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WO1996004379A9 - Procedes et compositions s'appliquant a l'expression d'un facteur de croissance derive des os et de la prostate - Google Patents

Procedes et compositions s'appliquant a l'expression d'un facteur de croissance derive des os et de la prostate

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Publication number
WO1996004379A9
WO1996004379A9 PCT/US1995/009261 US9509261W WO9604379A9 WO 1996004379 A9 WO1996004379 A9 WO 1996004379A9 US 9509261 W US9509261 W US 9509261W WO 9604379 A9 WO9604379 A9 WO 9604379A9
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WO
WIPO (PCT)
Prior art keywords
bone
bpgf
cells
prostate
protein
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PCT/US1995/009261
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English (en)
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WO1996004379A1 (fr
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Priority to AU31414/95A priority Critical patent/AU3141495A/en
Publication of WO1996004379A1 publication Critical patent/WO1996004379A1/fr
Publication of WO1996004379A9 publication Critical patent/WO1996004379A9/fr

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  • 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.
  • 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) .
  • 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 ) .
  • 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) .
  • this theory falls short as 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) .
  • prostate cancer cell growth may be under autocrine influences involving androgen-mediated regulation of TGF- ⁇ , 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) .
  • Prostatic osteoblastic factor a soluble substance found in benign hyperplastic and malignant prostatic tissue that stimulates osteoblasts, may well be a 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) .
  • 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 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.
  • BPGF bone and prostate derived growth factor
  • the present invention concerns the substantial purification of such prostate cell growth-promoting factor(s) from human bone tissues.
  • substantially purified human growth factor 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.
  • FIG. 1 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 a potential sources of prostate cell growth factors because of the frequent metastasis of prostate cancer to the axial skeleton.
  • 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.
  • 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 assays methods disclosed herein.
  • the method preferred by the present inventors to obtain a substantially purified human growth factor in accordance herewith is affinity chro atography, and in particular, affinity chromatography employing a heparin sepharose column.
  • affinity chromatography employing a heparin sepharose column.
  • a low salt containing buffer such as lOmM Tris/Hcl, ImM PMSF, pH 7.4
  • 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.
  • an increased salt concentration such as 1 M or 2 M NaCl
  • a buffered salt gradient for example, of 0-3 M NaCl.
  • 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.
  • 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.
  • 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.
  • 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.
  • assays may be directed to analyzing the growth of prostate cancer cells, rather than normal prostate cells, simply as a matter of convenience.
  • assays include, but are not limited to, in vi tro assays such as the uptake and elution of crystal violet dye; the MTT assay for staining and quantitation of live cells in a culture dish; or the incorporation of radioactive, or non-radioactive labels, such as 3 H-thymidine, or bromodeoxy uridine, respectively, into TCA-precipitable cellular DNA.
  • a preferred in vi tro 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 of skill 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 6 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.
  • LNCaP cells for use in such an assay are particularly preferred as such cells have certain advantageous features.
  • LNCaP cells produce prostate specific antigen (PSA) , a human tissue-specific tumor marker, which can be as one method to monitor in vivo prostate cancer cell growth.
  • PSA prostate specific antigen
  • LNCaP cells are the only androgen-responsive human prostate cancer cells that can be consistently grown in vi tro . This is an important aspect of the invention that allows one to conduct parallel in vi tro and in vivo assays of various compounds using the same prostate cancer cell types.
  • suitable athymic mice such as 6-8 week old BALB/c mice
  • suitable athymic mice such as 6-8 week old BALB/c mice
  • suitable athymic mice such as 6-8 week old BALB/c mice
  • LNCaP cells an approximately equivalent number of cells of the cell type to be investigated
  • 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 5 to 5 x 10 6 cells per inoculant of each cell type is preferred, with the administration of 1 x 10 6 LNCaP cells and 1 x 10 6 of the subject cells being particularly preferred.
  • FBS foetal bovine serum
  • 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.
  • 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 adsorption 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.
  • 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, for example, Matrigel, or even agar or agarose, may be employed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BPGF-1 bone and prostate derived growth factor
  • DNA segment 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 “DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • BPGF bone and prostate derived growth factor
  • 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.
  • gene is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit.
  • 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.
  • 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 ID NO:1, corresponding to that isolated from a human bone stromal cell line, MS.
  • the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode BPGF that includes within its amino acid sequence the amino acid sequence of SEQ ID NO:2, corresponding to BPGF.
  • 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.
