, MI), although it is believed that any sponge-like matrix, such as Matrigel, or even agar or agarose, may be employed. One would prepare the matrix under sterile conditions by first pre-soaking it with collagen IV, for example, by exposure to 100 μg/ml collagen IV for 12 hours at 4°C, and then exposing it to the test compound(s). The adsorbed matrix would then be minced to allow subcutaneous inoculation, for example, using a polytron. A suitable control for an assay such as this would be inoculation with Gelfoam pre-soaked with collagen IV alone.

Since either of the above methods can be utilized to generate an animal bearing a human prostate cancer, the present invention further provides an important model for use in screening for compounds with the potential to inhibit the growth of human prostate cancer. To screen for a substance having the capability to inhibit, retard, or otherwise exert a negative effect on prostate cancer cell growth, one may administer the test substance either simultaneously with, or subsequent to, the administration of the cancer promoting agents, i.e., the LNCaP cells and the previously identified stimulatory cells or substances. One would then determine the effect of the candidate inhibitory substance by measuring the degree of tumor formation or regression, or the prevention or inhibition of tumor growth, observed in the presence of the candidate inhibitory substance and comparing it to the tumor status in the absence of the potentially inhibitory substance.

Recombinant DNA Methodology

Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding bone and prostate derived growth factor (BPGF-1) and the creation and use of recombinant host cells through the application of DNA technology that express bone and prostate derived growth factor.

As used herein, the term "isolated DNA" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding bone and prostate derived growth factor (BPGF) refers to a DNA segment that contains BPGF coding sequences, yet is isolated away from, or purified free from, total human genomic DNA. Included within the term "isolated DNA" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified BPGF gene refers to a DNA segment including BPGF coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide, or peptide encoding unit. As will be understood by those skilled in the art, this functional term includes both genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, or peptides.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case BPGF, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a bone and prostate derived growth factor that includes within its amino acid sequence an amino acid sequence in accordance with SEQ. I.D. NO.: 1, corresponding to that isolated from a human bone stromal cell line, MS. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode the BPGF amino acid sequence.

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ. I.D. NO.: 1. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ. I.D. NO.: 1 under relatively stringent conditions such as those described herein in Example 4.

It will also be understood that this invention is not limited to the particular nucleic acid sequence of SEQ. I.D. NO.: 1. Recombinant vectors and isolated DNA segments may therefore variously include the BPGF coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include BPGF-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acid sequences. The DNA segments of the present invention encompass biologically functional equivalent BPGF proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test BPGF mutants in order to examine growth promoting activity at the molecular level. If desired, one may also prepare fusion proteins and peptides, e.g. , where the BPGF coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection puφoses (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively). Encompassed by the invention are DNA segments encoding relatively small peptides, such as peptides of from about 15 to about 50 amino acids in length, more preferably from about 15 to about 30 amino acids in length, and also larger polypeptides up to and including proteins. Such DNA segments are exemplified by, but not limited to, DNA segments that have nucleic acid sequences in accordance with the sequence of SEQ. I.D. NO.: 1.

Recombinant vectors form important further aspects of the present invention. 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 control of a promoter. The promoter may be in the form of the promoter that is naturally associated with BPGF gene(s), e.g., in human cells, 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.

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a BPGF gene in its natural environment. Such promoters may include CMV promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. 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 skilled in the art of molecular biology, for example, see Sambrook et al. (1989). 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. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, CMV, adenovirus, the T7 RNA polymerase promoter system described by Tabor & Richardson (1985), and the maltose binding protein-fusion protein system (Guan et al, 1987; Nagai and Thogersen, 1987).

As mentioned above, in connection with expression embodiments to prepare recombinant BPGF proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire BPGF protein or peptide fragments thereof being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of BPGF peptides or epitopic core regions, such as may be used to generate anti-BPGF antibodies, also falls within the scope of the invention.

DNA segments that encode peptide antigens from about 15 to about 50 amino acids in length, or more preferably from about 15 to about 30 amino acids in length, are contemplated to be particularly useful. DNA segments encoding peptides will generally have a minimum coding length of about 45 to about 150, or to about 90 nucleotides. DNA segments encoding full length proteins may have a minimum coding length of about 1620 nucleotides for a protein in accordance with SEQ. I.D. NO.: 1.

In addition to their use in directing the expression of the BPGF protein, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments.

In still further embodiments, the present invention concerns the generation of antibodies, particularly monoclonal antibodies (mAbs), against the growth factor polypeptide(s) disclosed herein. Such mAbs will have utility in a variety of applications. These include, for example, the rapid purification of the growth factors by immunoaffinity chromatography and the clinical use of mAbs or mAb-conjugates in diagnostic, prognostic, imaging, and therapeutic strategies for the treatment of prostate cancer in men.

The in vivo human prostate cancer model disclosed herein is contemplated to be particularly useful in testing mAbs to identify those that are suitable for clinical use. For example, one may test the ability of mAbs or mAb-conjugates to inhibit prostate cancer growth or metastasis in the mouse model prior to clinical trials in human subjects. It will be understood, however, that mAbs which are not considered to meet the criteria for clinical use may nonetheless have utility in other embodiments, such as in growth factor purification by affinity column chromatography or in Western blotting, ELISA, or other immunological screening assays.

It is proposed that such anti-growth factor mAb generation may be achieved most readily through the use of a modified immunization protocol. It is contemplated that the initial immunization of an experimental animal, such a mouse, would be performed according to the standard practice in the art. However, for the booster inoculation, the use of the following method is proposed to be advantageous in that it will allow the optimal exposure of splenocytes to the booster antigen. The immunized mice should be surgically opened to expose the spleen, and a sterile solution of the growth factor antigens should be injected directly into the spleen. The mouse should then be sutured and allowed to recover.

Blood samples of the immunized mice may be analyzed for the presence of circulating antibodies to the growth factors, and those mice producing reasonable titers of circulating antibodies would be sacrificed and their spleens would be removed for cell fusion. A mouse myeloma cell line proposed to be of use for hybridization is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line, which is known to be HAT sensitive. Cells may be fused according to any of the methods known in the art, such as by using polyethylene glycol (PEG), and later screened for antibody production, for example, by employing an ELISA or immunoblot technique.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a BPGF protein or peptide that has an amino acid sequence essentially as set forth by a contiguous sequence selected from those disclosed herein. The proteins encoded may be full length proteins, as represented by the 724 amino acids of the BPGF protein.

Any of the DNA segments of BPGF may encode peptides of from about 15 to about 50, or more preferably from about 15 to about 30, amino acids in length. Peptides may, of course, be of any length in this range, such as 16, 17, 18, 19, or 20 amino acids, or about 25, about 30, about 35, about 40, about 45, or about 50 amino acids in length, with "about" in this one context meaning a range of from 1 to 4 amino acids longer or shorter than the stated length.

Accordingly, the DNA segments encoding such peptides will have coding lengths, excluding any regulatory sequences, of between about 45 to about 150, or preferably of 45 to about 90, base pairs, with any length within or around these general guidelines being contemplated.

The term "biologically functional equivalent" is well understood in the art and is further defined in detail later herein. Accordingly, sequences that have between about 70% and about

80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% of amino acids that are identical or functionally equivalent to an amino acid that encodes BPGF-1.

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth by a contiguous sequence from the sequence SEQ. I.D. NO.: 1, preferably as set forth by a contiguous sequence from coding regions of SEQ. I.D. NO.: 1. The term "essentially as set forth by a contiguous sequence from SEQ. I.D. NO.: 1" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a contiguous portion of SEQ. I.D. NO.: 1 and has relatively few codons that are not identical to, or functionally equivalent with, the codons of SEQ. I.D. NO.: 1. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids. Although such information is generally known to those of skill in the art, Table 1 is provided herein to clearly set forth this information.

TABLE 1

Amino Acids Codon-

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG ecu

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Tφ w UGG

Tyrosine Tyr Y UAC UAU It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' nucleic acid sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the production of a BPGF protein where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. The addition of 5' nucleic acid sequences that constitute regulatory regions, such as promoters and/or enhancers, is particularly contemplated by the inventors and the addition of any such sequence to any of those coding regions described above thus falls within the scope of the present invention.

Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ. I.D. NO.: 1, or contiguous stretches thereof. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ. I.D. NO.: 1 under relatively stringent conditions, such as those described in Example 4.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length being limited only by the desired ease of preparation and use in the intended recombinant DNA protocol.

DNA segments of the invention may include within their sequence a nucleic acid sequence that is essentially as set forth in any contiguous stretch of the sequences of SEQ. I.D. NO.: 1. However, in certain embodiments, it is contemplated that stretches from the coding regions will be preferred. As such, the invention provides protein and peptide-encoding segments of DNA that may be taken from any contiguous stretch of the coding sequences, such as from position 694 to position 2314 of SEQ. I.D. NO.: 1, for BPGF peptides and proteins.

It will also be understood that the length and content of the nucleic acid and amino acid sequences disclosed herein is virtually unlimited, so long as the sequences are isolated free from their natural environment and contain BPGF protein or DNA sequences. Recombinant vectors and DNA segments may therefore include BPGF protein encoding regions in combination with other functional sequences.

It is contemplated that nucleic acid segments of the present invention will have numerous uses, for example, in connection with the expression of peptides or proteins, such as antigens, and also as probes and primers. Probes and primers based upon, or designed from,

SEQ. I.D. NO.: 1 , will have use in various hybridization embodiments, regardless of whether they encode proteins or peptides or whether they are derived from non-coding segments. Nucleic acid segments that incoφorate at least a 10-14 or 20 nucleotide-long stretch that corresponds to a sequence within SEQ. I.D. NO.: 1 may be employed as a selective hybridization probe.

Such probes may be used for the detection of BPGF sequences in selected samples or to screen clone banks to identify clones that comprise corresponding or related sequences. The detection of BPGF sequences in samples, particularly in clinical samples, represents an important utility of the present invention, as detection of BPGF is important in and of itself, and also as diagnosis of an increased risk of bone metastases, and is a first element in designing an appropriate treatment regimen for a given disease or disorder.

Thus, this invention also provides molecular biological methods for detecting BPGF in a suspected sample, including a clinical sample, as may be employed in the diagnosis of cancer or other neoplastic disease. Samples that may be analyzed include those such as biopsies of prostate, bone, or other tissues in which BPGF has activity.

To conduct such a diagnostic method, one would generally obtain nucleic acids from the sample and contact the nucleic acids with a nucleic acid segment that encodes a BPGF protein or peptide under conditions effective to allow hybridization of substantially complementary nucleic acids, and then one would detect the presence of any hybridized substantially complementary nucleic acid complexes that formed. The presence of a substantially complementary nucleic acid sequence in a sample, or a significantly increased level of such a sequence in comparison to the levels in a normal or "control" sample, will thus be indicative of a sample that may show enhanced proliferative ability. Where a substantially complementary nucleic acid sequence, or a significantly increased level thereof, is detected in a clinical sample from a patient suspected of having prostate cancer, this will be indicative of a patient that may be susceptible to increased bone metastatic growth. As used herein, the term "increased levels" is used to describe a significant increase in the amount of BPGF nucleic acids detected in a given sample in comparison to that observed in a control sample, e.g., an equivalent sample from a normal healthy subject.

A variety of hybridization techniques and systems are known that can be used in connection with detecting BPGF, including diagnostic assays such as those described in Falkow et al, U.S. Patent 4,358,535. Short coding or non-coding nucleic acid segment probes may also be employed as primers in connection with diagnostic PCR technology, as well as for use in any of a number of other PCR applications, including PCR-based cloning and engineering protocols. In general, the "detection" of a BPGF sequence is accomplished by attaching or incoφorating a detectable label into the nucleic acid segment used as a probe and "contacting" a sample with the labeled probe. In such processes, an effective amount of a nucleic acid segment that comprises a detectable label (a probe) is brought into direct juxtaposition with a composition containing target nucleic acids. Hybridized nucleic acid complexes may then be identified by detecting the presence of the label, for example, by detecting a radio, enzymatic, fluorescent, or even chemiluminescent label.

Many suitable techniques for use in the detection of BPGF nucleic acids will be known to those skilled in the art. These include, for example, in situ hybridization, Southern blotting, and Northern blotting. In situ hybridization describes the techniques wherein the target nucleic acids contacted with the probe sequences are those located within one or more cells, such as cells within a clinical sample or even cells grown in tissue culture. As is well known in the art, the cells are prepared for hybridization by fixation, e.g., chemical fixation, and placed in conditions that allow for the hybridization of a detectable probe with nucleic acids located within the fixed cell. Alternatively, target nucleic acids may be separated from a cell or clinical sample prior to contact with a probe. Any of the wide variety of methods for isolating target nucleic acids may be employed, such as cesium chloride gradient centrifugation, chromatography (e.g., ion, affinity, magnetic), phenol extraction, and the like. Most often, the isolated nucleic acids will be separated, e.g., by size, using electrophoretic separation, followed by immobilization onto a solid matrix, prior to contact with the labelled probe. These prior separation techniques are frequently employed in the art and are generally encompassed by the terms "Southern blotting" and "Northern blotting."

Nucleic acid molecules having contiguous stretches of 10-14, 20, 30, 50, or even 100- 200 nucleotides or so that correspond to, or are complementary to, sequences from SEQ. I.D. NO.: 1 will have utility as hybridization probes or primers. These probes will be useful in a variety of hybridization embodiments, which also include Southern and Northern blotting in connection with analyzing BPGF expression in various mammalian cells. The total size of the fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Fragments generally finding use in hybridization embodiments may have lengths of complementary regions that vary between about 10-14 or 20 and about 100 nucleotides, or even up to the full length sequence of

1620 nucleotides (SEQ. I.D. NO.: 1), according to the complementary sequences one wishes to detect.

Recombinant Host Cells The present invention also concerns recombinant host cells that include one or more

DNA segments that comprise an isolated BPGF gene as described herein. It is contemplated that virtually any cell may be employed as a recombinant host cell, but that certain advantages may be found in using a bacterial host cell, such as, for example, in the ease of cell growth and manipulation. Examples of preferred bacteria for use as recombinant host cells include, for example, E. coli. However, expression in eukaryotic cells is also contemplated, and exemplary cell lines that may be used include all those typically employed for eukaryotic expression, such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN, and MDCK cell lines.

The recombinant host cells of the invention may be employed to either propagate the vector and/or to express the various peptides and proteins described herein, allowing the encoded components to be obtained essentially free of other human or mammalian components.

That is, one may prepare such peptides or proteins by recombinant expression using a host cell other than human or mammalian, and/or produce the peptides or proteins at high levels so that their isolation directly results in a significantly enriched preparation. Preferred recombinant host cells are those capable of expressing peptides and proteins with sequences essentially as set forth in SEQ. I.D. NO.: 1. Depending on the host system employed, one may find particular advantages where

DNA segments of the present invention are incoφorated into appropriate vector sequences that may, e.g., improve the efficiency of transfection of host cells. Where bacterial host cells are employed, it is proposed that virtually any vector known in the art to be appropriate for the selected host cell may be employed. Thus, in the case of E. coli, one may find particular advantages through the use of plasmid vectors such as pBR322, or bacteriophages such as λGEM-11. Further examples will be known to those skilled in the art, as exemplified in Sambrook et al. (1989).

The recombinant host cells may be employed in connection with "overexpressing" BPGF proteins or peptides, that is, increasing expression over the natural expression levels in human or other mammalian cells, and may lead to the production of large quantities of proteins.

Overexpression may be assessed by a variety of methods, including radio-labelling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide, in comparison to the level in natural human or mammalian cells, is indicative of overexpression.

Various methods may be used to obtain or collect BPGF proteins or peptides from cells, whether native or recombinant. For example, one method involves applying dialyzεd bone fibroblast conditioned medium to a heparin sepharose affinity column, which was previously equilibrated in 10 mM Tris-HCl buffer containing 0.1 mM PMSF, pH 7.5. The column was washed with a 3 -bed volume of 10 mM Tris-HCl buffer and eluted with a linear salt gradient of NaCl (0 to 3 M NaCl). Two ml fractions were collected and dialyzed against distilled water containing 0.1 mM PMSF at 4°C. Protein concentrations are monitored by a spectrophotometer at 280 nM. Fifty ml of each fraction was used for the assessment of the stimulatory effect on the prostatic epithelial cells by soft agar colony forming assay. Antibody Generation and Kits for Antigen Detection

The present invention further provides protein or peptide compositions, free from total bacterial cells, comprising purified BPGF protein or peptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ. I.D. NO.: 1. Such compositions may be obtained from natural or recombinant sources and may include proteins or peptides, proteins and peptides, or BPGF compositions alone, obtainable from recombinant hosts. The compositions may include full length BPGF proteins and/or various peptides that include sequences in accordance with about 15 to about 50, or more preferably to about 30, amino acid long sequence from SEQ. I.D. NO.: 1. The present invention thus also provides methods of generating an immune response, which methods generally comprise administering to an animal, including a human subject, a pharmaceutically acceptable composition comprising an immunologically effective amount of a BPGF protein or peptide composition. The composition may include partially or significantly purified BPGF proteins or peptides, obtained from natural or recombinant sources. Smaller peptides that include reactive epitopes, such as those between about 30 and about 50 amino acids in length, will often be preferred.

By "immunologically effective amount" is meant an amount of a bone and prostate derived growth factor protein or peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response) and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments.

Another means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically acceptable composition comprising an immunologically effective amount of a BPGF factor nucleic acid composition (i.e., an amount capable of stimulating a B cell and/or T cell response). The stimulation of specific antibodies and CTL (cytotoxic T lymphocyte) responses upon administering to an animal a nucleic molecule is now well known in the art, as evidenced by articles such as Tang et al. (1992) and Fynan et al. (1993), each incoφorated herein by reference. Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies useful in the detection of BPGF detection, may comprise whole growth factor proteins or antigenic peptide fragments from these proteins. As such, antigenic functional equivalents of the proteins and peptides described herein also fall within the scope of the present invention. An "antigenically functional equivalent" protein or peptide is one that incoφorates an epitope that is immunologically cross-reactive with one or more epitopes of the bone and prostate growth factor proteins. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity. The identification or design of BPGF factor epitopes and/or functional equivalents that are suitable for use in immunoformulations, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of Hopp, as enabled in U.S. Patent 4,554,101, incoφorated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences; for example, Chou and Fasman (1974a,b; 1978a,b; 1979) address this subject. The amino acid sequence of these "epitopic core sequences" may then be readily incoφorated into peptides, either through the application of peptide synthesis or recombinant technology. To confirm that a protein or peptide is immunologically cross-reactive with one or more epitopes of the BPGF protein is also a straightforward matter. This can be readily determined using specific assays, e.g., of a single proposed epitopic sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments. The screening assays may be employed to identify either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, i.e., based upon competition for binding sites between antibodies and antigens.

Suitable competition assays that may be employed include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting, and the like. In any of the competitive assays, one of the binding components, generally the known element, such as BPGF protein or peptide, or the known antibody, such as a polyclonal antibody as set forth in

SEQ. I.D. NO.: 2, SEQ. I.D. NO.: 3 or SEQ. I.D. NO.: 4, will be labeled with a detectable label, and the test components, which generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen. As an exemplary embodiment, to conduct a competition study between BPGF and any test antigen, one would first label BPGF with a detectable label, such as biotin or an enzymatic, radioactive, or fluorogenic label, to enable subsequent identification. One would then incubate the labelled antigen with the other (test) antigen to be examined at various ratios (e.g., 1:1, 1:10 and 1 : 100) and, after mixing, one would then add the mixture to a known antibody. Preferably, the known antibody would be immobilized, e.g., by attaching it to an ELISA plate. The ability of the mixture to bind to the antibody would be determined by detecting the presence of the specifically bound label. This value would then be compared to a control value in which no potentially competing (test) antigen was included in the incubation.

The assay may be any one of a range of immunological assays based upon hybridization, and the reactive antigens would be detected by means of detecting their label, e.g., using streptavidin in the case of biotinylated antigens, using a chromogenic substrate in connection with an enzymatic label, or simply detecting a radioactive or fluorescent label. An antigen that binds to the same antibody as BPGF, for example, will be able to effectively compete for binding to the anti-peptide polyclonal antisera and thus will significantly reduce BPGF binding, as evidenced by a reduction in the amount of label detected.

In further embodiments, the invention concerns relatively purified antibodies that bind to, or have binding affinity for, BPGF proteins or peptides. Such relatively purified antibodies may be polyclonal or monoclonal and are distinct from those compositions that may be found in nature, e.g., as represented by the sera of an individual afflicted with prostate cancer, by virtue of their increased degree of purity. Even a polyclonal antibody raised in response to immunization with a purified, or enriched BPGF protein composition will be significantly distinct from the sera of an infected individual that contains a great diversity of antibodies.

Particular techniques for preparing antibodies in accordance with the invention are disclosed herein. However, it is proposed by the inventors that any of the current techniques known in the art for the preparation of antibodies in general may be employed, through the application of either monoclonal or polyclonal technology, and as represented by the generation of the anti-peptide polyclonal antisera. Antibodies that are cross-reactive with the anti-peptide antisera are also encompassed by the invention, as may be identified by employing a competition binding assay, such as those described above in terms of antigen competition.

Antibodies of the invention may also be linked to a detectable label, such as a radioactive, fluorogenic, or nuclear magnetic spin resonance label. Biolabels such as biotin and enzymes that are capable of generating a colored product upon contact with a chromogenic substrate are also contemplated. Exemplary enzyme labels include alkaline phosphatase, hydrogen peroxidase, and glucose oxidase enzymes.

In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is contemplated that the BPGF proteins or peptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, e.g., the peptide antibodies, may be employed to detect BPGF proteins or peptides. Either type of kit may be used in the immunodetection of compounds, present within clinical samples, that are indicative of prostate cancer or bone metastases. The kits may also be used in antigen or antibody purification, as appropriate.

In general, immunodetection methods will comprise first obtaining a sample suspected of containing such a protein, peptide, or antibody, such as a biological sample from a patient, and then contacting the sample with a first protein or peptide that is BPGF, or a first antibody that binds to a BPGF protein or peptide, as the case may be, under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed.

Contacting the chosen sample with BPGF protein or peptide, or antibody thereto, under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the protein, peptide, or antibody composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens or antibodies to form immune complexes with, i.e., to bind to, any antibodies or antigens present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot, or Western blot, will generally be washed to remove any non-specifically bound antigen or antibody species, allowing only those specifically bound species within the immune complexes to be detected. The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as Nakamura et al. (1987), incoφorated herein by reference. Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, florigenic, biological, or enzymatic label, with enzyme tags such as alkaline phosphatase, horseradish peroxidase, and glucose oxidase being suitable. The antigen (e.g., BPGF) or antibody (e.g., anti-peptide antibodies) employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen or antibody present in the composition to be determined. Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein, peptide, or antibody. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labelled secondary antibodies or ligands, and the remaining bound label is then detected.

For diagnostic puφoses, it is proposed that virtually any sample suspected of containing BPGF proteins, peptides, or antibodies sought to be detected, as the case may be, may be employed. Exemplary samples include clinical samples obtained from a patient, such as blood or serum samples, prostate or bone biopsy samples, or other tissue samples. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.

In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of BPGF factor proteins, peptides, and/or antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable BPGF protein or peptide or a first antibody that binds to a the BPGF protein or peptide together with an immunodetection reagent and a means for containing the protein, peptide, or antibody and reagent. The immunodetection reagent will typically comprise a label associated with the protein, peptide, or antibody or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first protein, peptide, or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g., for protocols where the first reagent is a protein that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antigen or antibody may be placed, and preferably suitably allocated. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as injection or blow-molded plastic containers in which the desired vials are retained.

Antisense RNA technology has been developed as one approach to inhibiting gene expression, particularly oncogene expression. An "antisense" RNA molecule is one which contains the complement of, and can therefore hybridize with, protein-encoding RNAs of the cell. It is believed that the hybridization of antisense RNA to its cellular RNA complement can prevent expression of the cellular RNA, perhaps by limiting its translatability. While various studies have involved the processing of RNA or direct introduction of antisense RNA oligonucleotides to cells for the inhibition of gene expression (Brown, et al, 1989; Wickstrom, et al, 1988; Smith, et al, 1986;), the more common means of cellular introduction of antisense RNA has been through the construction of recombinant vectors which will express antisense RNA once the vector is introduced into the cell.

A principal application of antisense RNA technology has been in connection with attempts to affect the expression of specific genes. For example, Delauney et al (1988) have reported the use of antisense transcripts to inhibit gene expression in transgenic plants. These authors report the down-regulation of chloramphenicol acetyl transferase activity in tobacco plants transformed with CAT sequences through the application of antisense technology. Antisense technology has also been applied in attempts to inhibit the expression of various oncogenes. For example, Kasid et al. (1989) report the preparation of a recombinant vector construct employing Craf-l cDNA fragments in an antisense orientation, brought under the control of an adenovirus 2 late promoter. These authors report that the introduction of this recombinant construct into a human squamous carcinoma resulted in a greatly reduced tumorigenic potential relative to cells transfected with control sense transfectants. Similarly, Prochownik et al (1988) have reported the use of Cmyc antisense constructs to accelerate differentiation and inhibit G₁ progression in Friend Murine Erythroleukemia cells. In contrast, Khokha et al. (1989) disclose the use of antisense RNA to confer oncogenicity on 3T3 cells through the use of antisense RNA to reduce murine tissue inhibitor or metalloproteinase levels.

In certain embodiments of the invention, inhibition or suppression of BPGF- 1 gene expression is desired and antisense molecules will be employed. By preparing a construct that encodes an RNA molecule that is an antisense or "complementary" configuration with respect to the RNA readouts of BPGF- 1, the construct will act to inhibit or suppress the ultimate expression of the target gene, presumably by binding to the target RNA and thereby preventing its translation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A, IB, 1C, ID, IE, and IF. Histomoφhological and immunohistochemical characterization of fibroblast-induced LNCaP chimeric tumors. Hematoxylin and eosin-stained sections shown in FIGs. 1 A-IC reveal differences between LNCaP/rUGM tumors in male (FIG. 1 A) and female (FIG. IB) hosts, the former a carcinosarcoma, the latter a pure sarcoma with no epithelial component. LNCaP/MS tumors shown in FIG. 1C formed only in male hosts and histologically are vascular carcinomas with a minor mesenchymal component. FIGs. ID- IF show immunohistochemical staining with monoclonal antibodies against PSA, demonstrating intense and generalized staining of the epithelial cells in only male LNCaP/rUGM (FIG. ID) and LNCaP/MS (FIG. IF) tumors but not of the sarcomatoid LNCaP/rUGM tumors (FIG. IE) in female mice. LNCaP/3T3 tumors are sarcomas histologically similar to LNCaP/rUGM tumors in females and also stained negatively for PSA. FIGs. 2A and 2B. Southern and Northern analysis of fibroblast-induced LNCaP chimeric tumors. Portions of tumors removed at the time of sacrifice were processed separately for DNA and RNA isolation, as described in the detailed examples. Controls consisted of human bladder cancer (+) and rUGM (-) cell DNA. Various concentrations of DNA were loaded and probed for repetitive Alu sequences to identify human cells. RNA, 20μg, was loaded and probed with a complementary DNA probe for PSA. FIG. 2A is a Southern dot blot of LNCaP/rUGM tumors demonstrating variably positive Alu in 6 or 7 tumors from male hosts

(lanes 1-7) and 0 or 3 tumors from female hosts (lanes 1-3). Northern analysis of corresponding tumors (FIG. 2A, bottom) demonstrates PSA expression only in tumors from male hosts. PSA expression did not correlate with Alu expression, likely resulting from varied tissue selection from a heterogenous tumor. FIG. 2B is a Southern analysis of LNCaP/3T3 tumors revealing no human component in these tumors (lanes a-e), while all LNCaP/MS tumors were positive for

Alu (lanes f-j). Northern analysis of LNCaP/MS tumors (FIG. 2B, bottom) demonstrates that all are strongly positive for PSA.

FIGs. 3A and 3B. Differences in serum PSA levels (ng/ml) in animals with various LNCaP chimeric tumors paralleled their differences in histomoφhology. Mice bearing tumors characterized as carcinosarcomas (LNCaP/rUGM) or carcinomas (LNCaP/MS or LNCaP/NbF-

1) (FIG. 3 A) had elevated serum PSA levels (FIG. 3B), while mice bearing sarcomas or no tumors had undetectable serum PSA levels (< 0.3 ng/ml). MS bone fibroblasts were the most reliable inducer of LNCaP carcinoma formation and resulted in the highest PSA levels, with a median of 68.1 ng/ml. FIGs. 4A and 4B. LNCaP cells are androgen sensitive in vitro. In FIG. 4A, LNCaP cells were stimulated in vitro by androgens with a 182 and 142% increase in cell growth with 1.0 nM testosterone (T) and 0.1 nM dihydrotestosterone (DHT), respectively. No mitogenic response was observed for rUGM cells using androgens in concentrations ranging from 0.1 to 100 nM. In FIG. 4B, androgen receptor assays demonstrated the presence of a substantial amount (B_max = 332 fmol/mg protein) of high-affinity (K_d = 0.22 nM) androgen receptors in

LNCaP cells. Points, averages of 6 replicated determinations from 3 separate studies; bars, SE ranging from 3-9%.

FIGs 5 A and 5B. Effect of defmed growth factors on LNCaP cell growth in vitro. The growth of LNCaP cells are stimulated in vitro by bFGF in a concentration-dependent manner as shown in FIG. 5 A, producing a 180% increase in cell number over 9 days. Both TGFα and

EGF had no significant effect on LNCaP growth in vitro using concentrations from 0.1 to 50 ng/ml. A 50% reduction in LNCaP cell growth was produced by 0.1 ng/ml TGFβ, as shown in FIG. 5B. Points, averages of 6 replicated determinations from 3 separate studies; bars, SE ranging from 3-9%.

FIGs. 6A and 6B. Stimulation of LNCaP cell growth in vitro by prostate- and bone- derived conditioned media. In FIG. 6 A, LNCaP cells are stimulated up to 210% in a concentration-dependent manner from 0.1- to 1.0-fold by rUGM conditioned media and are also stimulated by NbF-1 and MS conditioned media, but not by 3T3, CCD16, or NRK conditioned media. In FIG. 6B, a bidirectional paracrine-mediated stimulatory pathway exists between LNCaP cells and rUGM and MS fibroblasts. rUGM cells are stimulated up to 400% in a concentration-dependent manner from 0.1 - to 2-fold by LNCaP conditioned media and also less so by NbF-1, MS, 3T3, and CCD16 conditioned media. No autocrine growth loop was demonstrated as evidenced by lack of stimulation of LNCaP conditioned media on LNCaP cells or rUGM conditioned media on rUGM cells. Columns, averages of 6 replicated determinations from 3 separate studies; bars, SE ranging from 2-7%. FIGs. 7A, 7B, 7C, and 7D. rUGM and MS conditioned media stimulate LNCaP tumor growth in vivo. Gelfoam, a solid form of slowly absorbable gelatin, was used as a reservoir for delivery of biologically active factors to determine whether LNCaP tumor growth could be induced by fibroblast conditioned media in vivo in the absence of stromal cells. Gelfoam was adsorbed with 100 μg/ml collagen IV for 12 hours followed by EGF, bFGF, or stromal conditioned media. LNCaP cells, 2 x 10 , were inoculated s.c. with treated Gelfoam, except at some control sites, where ECGF-treated Gelfoam was injected alone to detect angiogenesis. Angiogenesis was visible after 3 weeks when Gelfoam plus collagen IV adsorbed with ECGF was injected, as shown in FIG. 7B. At rUGM conditioned media-treated sites, 5 of 10 (50%) tumors formed by 10 weeks (mean tumor volume, 278 mm ). With MS conditioned media- treated Gelfoam, 3 tumors formed at 8 sites (38%). bFGF was also tested because of its in vitro mitogenic activity and induced tumor formation at 3 of 5 sites (60%). All tumors were histologically carcinomas and stained intensely and uniformly for PSA, as shown in FIG. 7C. Southern blot analysis for Alu and corresponding Northern analysis for PSA expression are both positive, as shown in FIG. 7D. FIG. 8. Heparin affinity column chromatography of human bone stromal conditioned media. 560 mg total MS conditioned media protein was loaded onto the column. The column was washed with 10 mM Tris-HCl, 1 mM PMSF, pH 7.4, before eluting with a continuous salt gradient of 0 to 3 M NaCl. The tumor-inducing activity was recovered prior to elution with 2 M NaCl.

FIG. 9. SDS-PAGE analysis of the active fractions (1 M NaCl-eluted fractions) from heparin sepharose chromatography. Track 1 , high-molecular weight markers; track 2, control media; track 3, active fractions.

FIG. 10. Effect of various growth factor antibodies antagonizing the efficiency of soft agar colony formation of prostatic epithelial cells induced by partially purified bone stromal conditioned media. FIG. 11. Identification of a human growth factor polypeptide with an apparent molecular weight on SDS/PAGE of approximately 157 kD. This polypeptide, present within human bone marrow, is identified by its reactivity with the mAb MS 329 in Western blot analyses. BM, bone sample; TM, control media sample. The M_rs of the molecular weight standards are indicated to the left. FIG. 12. BPGF-1 Nucleotide Sequence.

FIGs. 13A and 13B. Schematic Representation of the BPGF-1 Sequence and Limited Restriction Enzyme Map. The solid box represents the ORF starting from the ATG 694 and ending to TGA 2314.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Previous studies demonstrated that a cell-cell recombination model, in which co- inoculation of nontumorigenic epithelial and organ-specific mesenchymal cells (e.g., bone and prostate), results in solid tumor formation in vivo (Camps et al, 1990, Gleave et al, 1991, Chung et α/.,1989). Soluble factors secreted by the prostate and bone stromal cells enhanced anchorage-independent growth of prostate epithelial cells in vitro and tumor growth in vivo

(Chung et al, 1992, Gleave et al, 1991). Alternatively, by co-inoculating nontumorigenic prostatic epithelial cells with selective growth factor(s) and extracellular matrix immobilized on Gelfoam also results in solid tumor formation in vivo (Chung et al, 1992). In the present invention, the identification and cloning of a human bone mesenchymal derived factor, or bone and prostate derived growth factor (BPGF-1), is shown. Prostatic carcinoma cells show an increased responsiveness to the growth-promoting activity of BPGF- 1. Sequence analysis revealed that BPGF-1 shares no homology with the known sequences in the GenBank. Suφrisingly, BPGF-1 is deferentially expressed in human bone and prostate cells, not in cells or tissues derived from other organs.

Isolation of the BPGF-1 cDNA clones

The inventors screened a cDNA expression library with a polyclonal antibody specifically against MS-1 fraction and isolated the clones that encode a BPGF-1 protein. The full-length cDNA sequence of clone BPGF-1 (3171 bp) corresponds well with the observed size of the BPGF-1 transcript of about 3.3 kb, suggesting that virtually the complete mRNA sequence has been isolated in cDNA form. Northern blotting using BPGF-1 cDNA as a probe detected two mRNA transcripts with approximately equal intensity in human bone and prostate cells. There is one single BPGF-1 gene, as evidenced by Southern blot analysis.

Differential Expression of BPGF-1 Gene From previous studies, it has been observed that organ-specific stromal (bone and prostate) cells, but not cells from the lungs or kidneys, accelerated human prostatic carcinoma growth both in vitro and in v/vø(Gleave et al, 1991). These results imply the possible existence of a tissue-specific factor or factors that stimulate prostatic carcinoma cell growth. The present invention shows the existence of BPGF- 1 that is tissue specific and expressed predominantly in the bone, seminal vesicles, and prostate, with substantially higher levels in the bone than in the prostate (at least 50 times higher in the bone than in the prostate), although it is also present in minute quantities in other tissues. It is likely that the expression of BPGF- 1 is qualitatively and quantitatively different in various tissues, along with the stimulation of prostatic carcinoma cell growth by BPGF-1, may contribute in part to the preference of prostatic carcinoma metastases in the bone.

The Biological Role of BPGF-1

Various factors are thought to be involved in mediating the proliferation of cells, the differentiation of cells, or both. Many soluble factors and their corresponding receptors have been implicated in normal and abnormal prostate growth and development (Davies et al, 1988;

Fiorelli et al, 1991; Chung et al, 1992). These include EGF and its receptor (Kishi et al, 1988; Fowler et al, 1988), TGF-α (Wilding et al, 1989a), TGF-β (Wilding et al, 1989b), FGF (Ikeda et al, 1987; Danielpour et al, 1989; Nakamoto et al, 1992), PDGF (Sitaras et al, 1988), IGF (Cohen et al, 1991), HGF/SF (Nakamura et al, 1986; Nakamura et al, 1989; Weidner et al, 1991 ; Montesano et al, 1991), KGF (Rubin et al, 1992), and NGF (Djakiew et al , 1991 ). Recent studies have shown that certain factors present in human bone marrow are able to stimulate human prostatic carcinoma cell growth (Gleave et al, 1991; Rossi et al, 1992; Chung et al, 1992). One of these factors has been further identified as transferrin (Rossi et al, 1992).

The identification of a cDNA clone encoding the BPGF-1 protein will help in understanding the interaction between the bone and the prostate. Rossi et al (1992) proposed that a high concentration of transferrin in the bone marrow may stimulate rapid proliferative growth of prostatic carcinoma cells once the cells have become lodged in the vertebral marrow cavity. The present results extend that finding to show that tissue-specific expression of BPGF- 1 in the bone and the prostate, coupled with the stimulation of prostatic carcinoma cells by BPGF-1, may account for the preferential colonization of the vertebrae by metastatic prostatic carcinoma cells.

The present disclosure presents the results from studies directed to identification and characterization of growth factors which promote prostate cell growth. Also examined is the question of whether fibroblast-specificity exists in affecting the growth of human prostate cancer, and in particular, of the lymph node derived prostate cancer cell line (LNCaP).

LNCaP cells were chosen for several reasons. Firstly, LNCaP cells have previously been shown to be nontumorigenic when injected subcutaneously in athymic mice with less than 4 x 10⁶ cells/inoculum (Horoszewicz et al, 1983). This observation was confirmed by the present inventors, and further extended by their discovery that LNCaP cells are nontumorigenic even at higher doses. Thus the inductive capabilities of specific fibroblasts can be examined following their co-administration to mice along with LNCaP. Secondly, the LNCaP cell line is the only prostate cell line that produces prostate specific antigen (PSA) (Papsidero et al, 1981), a human tissue-specific tumor marker used clinically to monitor in vivo prostate cancer cell growth (Stamey et al, 1987; Ford et al, 1985). Thirdly, LNCaP cells are androgen-responsive both in vivo (Sonnenschein et al, 1989) and in vitro (Schuurmans et al, 1989), which provides scope for the sex-dependent differences in chimeric tumor growth to be assessed. Furthermore, and importantly, of the androgen-responsive human prostate cancer models available, including PC82, HONDA, and LNCaP cell lines, only the LNCaP can be consistently grown in vitro (Isaacs, 1987). The inventors have exploited these properties in the development of parallel in vitro and in vivo cell-cell interaction assays. This allows, for the first time, the results from dual model systems using the same cell types and factors to be assessed.

Moreover, results from such coordinated in vitro and in vivo studies can be more confidently applied to the clinical situation.

The in vivo assay system disclosed herein is based upon the co-administration of LNCaP cells to athymic mice along with another cell type or composition. The effect of the cells or composition being analyzed can then be assessed by determining the degree of tumor growth in the co-inoculated animals and comparing it to the control growth observed (if any) in animals given either LNCaP cells or the test composition alone.

To analyze compositions other than intact cells, the inventors have developed a modified version of the assay. This is based upon the adsoφtion of concentrated substance(s) onto a solid matrix and the co-administration of the matrix and LNCaP cells to an experimental animal where the adsorbed matrix acts as a reservoir for the in vivo delivery of the test substance(s). It is contemplated that this method will be particularly useful for analyzing substances such as conditioned media from various cell types and known growth factors. The results disclosed herein demonstrate that certain fibroblasts can induce LNCaP tumor growth in vivo in a cell-type specific and androgen-dependent manner. Of the 6 fibroblast cell lines analyzed, bone fibroblasts, followed by the prostate-derived fibroblasts, were found to be the most effective in stimulating LNCaP cell growth both in vivo and in vitro. The presence of bidirectional paracrine pathways between LNCaP and fibroblast cells is illustrated in vivo by the development of sarcomas with the co-inoculation of LNCaP cells and nontumorigenic rUGM and 3T3 fibroblasts. Similar effects are also apparent in vitro as LNCaP and rUGM conditioned media produce bidirectional increases in growth in a paracrine-, but not autocrine-, mediated fashion. These observations suggest that LNCaP and fibroblast cells secrete factors that produce a more favorable microenvironment for tumorigenesis by reciprocally promoting growth, adherence or angiogenesis. LNCaP cells participated in chimeric tumor formation preferentially in males, demonstrating initial in vivo androgen-sensitive growth. These results, along with their in vitro androgen sensitivity, further support the view that the initial growth of LNCaP cells in vivo may be androgen-responsive (Sonnenschein et al, 1989).

Using this novel method, the LNCaP androgen-refractory cell lines C₄ and C₅ have been shown, for the first time, to be tumorigenic and to secrete high levels of PSA autonomously, i.e., in the absence of androgen. Both of these characteristics are typically found in human prostate cancer as it undergoes transformation to enter the hormonally refractory state.

Furthermore, the inventors' finding that hormonally refractory prostate cancer cells secrete specific autocrine protein factor(s) that induce PSA gene expression by the prostate cells is important because, at present, there have been no reports concerning this area of investigation. The identification of factor(s) produced by such refractory cells may have an impact on the development of new therapeutic approaches to address the problem of hormonally refractory prostate cancer cell growth.

Results from studies using a solid matrix adsorbed with conditioned media indicated that non-dialyzable factor(s) from bone fibroblast conditioned media samples alone could indeed induce LNCaP growth in vivo. This is the first demonstration that LNCaP tumor growth in vivo can be initiated by specific soluble growth factors derived from fibroblast cells. These results underscore the importance of growth factors in prostate cancer growth and progression. In further purifying the growth factors by employing heparin sepharose chromatography, it was determined that the substantially purified fraction contained novel polypeptides with apparent molecular weights on SDS/PAGE of: 227, 223, 218, 157, 90, 80, 48, and 20 kD.

These polypeptides were found to be distinct from bFGF by a number of criteria, including differential elution from heparin sepharose columns and distinct immunoreactivity. The presence of the novel 157 kD polypeptide within the active fractions was not initially detected, presumably as it was masked by an irrelevant and inactive polypeptide also present in the control media. Its presence was shown following the generation of an anti-growth factor mAb,

MS 329, which reacts with a 157 kD protein which is present in the active fractions, but absent from the control media (FIG. 11).

Skeletal tissues are known to produce various growth factors (Canalis et al., 1988;

Wergedal et al, 1986; Sampath et al, 1986; Globus et al, 1989; Hauschka et al, 1986), including bFGF (Globus et al, 1989). Osteoblasts are the principal source of synthesis and deposition of bone matrix and the site where bFGF is stored and mediates its mitogenic activity (Globus et al, 1989; Hauschka et al, 1986). bFGF promotes LNCaP cell growth and may also act in a paracrine fashion to stimulate metastatic cancer cell growth (Lu et al, 1989; Ensoli et al, 1989), but bFGF itself does not appear to be an active component of the growth factors disclosed herein. However, as anti-bFGF antibodies inhibited the bone fibroblast growth factor stimulatory action on prostate epithelial cells, albeit only slightly, the possibility remains that a bFGF-like protein may be responsible, in part, for the growth factor activity which stimulates prostate cell growth in vivo and in vitro.

EXAMPLE 1 Acceleration of Human Prostate Cancer Cell Growth In Vitro and In Vivo by Factors

Produced by Prostate and Bone Fibroblasts

Materials and Methods

Cell Lines and Cell Culture

LNCaP cells, passage 29, were obtained from Dr. Gary Miller (University of Colorado, Denver, CO) and grown in RPMI 1640 (Irvine Scientific, Santa Anna, C A) with 10% foetal bovine serum (FBS). Phenotypically, the cells resembled parental lines, as evidenced by the results of karyotypic analysis and androgen receptor analysis (see below).

The six nontumorigenic mesenchymal cell lines analyzed in this study are as follows: a foetal urogenital sinus mesenchyme-derived cell line (rUGM) from 18-day old Noble rat fetuses, developed as described by Chung et al, 1984. rUGM cells were maintained in DMEM (Gibco

Laboratories, Grand Island, NY), 5% calf serum (CS), and passages 14-16 were used. A human bone fibroblast cell line, MS, derived from an osteogenic sarcoma, was established by Dr. A. Y. Wang (The University of Texas M. D. Anderson Cancer Center, Houston, TX). MS cells were maintained in T-medium (80% DMEM, 20% F12K [Irvine Scientific], 3 g/1 NaHCO₃, 100 u ml penicillin G, 100 μg/ml streptomycin, 5 μg/ml insulin, 13.6 pg/ml triiodothyronine, 5 μg/ml transferrin, 0.25 μg/ml biotin, and 25 μg/ml adenine) with 5% FBS, and passages 29-33 were used. A rat prostatic fibroblast line, NbF-1, was established from a normal Noble rat ventral prostate gland as described previously (Chang & Chung, 1989). NbF-1 cells were maintained in DMEM and 5% CS, and nontumorigenic passages 18-22 were used. Normal adult human lung fibroblasts, CCD16 (American Tissue Culture Catalogue CCL 204), were supplied by Dr.

J. Roth (Dept. of Thoracic Surgery, UT M. D. Anderson Cancer Center, Houston, TX), and passages 14-16 were used. NIH-3T3 cells (ATCC #6587), derived from embryonic mouse tissue, were supplied by Dr. D. Becker (UT M.D. Anderson Cancer Center, Houston, TX) and maintained in DMEM with 5% CS. Normal rat kidney (NRK) fibroblasts (ATCC #6509) were grown in DMEM with 5% CS, and passages 10-12 were used. Conditioned media from LNCaP and all 6 fibroblast cell lines was collected and prepared as follows: Cells were cultured in 150 mm tissue culture dishes (Falcon, Becton Dickinson Laboratories, Lincoln Park, NJ) with T-medium, 2% TCM, a serum-free defined media supplement (Celox Co., Minnetonka, MN), and 1% FBS until 60-70% confluent; washed with PBS/EDTA; and changed to serum-free T-medium containing 2% TCM only. After 48 hours, the conditioned media was removed and filtered through a 0.2 μm filter (Nalge Co.,

Rochester, NY), and 0.1 mM phenylmethyl-sulfonylfloride (PMSF, Sigma) was added. Protein concentrations in the conditioned media were determined using a protein assay (Bio-Rad Laboratories, Richmond, CA), and ranged from 70-100% of control (T-medium and 2% TCM; 1.3 mg/ml). The conditioned media was dialyzed at 4°C against distilled water containing 0.1 mM PMSF using Spectra/Por 3 dialysis membranes (M_r > 3500 dalton, PGC Scientifics,

Gaithersburg, MD) for 96 hours, changing the water after 48 hours. The samples were lyophilized to dryness and reconstituted in T-medium to ten times concentration (lOx), filtered, and diluted to the desired working concentration (0.1 to 2x) with T-medium containing 2% TCM.

Assessment of In Vivo Tumor Growth

To determine the ability of specific fibroblasts to elicit LNCaP growth in vivo, 6-8 week old athymic nude mice (BALB/c strain, Charles River Laboratory, Wilmington, MA) of both sexes were co-inoculated subcutaneously with 1 x 10 LNCaP cells and 1 x 10 of one of the 6 fibroblast cell lines described above. Up to 5 x 10⁶ LNCaP cells and 2 x 10 of each of the fibroblast cell lines were injected alone as controls to assess their tumorigenicity. The cells were suspended in 0.1 ml of RPMI 1640 with 10% FBS prior to injection and inoculated via a 27-gauge needle. Tumors were measured twice weekly and their volumes were calculated by the formula L x W x H x 0.5236 (Janek et al., 1975). At the time of sacrifice, sternotomy was performed and a cardiac puncture was carried out to obtain serum for PSA analysis. Tumors were excised, weighed, and subjected to various moφhological and biochemical analyses (see below).

Further studies were performed to determine whether LNCaP tumor growth in vivo could be affected by soluble growth factors alone. LNCaP cells were injected along with a Gelfoam preparation (Upjohn, Kalamazoo, MI) adsorbed with type IV collagen (Collaborative

Research, Bedford, MA), endothelial cell derived growth factor (ECGF) (Collaborative Research), and ten-times concentrated rUGM or MS conditioned media. This novel matrix system was developed through modification of a previously described procedure (Thompson et al., 1988) and serves as a reservoir for delivery of biologically active factors in vivo. ECGF was chosen as a marker of physiologic response to determine whether it could retain its biologic activity during this procedure, and whether this angiogenesis alone would be sufficient to promote tumor formation. rUGM and MS conditioned media were used because these cells could induce LNCaP growth in vivo. Basic fibroblast growth factor (bFGF, Collaborative Research) was also used because of its mitogenic effect on LNCaP cells in vitro (see below). Under sterile conditions, Gelfoam, a solid gelatin sponge, was pre-soaked with 100 μg/ml collagen IV for 12 hours at 4°C, followed by either 1 μg/ml ECGF, bFGF, or ten-times concentrated stromal conditioned media, for 1 hour. The Gelfoam was then minced using a polytron to allow subcutaneous inoculation via an 18-gauge needle. Following subcutaneous injection of 0.1 ml Gelfoam, the same site was injected with 2 x 10 LNCaP cells using a 27- gauge needle. For controls, 2 x 10 LNCaP cells were inoculated with Gelfoam and collagen

IV, with or without ECGF. Tumor incidence and size was monitored as described above.

Histology and Immunohistochemistry

For routine histology, specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Eight micron fixed sections were cut and stained with hematoxylin and eosin (H&E). For immunohistochemical studies, specimens were deparaffinized with xylene, rehydrated with 70% ethanol, and treated with 0.1% trypsin for 10 min at 37°C. Sections were then incubated with monoclonal antibodies prepared against cytokeratin, PSA, or prostatic acid phosphatase (PAP) (Biogenex, Dublin, CA). An avidin-biotin complex method was used with all specimens using fast red TR or AEC as chromogens (Biogenex). Slides were counterstained with aqueous hematoxylin and mounted with glycerol for visual inspection and photography. Determination of Serum PSA Values

Animals were killed by cardiac puncture under methoxyflurane anesthesia. Blood was allowed to clot at 37°C and centrifuged, and the serum was stored at -20°C. PSA values were determined using a dual reactive enzymatic immunoassay kit with a lower limit of sensitivity of

0.4 ng/ml (Hybritech Inc., San Diego, CA).

DNA Isolation and Southern Blot Analysis

Tissue DNA was isolated from tumors as described by Davis (1986). DNA concentration was determined with a spectrophotometer. DNA specimens were applied to

Zetaprobe membranes (Bio-Rad) then baked at 80°C for 90 minutes prior to hybridization with a P-labeled human Alu repetitive sequences probe (Oncor, Gaithersburg, MD).

RNA Isolation and Northern Blot Analysis Total cellular RNA was prepared from frozen tissues by the 4 M guanidinium thiocyanate extraction method (Chomcjymski & Sacchi, 1987). Typical yields of total cellular RNA were about 300 μg/200 mg tissue as quantified spectrophotometrically using 40 μg RNA/A₂₆₀ unit. RNA was denatured in 50% formamide/18% formaldehyde at 55°C and fractionated by electrophoresis in a 0.9% denaturing formaldehyde agarose gel. Samples were transferred onto a Zetaprobe membrane (Bio-Rad) by capillary method, and the membrane was then baked for 2 hours at 80°C. Following this, the membrane was prehybridized in the presence of 1 M NaCl, 10% dextran sulfate, 1% SDS, and 200 μg/ml salmon sperm DNA for at least 2 hours at 65°C. Hybridization was carried out at 65°C overnight with a random-primer- labeled probe, as indicated. Finally, the membrane was washed under high stringency conditions (0.5 x SSC, 1% SDS at 65°C). Autoradiograms were prepared by exposing Kodak

X-Omat AR film to the membrane at -80°C with intensifying screens.

Mitogenic Assays

To determine the mitogenic activity of androgens (testosterone and dihydrotestosterone, Sigma) and conditioned media prepared from various types of fibroblasts on the growth of human LNCaP cells in vitro, a 96-well assay based on the uptake and elution of crystal violet dye by the cells in each well was employed (Gillies et al, 1987; Kanamarus & Yoshida, 1989). Various defined growth factors, including basic fibroblast growth factor (bFGF), transforming growth factors alpha and beta (TGFα, TGFβ) and epidermal growth factor (EGF) (Collaborative Research) were also tested.

Using 96 well plates, 3,000 LNCaP, 500 MS, or 200 rUGM cells were plated per well (Falcon) in T-medium containing 1% charcoal stripped CS and 2% TCM. Twenty-four hours later, the cells were downshifted to serum-free condition (see above) with various concentrations of androgens, growth factors, or conditioned media. To avoid stripping poorly adherent LNCaP cells with each media change, media was partially removed by gentle suction and 100 μl of fresh media was added in 50 μl aliquots. The medium was changed every 2 days; 7-10 days later the cells were fixed in 1% glutaraldehyde (Sigma) and stained with 0.5% crystal violet (Sigma). Plates were washed, air-dried, and the dye was eluted with 100 μl Sorensen's solution (9 mg trisodium citrate in 305 ml distilled H₂0, 195 ml of 0.1 N HC1, and 500 ml 90% ethanol). The absorbance of each well was measured by a Titertek Multiskan TCC/340 (Flow

Laboratories, McLean, VA) at 560 nm. Control studies demonstrated that absorbance is directly proportional to the number of cells in each well.

Androgen Receptor Assays Whole-cell androgen receptor assays were performed as described previously by Guthrie et al. (1990) with the following modifications. LNCaP cells were plated in T-medium plus 5% FBS in 6 well plates (Falcon) and downshifted to 0.4% charcoal-stripped calf serum 24 hours preceding the assay. Just prior to beginning the assay, this medium was removed and cells were washed twice with PBS/EDTA, and T-medium with various dilutions of H-R1881 (methyltrienolone 81.8 Ci/mmol, DuPont Co., Wilmington, DE) was added to appropriate wells. In some wells, unlabeled R1881 (200-fold of ³H-R1881) was added to determine the extent of nonspecific binding. Following a 90-minute incubation at 37°C, the media was removed, cells were washed with ice-cold PBS/EDTA, and 1 ml of 100% ethanol was added to each well. A 500 μl aliquot was added to a scintillation vial and counted with a scintillation counter (Beckman Instruments, Inc., Houston, TX). Results

Effect of Co-inoculated Fibroblasts on LNCaP Tumor Growth

The incidence of tumor formation in mice co-inoculated with LNCaP cells and various types of fibroblasts was compared (Table 2). The observation period for all injections was 3 months. LNCaP and all fibroblast cell lines were found to be nontumorigenic (0/20) with injections of up to 5 x 10 or 2 x 10 cells, respectively. No significant sex differences in tumor formation were observed in hosts co-inoculated with LNCaP and rUGM cells, with an overall tumor incidence of 61% for males and 50% for females. The average latency period for measurable tumor growth was 42 days in male and 45 days in female hosts. No difference in tumor volume or latency period was observed by increasing the rUGM inoculum from 1 x 10⁵ to 1 x 10 cells. Mean tumor volume was 322 + 106 mm . No sex differences in the incidence of tumor formation was observed in hosts co-inoculated with LNCaP and 3T3 cells (67%, mean tumor volume 420 mm ). In contrast, marked sex differences in tumor induction were observed with co-inoculation of LNCaP and human bone (MS) or LNCaP and rat prostatic (NbF-1) fibroblasts, as these tumors formed only in male hosts (62% and 17%, respectively). Mean tumor volume for LNCaP/MS and LNCaP/NbFl tumors was 238 ± 74 mm³ and 72 ± 52 mm³, respectively. Lung CCD16 and NRK fibroblasts did not induce chimeric tumor growth in either sex. The histomoφhology and relative content of LNCaP cells in the various fibroblast-induced tumors differed markedly, as characterized below. TABLE 2

Histomoφhology of tumors

Fibroblast Host Incidence of tumor Carcinosarcoma Sarcoma formation

MS Male 8/13 (62%) 8/13^a (62%) 0/13 (0%) Female 0/10 (0%) rUGM Male 31/51 (61%) 16/51 (31%) 15/51 (30%)

Female 18/36 (50%) 2/36 (6%) 16/36 (44%)

NbF-1 Male 3/18 (17%) 3/18 (17%) 0/18 (0%) Female 0/10 (0%)

CCD16 Male 0/20 (0%) Female 0/6 (0%) NRK Male 0/20 (0%)

Female 0/10 (0%)

^a All carcinomas with no sarcomatous component

In further studies of this kind, only nonirradiated human bone stromal (MS) cells were found to be active in promoting LNCaP tumor formation (Table 3).

TABLE 3

Fibroblast LNCaP Cell Incidence of tumor formation*

None 2 x l0⁶ 0/6 (0%)

^{MS l x l}°⁶ l x lO⁶ 15/17 (88%) (nonirradiated)

MS 1 x 10⁶** (irradiated) _{l χ l Q} ⁶ 0/8 (0%)

CCD16 1 x 10⁶ 1 x 10⁶ 0/6 (0%)

* Incidence of tumor formation was recorded 32-54 days after inoculation

** The cells were irradiated with 40 Gy before co-inoculation with LNCaP Cells

Characterization of the Chimeric Tumors

Chimeric tumors were characterized histomoφhologically, immunohistochemically, and biochemically. A difference in histomoφhology of LNCaP/rUGM chimeric tumors was noted between males and females: in males, 51% of tumors (or 31% of inoculation sites) were carcinosarcomas, with a predominantly epithelioid component separated by strips of mesenchymal cells (FIG. 1A), while 89% (16/18) of the tumors in females were pure sarcomas (FIG. IB). MS bone fibroblasts were found to be the most potent inducer of LNCaP tumor formation. All tumors were carcinomas composed of sheets of poorly-differentiated epithelial cells with minimal mesenchymal cells and formed at 62% of inoculated sites in male hosts (FIG. 1C); no tumors formed in female hosts. NbF-1 cells were also capable of inducing

LNCaP tumor growth in male hosts, but not as well as the MS or rUGM cells; three carcinomas formed from 18 inoculations (17%). LNCaP/3T3 tumors, however, were all sarcomas with no epithelial component. No tumors formed with co-inoculation of LNCaP with human lung CCD16 or NRK fibroblasts. The prostatic origin of the epithelial cells participating in the MS-, rUGM-, and NbF- induced tumor formation in male hosts was confirmed with immunohistochemical staining procedures using monoclonal antibodies directed against PSA, PAP, and cytokeratin. The epithelial component of these tumors stained intensely positive for PSA using fast red TR as the chromogen (FIGs. ID and IF) with no staining of the associated stromal component. The epithelial component of these tumors also stained positive for PAP and cytokeratin, but in an irregular and scattered manner compared to PSA. In contrast, sarcomas arising from LNCaP/rUGM inoculations in females and LNCaP/3T3 inoculations in both males and females stained negatively for PSA (FIG. IE), PAP, and cytokeratin. Biochemical characterization using Northern and Southern hybridization techniques corroborated the histo logic findings to confirm the human prostatic origin of the epithelial component of the chimeric tumors (FIGs. 2 A and 2B). The LNCaP/rUGM tumors in male hosts contained a predominantly human component as manifested by the presence of Alu- sequences in 6 (2 weakly) of 7 tumors examined, compared to none in female tumors (FIG. 2A). PSA expression was more variable in these tumors and did not correlate consistently with the histomoφhologic and Southern dot blot analysis, likely because of different sampling from a heterogenous carcinosarcoma. All LNCaP/MS tumors were strongly positive for PSA expression and human-specific Alu sequences on Northern and Southern analysis, respectively (FIG. 2B). None of the LNCaP/3T3 tumors that formed had any human prostate component (FIG. 2B).

Serum PSA Levels

Sera from male and female mice bearing chimeric tumors were assayed for PSA using the Hybritech enzyme immunoassay. Four control males injected with human bladder transitional cell carcinoma cells (Stamey et al, 1987) had undetectable PSA levels, as anticipated, since PSA is a human prostate marker. Significant differences in median serum PSA values were observed among the different fibroblast-induced tumors, as well as between male and female hosts, paralleling differences in their histomoφhology (FIGs. 3A and 3B). LNCaP/MS tumors were associated with consistently elevated serum PSA levels ranging from 25.1 ng/ml to 323 ng/ml, with a median of 68.1 ng/ml (n = 6). Similarly, male hosts bearing

LNCaP/NbF-1 tumors had elevated serum PSA (n = 4). However, nontumor-bearing females with LNCaP/MS and LNCaP/NbF-1 injections had undetectable serum PSA levels. Serum PSA values in males with LNCaP/rUGM tumors ranged from 0.4 to 348 ng/ml with a median of 16.1 ng/ml; 11 of 12 males had detectable levels and 3 had levels > 100 ng/ml. In all but one of the 8 females with LNCaP/rUGM tumors, serum PSA was undetectable. All animals with LNCaP/3T3 tumors, as well as those inoculated with LNCaP/CCD 16 or LNCaP/NRK cells, had undetectable serum PSA levels.

LNCaP Androgen Sensitivity and Androgen Receptor Content

To determine whether the LNCaP cell line was indeed androgen-sensitive, the in vitro mitogenic effects of testosterone and DHT in serum-free and chemically-defined medium were evaluated. Peak responses were seen with 5 10 M testosterone and 1 x 10 M DHT, producing 62% and 43% increases, respectively, in cell number over 9 days when compared to controls grown in serum- and hormone-free media (FIG. 4A). Whole cell androgen receptor assays revealed the presence of a substantial number of high affinity androgen receptors (K_ά = 0.23 nM; B_max = 332 fmol/mg protein, FIG. 4B).

Effect of Defined Growth Factors on LNCaP Cells In Vitro

To identify possible mitogens involved in LNCaP cell growth, the dose-response relationship between LNCaP cells and bFGF, EGF, TGFα, and TGFβ was investigated. Using concentrations ranging from 0.1 to 50 ng/ml, bFGF stimulated LNCaP cell growth 180% in a concentration-dependent manner compared to cells grown in serum-free media alone (FIG. 5A). Minimal increases in cell number compared to controls were seen with EGF and TGFα over a wide range of concentrations. TGFβ, at 1 ng/ml, inhibited LNCaP cell growth by 70%. Time course studies also revealed that bFGF (50 ng/ml) stimulated LNCaP cell growth in a linear fashion during a 9-day observation period (FIG. 5B).

Effect of Fibroblast-Conditioned Medium on the Growth of LNCaP Cells In Vitro

To determine whether the in vivo fibroblast specificity in inducing LNCaP growth could be explained by specific soluble growth factors produced by the fibroblasts, the mitogenic activity of conditioned media from MS, rUGM, NbF-1, 3T3, CCD16, and NRK cells on LNCaP growth in vitro was compared. The conditioned media from MS, rUGM, and NbF-1 cells stimulated LNCaP cell growth up to 210% compared to controls (FIG. 6 A), whereas 3T3, CCD16, and NRK conditioned media were ineffective. This paracrine effect was observed to be bidirectional, as LNCaP conditioned media stimulated rUGM cell growth up to 275% (FIG. 6B) and MS cell growth 225%. The bidirectional paracrine stimulation between LNCaP and rUGM or MS cells in vitro is dependent on the concentration of conditioned media. No autocrine stimulatory effect was observed on exposing LNCaP, rUGM, or MS cells to their own conditioned media.

Effect of MS- and rUGM-Conditioned Media and bFGF on LNCaP Growth In Vivo Since MS- and rUGM-conditioned media and bFGF stimulated LNCaP cell growth in vitro, possible growth-promoting effects in vivo were examined following the coating of these growth factor onto a solid Gelfoam matrix. Control subcutaneous injections of Gelfoam with collagen IV plus ECGF with no co-inoculated LNCaP cells was found to induce local neovascularization at 3 weeks (FIG. 7B), illustrating that certain growth factors could maintain their biological activity when injected subcutaneously with this technique. Co-inoculation of

2 x 10 LNCaP cells with Gelfoam adsorbed with collagen IV alone or with collagen IV and ECGF failed to induce LNCaP tumor formation.

However, when 2 x 10 LNCaP cells were inoculated with Gelfoam plus collagen IV adsorbed with either bFGF (1 μg/ml) or ten-times concentrated conditioned media from rUGM or MS cells, LNCaP tumors formed at 60%, 50%, and 38% of inoculated sites, respectively.

Tumor latency, growth rate, and size were similar and did not differ from those of chimeric tumors induced by co-injecting LNCaP cells with rUGM or MS fibroblasts. Animals bearing LNCaP tumors had an elevated serum PSA (median 73 ng/ml) and the tumors were histologically carcinomas staining positive for PSA (FIG. 7C). The human prostatic origin of these tumors was confirmed with Southern dot blot analysis for human Alu sequences and

Northern analysis for PSA mRNA expression (FIG. 7D). EXAMPLE 2

Isolation and Characterization of Growth-Promoting Factor(s) in the Conditioned Media of Cultured Human Bone Stromal Cells

As shown in Example 1 , accelerated LNCaP tumor growth still occurred in vivo when human bone stromal cells themselves were substituted by their conditioned media. Also, purified bFGF induced LNCaP tumor growth both in vitro and in vivo. These observations prompted further investigation of the properties of the conditioned media and raised the possibility that bFGF itself may be the active component of the conditioned media.

The MS conditioned media was dialyzed prior to further purification and analysis. First, a sample of conditioned media was subjected to affinity chromatography using a heparin sepharose column. The sample was loaded onto the column in the low salt-containing buffer 10 mM Tris/HCl, 1 mM PMSF, pH 7.4, to allow binding to the column, and the column was then washed with this buffer to remove any non-binding species. The components that bound to the column were then eluted using the above buffer containing an increasing gradient of NaCl, from 0-3 M. Following assays of the eluted material, it was determined that the peak of the active component(s) responsible for LNCaP tumor growth in vivo corresponded to the 1 M NaCl eluted fraction (FIG. 8). This is distinct from bFGF, which is known to elute at > 2.0 M NaCl (Story et al, 1987). As can be clearly seen in FIG. 8, even the trailing edge of the activity peak eluted prior to exposure to 2 M NaCl. The SDS/PAGE profile of this partially-purified heparin sepharose-eluted growth factor preparation was then determined and compared to the control media (TCM). Following silver stain analysis of SDS gels, several distinct polypeptide bands in the M_r range of 18 to 228 kDa were found to be present in this fraction, which were absent from the control (FIG. 9, track 3 vs. track 2). The presence of a further novel 157 kD polypeptide within the active fractions was not initially detected, presumably as it was masked by an irrelevant and inactive polypeptide also present in the control media. Its presence was shown following the generation of an anti- growth factor mAb, MS 329, which reacted with a 157 kD protein present in the active fractions and absent from the control media (FIG. 11). The activity of this fraction in stimulating prostatic cell growth and soft agar colony formation and in inducing in vivo LNCaP tumor growth was investigated and compared to that of other fractions. The soft agar colony formation assay is a standard in vitro assay to test for transformed cells, as only such transformed cells can grow in soft agar. 0.6% (w/v) agar was placed into the bottom of each well on a 24 well plate, and each well was seeded with 2,000 NbE-1 cells. A feeder layer of 0.3 to 0.4% (w/v) agar containing the potential growth factor substances to be analyzed was then placed on top of the cells. The number of soft agar colonies formed was recorded 3 to 4 weeks after seeding. The active fractions (1 M eluates) from the column were found to be particularly active in both assays, whereas control media, the 2 M NaCl eluate, and similar fractions eluted by 1 M NaCl from 3T3 cell conditioned media, were found to be completely inactive (Table 4). TABLE 4

Condition Incidence of Tumor Formation Soft Agar Colony Formation

Gelfoam & Collagen IV 0/6 (0%) 4 ± 1.5

+ MS 1.0 M NaCl Eluate _9/12 (₇₅o_/o) ± ₇.₂

+ MS 2.0 M NaCl Eluate ₀/24 (0%)

+ 3T3 1.0 M NaCl Eluate _ ₂ ± 0.7

+ TCM 1.0 M NaCl Eluate _{0/6 (0}o_{/o) 4 ± 0 7}

The properties of the partially purified heparin sepharose-eluted growth factor preparation were then further investigated. The mitogenic and tumor-forming activities were found to be trypsin and heat sensitive, but to be partly resistant to acid and reducing agent treatment (Table 5).

TABLE 5

Condition Remaining activity (%)*

None 100

Heat

70°C, 5 min 66 ± 4

100°C, 5 min 18 ± 3

HC1, IN 52 ± 4

Dithiothreitol 63 ± 3

(0.05M)

Trypsin (10 ug/ml) 0

* Defmed by [ H] thymidine incoφoration into cellular DNA

The above results indicated that bFGF was not responsible for the activity of the bone derived conditioned media. Further studies were conducted to confirm this using antibodies directed to pvirified growth factors. It was determined that the NbE-1 soft agar colony-forming efficiency of the active fraction could not be neutralized by antibodies to bFGF, KGF or HGF antibodies (FIG. 10). Moreover, immunoblotting of the active fraction with antibodies against bFGF, KGF, TGFβ HGF, and EGF also failed to reveal any immunoreactive bands. These results together suggest that the above factors are probably not the endogenous growth factors in the conditioned media, and, therefore, that certain unique growth factor(s) are largely responsible for such stimulatory activity.

It should be noted here, however, that the above results do not exclude the possible involvement of factors that may belong to one of the bFGF, KGF, TGFβ, HGF, or EGF families. In a separate study, the inventors have also shown that bFGF, HGF, and nerve growth factor (NGF) have certain growth-promoting effects on prostate cancer cells.

Due to the presence of several high M_r protein species in the active fraction, Western blot analyses of this fraction with antibodies raised against several (ECM) proteins, laminin, fibronectin, tenascin, and entactin were performed. The results demonstrated that this fraction reacted positively with fibronectin and tenascin, but not laminin and entactin antibodies. It is possible that the ECM proteins may act in concert with active growth factor(s), both of which may be necessary for inducing prostate cancer growth or progression. In extending these analyses to two fresh bone marrow aspirate samples obtained from patients with prostate cancer (with confirmed bony metastasis) and lung cancer (without metastasis), it was found that fresh bone marrow aspirates from both patients contained similar tenascin antibody -reactive proteins to the conditioned media. However, a l lO kD (pl lO) protein was found to be present in the bone marrow aspirate of the prostate but not the lung cancer patient. This pi 10 could represent a tenascin proteolytic fragment. In addition to tenascin, fibronectin antibody also reacted selectively with some common proteins (banded at > 228 kDa) present in both bone marrow samples and the conditioned media. The control media was found to be devoid of immunoreactivity with tenascin and fibronectin antibodies.

EXAMPLE 3

Further Characterization of Bone-Marrow Derived Growth Factors

This example demonstrates an approach which the inventors propose may be employed in the future characterization of the growth factors. The preferred approach recommended by the inventors involves the initial preparation of antibodies against the growth factor polypeptides.

To further characterize the biochemical nature of these human bone-derived growth factors, monoclonal antibodies (mAbs) will first be raised against the constituent polypeptides. The inventors propose the partially-purified growth factor preparation as a starting material for this procedure for the following reasons. First, the action of the conditioned media cannot be neutralized using a single commercially available antibody directed against any of the known growth factors. Second, the total number of bone stromal cell-associated proteins in the partially purified fractions is relatively small, and it will be possible to develop specific mAbs against all of these proteins. Most importantly, fresh bone marrow supernatant fractions contain proteins similar to those of the conditioned media.

It is proposed that such mAbs will have utility in a variety of different embodiments. They will be powerful tools for the further purification of the growth factors. From the data presented above, it seems likely that the interaction between the growth factors, the prostate cancer cells, and certain ECM proteins may be required for prostate cancer progression and acquired behaviors such as metastatic and androgen-independent properties. mAbs against such polypeptides are therefore potentially attractive diagnostic, prognostic, imaging, and therapeutic agents for the treatment of prostate cancer in men. Since mAbs may be obtained which bind specifically to the cancer cells or to cancer-specific antigens in circulation, such mAbs would also be a powerful diagnostic agent.

The conditioned media of the MS culture will be fractionated to prepare the partially purified growth factors against which mAbs are to be generated. An aliquot of this material will be loaded onto a heparin sepharose affinity column previously equilibrated as an appropriate buffer, such as Tris HC1 (pH 7.4). Proteins will be eluted from the column by a continuously increasing NaCl gradient, and the concentration, mitogenic activity, and soft agar colony-forming efficiency of all fractions eluted from the column will be determined. The biologically active fractions will be pooled and concentrated by a suitable method, such as dialysis and lyophilization, or desalting using dry sephadex gels or sephadex gel exclusion column chromatography followed by lyophilization.

It is proposed that B ALB/c mice of approximately 3 months in age will be immunized intraperitoneally (day 0) with 10 to 50 μg/mouse of the partially purified growth factors homogenized with Ribi mouse adjuvant system (Ribi, 1985). The mice will then be given two consecutive weekly intraperitoneal injections of the antigens mixed with Ribi mouse adjuvant (days 7 and 14). Approximately one month after the third injection, a booster inoculation of antigens alone will be given. Here the inventors contemplate that the novel booster method described below will be advantageously employed. It is proposed that the immunized mice will be surgically opened to expose the spleen and a sterile solution of 5 to 20 μg of the growth factor antigens will be injected directly into the spleen. The mice will then be sutured and allowed to recover. It is believed that this method will allow the optimal exposure of the splenocytes to the booster antigen.

Five to seven days after the booster injection, a small amount of blood from the tail of the immunized mice will be bled and tested for the presence of circulating antibodies to the growth factors by an enzyme-linked immunosorbent assay (ELISA). Those mice producing reasonable titers of circulating antibodies to the partially purified antigens will be sacrificed and their spleens will be aseptically removed for cell fusion.

The mouse myeloma cell line proposed to be of use for hybridization is the 8- azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line. The SP2/0 cell line has been selected for 8-azaguanine resistance and does not survive in medium containing hypoxanthine, aminoprotein, and thymidine (HAT). The cells will be fused as described by

Chan et al (1987). Immune splenocytes (10 cells) obtained from two hyperimmunized mice and 8-azaguanine-resistant SP2/0 mouse myeloma cells (10 cells) will be fused using 37% (v/v) polyethylene glycol 1500 (M.W. 500-600 M.A. Bioproducts, Inc.). Fused cells will be maintained for two days in growth medium that has been conditioned by SP2/0 cells, and then plated in five or six 96-well microtiter plates in growth medium containing HAT (selection medium) and screened for antibody production at the end of two weeks by indirect ELISA.

For the screening, purified growth factors, or partially purified growth-promoting factor(s) obtained from the conditioned media, or bone marrow supernatant fractions may be used as target antigens, and media plus NaCl may be used as a control. The target antigens (50 ng/50μl/well) will be immunobilized onto the bottoms of the 96-well microtiter plates by slow evaporation at 4°C overnight. The culture medium from the wells propagating the splenocyte- myeloma (hybridoma) cells growing in the selection medium will be assayed for secreted antibodies that react with the immobilized antigens (either bone marrow supernatant fractions, or bone stromal cell-conditioned media, or purified growth factors may be used). The isotypes of the immunoglobulin(s) produced by cloned hybridoma cell clones may also be determined by ELISA, employing a commercial isotyping kit. The specificity of the mAbs may be determined by their reactivity with various antigens, as examined by ELISA and confirmed by Western blot analysis. After the mAbs are characterized, they may be produced in the form of mouse ascites fluid, purified and used to antagonize the soft agar colony forming efficiency of NbE-1 cells which are stimulated by the partially purified growth factors. This assay is proposed to be a reproducible, convenient and rapid assay method. Soft agar colony-forming efficiency is known to correlate directly with LNCaP tumorigenicity in vivo. A mAb, termed MS 329, has been produced which has reactivity with a 157 kD growth factor polypeptide (FIG. 11).

Based on previous experience (Chi et al, 1987; Drewinko et al, 1986; Zhang et al, 1989), the inventors further propose that it will be possible to identify specific mAbs that may have diagnostic and prognostic values in predicting human prostate cancer metastasis to the bone, imaging the prostatic metastasis, and inhibiting tumor-stromal interaction. The criteria to be used in assaying for such mAbs are proposed to include tests for, e.g., specific reaction with a defined protein band of conditioned media in immunoblots or in immunohistochemical assays, and/or competition for the binding of the putative growth promoting factor(s) with the cell membrane fraction prepared from prostate cancer cell lines.

Once specific mAb(s) that meet the above criteria have been identified, the inventors contemplate their use in diagnosis, prognosis, imaging, and therapy. This approach is advantageous because, unlike any anti-PSA antibodies, the mAbs against cancer-specific antigens may not be trapped in the blood compartment, and they would therefore more efficiently block prostate cancer and bone cellular interactions.

In addition, the inventors propose that the levels of these growth factors may correlate positively with prostate cancer progression. To investigate this, it is proposed that bone marrow aspirates will be obtained initially from late stages of the untreated prostate cancer patients (Stage Dl, D2) and prostate cancer patients treated with hormonal therapy, or failed hormonal therapy and chemotherapy. The concentration of growth factors in such samples may be analyzed by ELISA or radioimmunoassay (RIA) and compared to the number of prostate cancer cells present in bone marrow.

The inventors propose that the concentrations of growth factors will correlate with the proliferative potential and aggressiveness of the prostate tumor in vivo and inversely with patients' survival, and may also predict the length of period of remission and disease-free survival. The concentration of these growth factors may also serve as a valuable index to predict cancer progression prior to the manifestation of clinical symptoms. It is believed that the ELISA or RIA assay contemplated by the inventors will be extremely sensitive. Based on immunoblot analysis of the growth factors, the sensitivity of this assay is estimated to be in the ng range. This sensitivity of assay could be used effectively to diagnose prostate cancer, or to predict the progression of prostate cancer and its response to various therapies in very small volumes of bone marrow aspirates. Similarly, the assay will be refined as a diagnostic tool for the early detection of the onset of prostate cancer.

In further embodiments, it is proposed that the mAb(s) will have utility in radio-imaging protocols. mAb(s) labeled with indium 111 (100) can be administered to mice previously inoculated with LNCaP and bone fibroblasts for the development of LNCaP tumors. In this manner the tumor can be imaged, the sensitivity determined, and the distribution of mAb-In 111 complex in this model of prostate cancer examined. mAb(s) previously labeled with [ I] or mAb-immunotoxins such as mAb-ricin A chain (Pearson et al , 1990) could be delivered through continuous infusion to mice which bear experimental LNCaP tumors and the outcome monitored.

The specific mAbs could also be employed in the rapid purification of the growth factor polypeptides following the creation of an mAb-affinity column. This could be achieved by conjugating a specific mAb to cyanogen bromide (CNBr)-activated sepharose CL4B (Pharmacia) (Chan et al, 1986; Li et al, 1987). As such, the antibodies would first be attached to the CNBr-sepharose, and the antisera-bound matrix would then be poured into a column and washed with a suitable wash buffer. An aqueous mixture including the growth factor polypeptides could then be passed over the column under conditions which allow for immunocomplex formation between components in the mixture and the sepharose-bound antibodies. The column would then be washed extensively to remove non-specifically bound material and the specifically-bound antigens eluted from the column in a substantially purified state. Such an affinity column could also be used to isolate and characterize growth-promoting component(s) from human bone marrow aspirates obtained from prostate cancer patients. In such embodiments, bone marrow aspirates (~10 ml per patient, at 20 to 30 mg protein/ml) could be obtained from prostate cancer patients, from female breast cancer patients (with or without bony metastasis), and from healthy normal male and female donors and analyzed. From such investigations, the sex-dependent differences and disease specificity of the growth factors that appear to promote human prostate tumor growth could be investigated.

EXAMPLE 4

Isolation and Characterization of a Bone and Prostate Stromal Cell-Derived Growth Factor

Experimental Procedures Used

Cells and Media

The human bone fibroblast cell line, MS, derived from an osteogenic sarcoma, was established by Dr. A. Y.Wang (The University of Texas M.D.Anderson Cancer Center, Houston, TX). MS cells were maintained in T-medium (80% DMEM, 20% F12K, 3 g/ml

NaHC0₃, 100 units/ml penicillin G, 100 μg/ml streptomycin, 5 μg/ml insulin, 13.6 pg/ml triiodothyronine, 5 μg/ml transferrin, 0.25 μg/ml biotin, and 25 μg/ml adenine) with 5% FBS; passages 29-33 were used. The rat prostate epithelial cell line, NbE-1, was established from a normal Noble rat ventral prostate gland using procedures previously described (Chung et al. , 1989). PC-3 cells, human prostate adenocarcinoma, were derived from bone marrow metastases. LNCaP cells, passage 29 of the original line were kindly supplied by Dr. G. Miller

(University of Colorado, Denver, CO). All cells were maintained in T-medium with 5% FBS, unless otherwise stated.

Construction of the Human MS cDNA Expression Library Total cellular RNAs were extracted from MS cells using the RNAzol B method, a single-step purification protocol as described previously (Chomcyzynski and Sacchi, 1987). The Poly(A) mRNA was purified by two cycles of oligo (dT)-cellulose column chromatography according to the manufacture's procedures (Pharmacia LKB Biotechnology). 5 μg of MS Poly(A) mRNA was used to construct MS cDNA expression library in the λZAP II vector (Stratagene). Doubles-strands of cDNA primed with a oligo(dT)₁₂.₁₈ was synthesized as described by the manufacture's protocols (Pharmacia LKB Biotechnology). An EcoRl adaptor with an internal Notl site was added to the cDNA and ligated to the λZAPII/EcoRl vector. The final expression library contained a total of 2 x 10 clones, with more than 95% of the clones containing the cDNA inserts.

Heparin Sepharose- Affinity Column Chromatography

Dialyzed bone fibroblast conditioned medium was applied to a heparin sepharose affinity column (heparin-sepharose CL-6, Pharmacia LKB Biotechnology, Piscataway, NJ), 1.0 x 9 cm, which was previously equilibrated in 10 mM Tris-HCl buffer containing 0.1 mM PMSF, pH 7.5. The column was washed with a 3-bed volume of 10 mM Tris-HCl buffer and eluted with a linear salt gradient of NaCl (0 to 3 M NaCl). Two ml fractions were collected and dialyzed against distilled water containing 0.1 mM PMSF at 4°C. Protein concentrations were monitored by a spectrophotometer at 280 nM. Fifty ml of each fraction was used for the assessment of the stimulatory effect on the prostatic epithelial cells by soft agar colony forming assay. Clonin of the BPGF-1

The MS cDNA expression library was screened with the polyclonal antibody against MSI fraction. Screening of cDNA expression library was performed as described by the manufacturer's protocol (Stratagene). The cDNA clones were sequenced by the standard dideoxy chain termination method using sequenase (United States Biomedical). Two strands of the templates were sequenced using T7, T3, and internal primers generated from the sequence. Overlapping sequences were assembled into a contiguous sequence using the computer program Mac Vector®.

Northern Blot analyses

Total RNA was prepared from variety of human tissues and cells as described above. 20 μg of total RNA was subjected to Northern blot analysis by electrophoresis on 0.9% agarose formaldehyde and then transferred onto a Zetaprobe membrane. Membranes were baked at 80°C for 2 hours, prehybridized in a hybridization buffer (Amersham), and then hybridized with ³ p random primer-labeled cDNA containing entire insert of BPGF- 1 (1 X 10⁶ dpm/ml hybridization buffer). The membranes were incubated at 65°C overnight. After hybridization, the membranes were washed in 2 X SSC at room temperature for 30 min; then, the membranes were washed under high stringent conditions ( 0.1 X SSC, 1% SDS) at 65°C for 30 min. Autoradiography was performed using O-MAX films with the intensifying screen at -80°C.

Southern Blot Analyses

High-molecular-weight DNA was isolated using the procedure described before (Davis et al, 1987). The DNA was digested with restriction endonucleases overnight at 37°C and then fractionated by electrophoresis in a 0.8 % agarose gel containing 1 X TAN buffer (40 mM Tris- HC1, 18 mM NaCl, 20 mM sodium acetate, and 2 mM EDTA). After alkaline denaturation,

DNA was transferred onto a Zetaprobe membrane (Bio-Rad). The membranes were baked for 2 hours at 80°C, prehybridized in a hybridization buffer (Amersham) at 65 °C, and then hybridized with ³²P random primer-labeled cDNA containing entire insert of BPGF- 1 overnight at the same temperature. After hybridization, the membranes were washed in 2 X SSC at room temperature for 20 min, and then the membranes were washed twice under high stringent conditions (0.1 X SSC, 1% SDS) at 65°C for 30 min. Autoradiography was performed using O- MAX film with the intensifying screen at -80°C.

Western Blot Analysis Following SDS-PAGE (7.5% gel) (Laemmli, 1970) and electrotransfer to nitrocellulose membrane, membranes were blocked with 5% powdered milk in TBS, 0.1% Tween 20 for three hours. Membranes then incubated with primary antibodies diluted in the blocking solution with 3% powder milk for one hour. After washing with TBS, 0.1% Tween 20, membranes were incubated in the blocking solution with 3% powdered milk for one hour with proper secondary antibodies (Horseradish peroxidase conjugated streptavidin). The protein was detected with

ECL Western blotting reagents and exposed to Hyperfilm ECL (Amersham Life Science).

Generation of Anti-Peptide Antibodies

Three synthetic peptide (CSPLTGSTQGQGGPP, CGTWKPPSTSSSPTSP, GPEASRPPKLHPG) corresponding to the BPGF-1 amino acid sequences were coupled to keyhole limpet hemocyanin and used to immunize mice. Antisera from these mice were used for Western blot analysis.

Transfection of PC-3 and COS-1 cells Transfections were performed as previously described (Sambrook et al, 1989). The entire open reading frame of the BPGF-1 was subcloned into mammalian expression vector pCDNAl/neo (Invetrogen) under control of the CMV promoter. 20 μg of DNA was used to transfect PC-3 and COS-1 cells, respectively.

Preparation of Conditioned Media from the Transfected cells for the Growth Assay

After 24-hour transfection, the cells were washed twice with PBS and changed to serum- free T-media containing 1% TCM (a serum-free defined media supplement, Celex Co., Minnetonka, MN). The conditioned media was collected after 48 hours and filtered through a 0.2 μM filter, and then it was dialyzed with a 10,000 molecular weight cut-off membrane (Spectrum Medical Industries, Inc., Los Angeles, CA) for 72 hours at 4°C against distilled water containing 0.0 lμM phenylmethylsulfonyl fluoride; the water was changed every 24 hours. Samples were lyophilized and reconstituted to a ten-fold concentration with T-medium, filtered, and diluted to the desired working concentration with T-media containing 1% TCM. For the growth assay, PC-3 and NbE-1 cells were seeded in 96-well plates with T- medium containing 1% TCM and 2% FBS. These cells were then washed with PBS twice and the desired condition media were added. The media were changed every other day. Cell numbers were determined by crystal violet assay (Gillies et al, 1987). The cells were grown in 96-well plates for another three, five, and seven days prior to fixation in 1% glutaraldehyde. The fixed cells were stained using 0.5% (w/v) crystal violet. Sorenson's solution was added to hydrolyze the cells and release trapped dye. Quantitation was performed using a Titertek

Multiscan 96-well plate reader at 560 nm.

Anchorage-Independent Growth

NbE-1 cells were trypsinized and washed in PBS; then, they were plated at 2000 cells/well in 12- well plates in 0.4% agar in the conditioned medium over a 1 ml layer of T- medium with 1% agar. The colonies were scored after growing three to four weeks.

Regulation of BPGF Expression in LNCaP cells by Growth Factors

LNCaP cells were cultured in 65-mm tissue culture dishes with T-medium containing 2% TCM and 2% FBS until 80% confluence was reached. These cells were washed with PBS twice and continuance cultured in serum-free T medium with 2% TCM. After 48 hours, fresh medium was added, and some cultures were supplemented with variety of growth factors. The total cellular RNA was prepared 48 hours after treatment and subjected to Northern blot analysis.

Expression of BPGF- 1 in Bacteria E. coli

The entire cDNA encoding BPGF-1 was ligated to inducible E. coli expression vector pTrcHis B (Invitrogen). The recombinant DNA was then transformed into E. coli strain TOP 10 (Invitrogen). The fused protein was induced by IPTG (1 mM) and separated in SDS-PAGE and visualized by Western blot analysis using polyclonal antibody against MS-1 fraction. Results

In an effort to isolate bone stromal cell-derived growth factors, serum-free conditioned medium from human bone stromal cells was fractionated by heparin affinity chromatography. Bone stromal cell conditioned medium contains factors that were able to stimulate human prostate tumor growth both in vitro and in vivo (Chung et al. , 1992) and that certain heparin- bound growth factors were implicated in both benign and malignant growth of human prostate cancer cells (Gleave et al, 1991; Chung et al, 1992).

Dialyzed conditioned medium from human bone fibroblasts was applied to a heparin sepharose affinity column. Bound proteins were eluted with a continuous linear sodium chloride gradient from 0 to 3 M. The elution profile and distribution of mitogenic activity are shown in

FIG. 8. Prostate tumor-inducing activity was eluted predominantly in the 1.0 M NaCl fraction of the bone fibroblast cell conditioned medium. Specificity of tumor-inducing activity was demonstrated in studies where similar fractions eluted at 2.0 M NaCl or identical fractions eluted from NIH 3T3 cell conditioned medium, or control serum-free TCM medium failed to exert prostate tumor-inducing activity as analyzed by both in vivo tumor growth and in vitro soft agar colony formation (see Table 6).

TABLE 6

Condition Incidence of Soft Agar Tumor Formation Colony Formation

Gelfoam & Collagen 0/6 (0%) 4 ± 1.5 IV*

+MS 1.0 M NaCl 9/12 (75%) 121 ± 7.2 Eluate

+MS 2.0 M NaCl 0/24 (0%) Eluate

+3T3 1.0 M NaCl 2 ± 0.7 Eluate

+TCM 1.0 M NaCl 0/6 (0%) 4 ± 0.7 Eluate

To further characterize the MS-1, a polyclonal antibody specifically against MS-1 proteins was raised. This polyclonal antibody was used as a reagent to clone a bone and prostate derived growth factor by screening a cDNA expression library from a human bone stromal cell line, MS. Screening of cDNA Library

A cDNA expression library was constructed using mRNA isolated from a human bone stromal cell line, MS. Total RNA was isolated from MS cells. The mRNA was isolated using oligo (dT)-cellulose chromatography and 5 μg of mRNA was used for the reverse transcription and the synthesis of cDNA using a cDNA synthesis system. EcoRI adapters were ligated to cDNAs, and a library was constructed in λZAPII using a Gigapack Gold packaging kit (Stratagene, CA). Approximately one million clones from the library were screened with the polyclonal antibody against the MS-1 fraction. Ten positive clones were identified that remained positive upon secondary and tertiary screening. The range of DNA inserts of the clones is 1.5 to 4.0 kb. Partial DNA sequencing was performed in all isolated clones, and it was found that three of the cDNA clones shared a significant degree of homology. The clone (BPGF-1) containing the largest insert from the three highly related clones was about 3.2 kb, and this clone was chosen for further analysis.

Sequencing of BPGF- 1

DNA sequence analysis was conducted using a combination of a controlled unidirectional Erase-a-base deletion system using exonuclease III, SI nuclease, and synthetic oligonucleotide primers. Templates were sequenced from both strands by the dideoxynucleotide chain-termination method using sequenase. The BPGF-1 cDNA comprised

3171 nucleotides (GenBank accession number will be obtained upon submission for publication) (FIG. 12). The BPGF-1 gene map is presented in FIGs. 13A and 13B.

* * *

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the composition, 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.

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SEQUENCE LISTING

( 1) GENERAL INFORMATION :

(i) APPLICANT:

(A) NAME: Board of Regents, The University of Texas

System

(B) STREET: 201 W. 7th Street

(C) CITY: Austin

(D) STATE: TX

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP) : 78701

(ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR THE EXPRESSION OF A BONE AND PROSTATE DERIVED GROWTH FACTOR

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/873,266

(B) FILING DATE: 10-JUN-1997

(2) INFORMATION FOR SEQ ID NO : 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3402 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TCACACAGGA AACAGCTATG ACCATGATTA CGCCAAGCTC GAAATTAACC CTCACTAAAG 60

GGAACAAAAG CTGGAGCTCC ACCGCGGTGG CGGCCGCTCT AGAACTAGTG GATCCCCCGG 120

GCTGCAGGAA TTCGCGGCCG CTGCTCGCGG TTCCCGGCGC TAACGCGGCC CCGCGGTCGG 180

CGCTCTATTC GCCTTCCGAC CCGCTGACGC TGCTGCAGGC GGACACGGTG CGCGGCGCGG 240

TGCTGGGCTC CCGCAGCGCC TGGGCCGTGG AGTTCTTCGC CTCCTGGTGC GGCCACTGCA 300

TCGCCTTCGC CCCGACGTGG AAGGCGCTGG CCGAAGACGT CAAAGCCTGG AGGCCGGCCC 360

TGTATCTCGC CGCCCTGGAC TGTGCTGAGG AGACCAACAG TGCAGTCTGC AGAGACTTCA 420

ACATCCCTGG CTTCCCGACT GTGAGGTTCT TCAAGGCCTT TACCAAGAAC GGCTCGGGAG 480

CAGTATTTCC AGTGGCTGGT GCTGACGTGC AGACGCTGCG GGAGAGGCTC ATTGACGCCC 540 TGGAGTCCCA TCATGACACG TGGCCCCCAG CCTGTCCCCC ACTGGAGCCT GCCAAGCTGG 600

AGGAGATTGA TGGATTCTTT GCGAGAAATA ACGAAGAGTA CCTGGCTCTG ATCTTTGAAA 660

AGGGAGGCTC CTACCTGGGT AGAGAGGTGG CTCTGGACCT GTCCCAGCAC AAAGGCGTGG 720

CGGTGCGCAG GGTGCTGAAC ACAGAGGCCA ATGTGGTGAG AAAGTTTGGT GTCACCGACT 780

TCCCCTCTTG CTACCTGCTG TTCCGGAATG GCTCTGTCTC CCGAGTCCCC GTGCTCATGG 840

AATCCAGGTC CTTCTATACC GCTTACCTGC AGAGACTCTC TGGGCTCACC AGGGAGGCTG 900

CCCAGACCAC AGTTGCACCA ACCACTGCTA ACAAGATAGC TCCCACTGTT TGGAAATTGG 960

CAGATCGCTC CAAGATCTAC ATGGCTGACC TGGAATCTGC ACTGCACTAC ATCCTGCGGA 1020

TAGAAGTGGG CAGGTTCCCG GTCCTGGAAG GGCAGCGCCT GGTGGCCCTG AAAAAGTTTG 1080

TGGCAGTGCT GGCCAAGTAT TTCCCTGGCC GGCCCTTAGT CCAGAACTTC CTGCACTCCG 1140

TGAATGAATG GCTCAAGAGG CAGAAGAGAA ATAAAATTCC CTACAGTTTC TTTAAAACTG 1200

CCCTGGACGA CAGGAAAGAG GGTGCCGTTC TTGCCAAGAA GGTGAACTGG ATTGGCTGCC 1260

AGGGGAGTGA GCCGCATTTC CGGGGCTTTC CCTGCTCCCT GTGGGTCCTC TTCCACTTCT 1320

TGACTGTGCA GGCAGCTCGG CAAAATGTAG ACCACTCACA GGAAGCAGCC AAGGCCAAGG 1380

AGGTCCTCCC AGCCATCCGA GGCTACGTGC ACTACTTCTT CGGCTGCCGA GACTGCGCTA 1440

GCCACTTCGA GCAGATGGCT GCTGCCTCCA TGCACCGGGT GGGGAGTCCC AACGCCGCTG 1500

TCCTCTGGCT CTGGTCTAGC CACAACAGGG TCAATGCTCG CCTTGCAGGT GCCCCCAGCG 1560

AGGACCCCCA GTTCCCCAAG GTGCAGTGGC CACCCCGTGA ACTTTGTTCT GCCTGCCACA 1620

ATGAACGCCT GGATGTGCCC GTGTGGGACG TGGAAGCCAC CCTCAACTTC CTCAAGGCCC 1680

ACTTCTCCCC AAGCAACATC ATCCTGGACT TCCCTGCAGC TGGGTCAGCT GCCCGGAGGG 1740

ATGTGCAGAA TGTGGCAGCC GCCCCAGAGC TGGCGATGGG AGCCCTGGAG CTGGAAAGCC 1800

GGAATTCAAC TCTGGACCCT GGGAAGCCTG AGATGATGAA GTCCCCCACA AACACCACCC 1860

CACATGTGCC GGCTGAGGGA CCTGAGGCAA GTCGACCCCC GAAGCTGCAC CCTGGCCTCA 1920

GAGCTGCACC AGGCCAGGAG CCTCCTGAGC ACATGGCAGA GCTTCAGAGG AATGAGCAGG 1980

AGCAGCCGCT TGGGCAGTGG CACTTGAGCA AGCGAGACAC AGGGGCTGCA TTGCTGGCTG 2040

AGTCCAGGGC TGAGAAGAAC CGCCTCTGGG GCCCTTTGGA GGTCAGGCGC GTGGGCCGCA 2100

GCTCCAAGCA GCTGGTCGAC ATCCCTGAGG GCCAGCTGGA GGCCCGAGCT GGACGGGGCC 2160

GAGGCCAGTG GCTGCAGGTG CTGGGAGGGG GCTTCTCTTA CCTGGACATC AGCCTCTGTG 2220

TGGGGCTCTA TTCCCTGTCC TTCATGGGCC TGCTGGCCAT GTACACCTAC TTCCAGGCCA 2280 AGATAAGGGC CCTGAAGGGC CATGCTGGCC ACCCTGCAGC CTGAACCACC TGGGGAGGAG 2340

GCGGGAGAGG GAGCTGCCAT CTCTAGGCAC CTCAAGCCCC CTGACCCCAT TCCCTCCCCT 2400

CCCACCCCTT GCTCCTTGTC TGGCCTAGAA GTGTGGGAAA TTCAGGAAAA CGAGTTGCTC 2460

CAGTGAAGCT TCTTGGGGTT GCTAGGACAG AGAGCTCCTT TGACACAAAA GACAGGAGCA 2520

GGGTCCAGGT TCCCCTGCTG TGCAGGGAGG GCAGCCCCGG GCAGTGGGCA TAGGGCAGCT 2580

CAGTCCCTGG CCTCTTAGCA CCACATTCCT GTTTTTCAGC TTATTTGAAG TCCTGCCTCA 2640

TTCTCACTGG AGCCTCAGTC TCTCCTGCTT GGTCTTGGCC CTCAACTGGG GCAAGTGAAG 2700

CCAGAGGAGG GTCCCCCAGC TGGGTGGGCT GGAATGGAAC TCCTCACTAG CTGCTGGGGC 2760

TCCGCCCACC CTGCTCCCTT CCGGACAATG AAGAAGCCTT TGCACCCTGG GAGGAAGGAC 2820

CACCCCGGGC CCTCTATGCC TGGCCAGCCT CCAGCTCCTC AGACCTCCTG GGTGGGGTTT 2880

GGCTTCAGGG TGGGGTTTGG AAGCTTCTGG AAGTCGTGCT GGTCTCCCAG GTGAGGCAAG 2940

CCATGGTTGC TGGGCTGTAG GGTGAGTGGC TTGCTTGGTG GGACCTGACG AGTTGGTGGC 3000

ATGGGAAGGA TGTGGGTCTC TAGTGCCTTG CCCTGGCTTA GCTGCAGGAG AAGATGGCTG 3060

CTTTCACTTC CCCCCATTGA GCTCTGCTCC CTCTGAGCCT GGTCTTTTGT CCTTTTTTAT 3120

TTTGGTCTCC AAGATGAATG CTCATCTTTG GAGGGTGCCA GGTAGAAGCT AGGGAGGGGA 3180

GTGTCTTCTC TCTCCAGGTT TCACCTTCCA GTGTGCAGAA GTTAGAAGGG TCTGGCGGGG 3240

GCAGTGCCTT ACACATGCTT GATTCCCACG CTACCCCCTG CCTTGGGAGG TGTGTGGAAT 3300

AAATTATTTT TGTTAAGGCA AAAAAAAAAA AAAGCGGCCG CGAATTCGAT ATCAAGCTTA 3360

TCGATACCGT CGACCTCGAG GGGGGCCCGG TACCCAATCG CC 3402

(2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2:

Cys Ser Pro Leu Thr Gly Ser Thr Gin Gly Gin Gly Gly Pro Pro 1 5 10 15

(2) INFORMATION FOR SEQ ID NO : 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Cys Gly Thr Trp Lys Pro Pro Ser Thr Ser Ser Ser Pro Thr Ser Pro 1 5 10 15

(2) INFORMATION FOR SEQ ID NO : 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4:

Gly Pro Glu Ala Ser Arg Pro Pro Lys Leu His Pro Gly 1 5 10

Claims

1. An isolated DNA comprising a sequence region consisting of a bone and prostate derived growth factor 1, or BPGF-1 coding region.

2. The isolated DNA according to claim 1, wherein the segment comprising the coding region codes for human BPGF-1.

3. The isolated DNA of claim 2, wherein the human BPGF-1 encodes an amino acid sequence coding for BPGF-1.

4. The isolated DNA according to claim 1 further defined as being a cDNA sequence which is complementary to human BPGF-1.

5. The isolated DNA of claim 3, comprising a BPGF-1 gene that includes a nucleic acid sequence essentially as set forth by a contiguous sequence from the sequence between position 694 and position 2314 of SEQ. I.D. NO.:l.

6. The isolated DNA of claim 1, wherein the BPGF-1 coding region is positioned under the control of a promoter.

The isolated DNA of claim 6, wherein the promoter is a recombinant promoter.

8. A nucleic acid segment that comprises at least a 10 nucleotide long contiguous stretch that corresponds to a contiguous nucleic acid sequence of SEQ. I.D. NO.:l .

9. The nucleic acid segment of claim 8, further defined as comprising at least a 20 nucleotide long contiguous stretch that corresponds to a contiguous nucleic acid sequence of SEQ. I.D. NO.: 1.

10. The nucleic acid segment of claim 9, further defined as comprising at least a 30 nucleotide long contiguous stretch that corresponds to a nucleic acid sequence of SEQ. I.D. NO.:l .

11. The nucleic acid segment of claim 10, further defined as comprising at least a 50 nucleotide long contiguous stretch that corresponds to a nucleic acid sequence of SEQ. I.D. NO.:l.

12. The nucleic acid segment of claim 11, further defined as comprising at least a 100 nucleotide long contiguous stretch that corresponds to a nucleic acid sequence of SEQ. I.D. NO.:l.

13. The nucleic acid segment of claim 8, further defined as a segment of up to 10,000 nucleotides.

14. The nucleic acid segment of claim 13, further defined as a segment of up to 5000 nucleotides.

15. The nucleic acid segment of claim 14, further defined as a segment of up to 2000 nucleotides.

16. The nucleic acid segment of claim 15, further defined as a segment of up to 50 nucleotides.

17. A vector comprising the isolated DNA of claim 1.

18. The DNA segment of claim 17, wherein the vector is a recombinant vector.

19. The DNA segment of claim 18, wherein the recombinant vector is adapted for transfer in a eukaryotic host.

20. The DNA segment of claim 18, wherein the recombinant vector is adapted for transfer in a eukaryotic host.

21. A recombinant host cell comprising a DNA segment that encodes the isolated BPGF-1 of claim 1.

22. The recombinant host cell of claim 21 , further defined as a bacterial host cell.

23. The recombinant host cell of claim 22, wherein the bacterial host cell is E coli.

24. The recombinant host cell of claim 21 , wherein the DNA segment is introduced into the cell by means of a recombinant vector.

25. The recombinant host cell of claim 24, wherein the host cell expresses the DNA segment to produce the encoded protein or peptide.

26. The recombinant host cell of claim 25, wherein the expressed protein or peptide includes an amino acid sequence coding for BPGF-1.

27. A method of using an isolated DNA that encodes an isolated bone and prostate derived growth factor, comprising:

(a) preparing a recombinant vector in which a bone and prostate derived growth factor- encoding DNA segment is positioned under the control of a promoter;

(b) introducing the recombinant vector into a recombinant host cell;

(c) culturing the recombinant host cell under conditions effective to allow expression of an encoded bone and prostate derived growth factor protein or peptide; and

(d) collecting the expressed bone and prostate derived growth factor protein or peptide.

28. A method of providing human bone and prostate derived growth factor comprising:

(a) providing a recombinant host bearing a recombinant DNA segment encoding a human bone and prostate derived growth factor and capable of expressing the factor; (b) culturing the recombinant host under conditions appropriate for the production of human bone and prostate derived growth factor; and

(c) separating the human bone and prostate derived growth factor from the recombinant host.

29. A protein or peptide composition, free from total bacterial cells, comprising a purified bone and prostate derived growth factor protein or peptide that includes an amino acid sequence coding for BPGF- 1.

30. The composition of claim 29, prepared by the method of claim 28.

31. The composition of claim 29, wherein the protein or peptide is a recombinant protein or peptide.

32. An antibody having binding affinity for a factor having the ability to stimulate the growth of prostate cells.

33. A purified antibody that binds to a bone and prostate derived growth factor protein or peptide.

34. The antibody of claim 35, wherein the antibody is linked to a detectable label.

35. The antibody of claim 36, wherein the antibody is linked to a radioactive label, a flurogenic label, a nuclear magnetic spin resonance label, biotin, or an enzyme that generates a colored product upon contact with a chromogenic substrate.

36. The antibody of claim 37, wherein the antibody is linked to an alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.

37. A method for detecting the presence of bone and prostate derived growth factors in a clinical sample, comprising:

(a) obtaining a clinical sample suspected of containing bone and prostate derived growth factors;

(b) contacting the sample with a first bone and prostate cell derived growth factor or a first antibody that binds to bone and prostate cell derived growth factors under conditions effective to allow the formation of immune complexes; and

(c) detecting the immune complexes formed.

38. The method of claim 39, wherein the first antibody is to the synthetic peptides as set forth in SEQ. I.D. NO.:3, SEQ. I.D. NO.:4, or SEQ. I.D. NO.:5.

39. The method of claim 39, wherein the first protein, peptide or antibody is linked to a detectable label and the immune complexes are detected by detecting the presence of the label.

40. The method of claim 39, wherein the immune complexes are detected by means of a second antibody linked to a detectable label, the second antibody having binding affinity for the first protein, peptide, or antibody.

41. An immunodetection kit comprising, in suitable containers, a bone and prostate derived growth factor protein or peptide, or a first antibody that binds to a bone and prostate derived growth factor protein or peptide, and an immunodetection reagent.

42. The immunodetection kit of claim 43, wherein the immunodetection reagent is a detectable label that is linked to the protein, peptide or the first antibody.

43. The immunodetection kit of claim 43, wherein the immunodetection reagent is a detectable label that is linked to a second antibody that has binding affinity for the protein, peptide, or the first antibody.

44. A method of generating an immune response, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a bone and prostate derived growth factor composition.

45. The method of claim 46, wherein the composition comprises an immunologically effective amount of a bone and prostate derived growth factor nucleic acid composition.

46. The method of claim 47, wherein the nucleic acid composition comprises an antisense molecule.