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • 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 ID NO:l under relatively stringent conditions such as those described herein in example 4.
  • 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 acids 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.
  • 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.
  • 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 purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively) .
  • DNA segments encoding relatively small peptides such as, for example, peptides of from about 15 to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; and also larger polypeptides up to and including proteins corresponding to the full-length sequences set forth in SEQ ID NO:2.
  • DNA segments are exemplified by, but not limited to, DNA segments that have nucleic acid sequences in accordance with the sequence of SEQ ID NO:l.
  • 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.
  • 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.
  • CMV promoters normally associated with other genes
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al . (1989) .
  • 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 & Thogersen, 1987) .
  • DNA segments encoding the entire BPGF protein or peptide fragments thereof being most preferred.
  • DNA segments encoding the entire BPGF protein or peptide fragments thereof being most preferred.
  • 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 in the order of about 45 to about 150, or to about 90 nucleotides.
  • DNA segments encoding full length proteins may have a minimum coding length in the order of about 1620 nucleotides for a protein in accordance with SEQ ID NO:l.
  • 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.
  • the present invention concerns the generation of antibodies, and particularly, monoclonal antibodies (mAbs) against the growth factor polypeptide (s) disclosed herein.
  • mAbs monoclonal antibodies
  • 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 man.
  • 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.
  • the initial immunization of an experimental animal such a mouse
  • an experimental animal such a mouse
  • 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 be injected directly into the spleen. The mouse would then be sutured and allowed to recover.
  • mice 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 will 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.
  • PEG polyethylene glycol
  • the invention concerns isolated DNA segments and recombinant vectors incorporating 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.
  • sequence essentially as set forth by a contiguous sequence from SEQ ID NO:2 means that the sequence substantially corresponds to a contiguous portion of SEQ ID NO:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2.
  • biologically functional equivalent is well understood in the art and is further defined in detail later herein.
  • sequences that have between about eg., 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 the amino acids of SEQ ID NO:2 will be sequences that are "essentially as set forth in SEQ ID NO:2" .
  • 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 ID NO:l, preferably, as set forth by a contiguous sequence from coding regions of SEQ ID NO:l.
  • the term "essentially as set forth by a contiguous sequence from SEQ ID N0:1" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a contiguous portion of SEQ ID NO:l and has relatively few codons that are not identical to, or functionally equivalent with, the codons of SEQ ID NO:l.
  • 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.
  • nucleic acid sequences that constitute regulatory regions such as promoters and/or enhancers
  • 5' nucleic acid sequences that constitute regulatory regions such as promoters and/or enhancers
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • 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 ID N0:1 under relatively stringent conditions such as those described in example 4.
  • nucleic acid segments of the present invention 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 ID NO:l. 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 ID NO:1, for BPGF peptides and proteins.
  • 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.
  • 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 ID NO:l, 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 incorporate at least a 10-14 or 20 nucleotide long stretch that corresponds to a sequence within SEQ ID NO:l 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.
  • This invention thus 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 biopsy of prostate or bone, or other tissue in which BPGF has activity
  • 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 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.
  • 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.
  • 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 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.
  • the "detection" of a BPGF sequence is accomplished by attaching or incorporating a detectable label into the nucleic acid segment used as a probe and "contacting" a sample with the labeled probe.
  • 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.
  • BPGF nucleic acids 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.
  • 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.
  • 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 of 100-200 nucleotides or so, that corresponds to, or are complementary to, sequences from SEQ ID NO:l 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 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 sequences of 1620 nucleotides (SEQ ID NO:l), according to the complementary sequences one wishes to detect .
  • Recombinant Host Cells 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 sequences of 1620 nucleotides (SEQ ID NO:l), according to the complementary sequences one wishes to detect .
  • 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 level's 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 ID NO:l.
  • DNA segments of the present invention are incorporated into appropriate vector sequences that may, e.g., improve the efficiency of transfection of host cells.
  • bacterial host cells it is proposed that virtually any vector known in the art to be appropriate for the selected host cell may be employed.
  • plasmid vectors such as pBR322, or bacteriophages such as ⁇ GEM-11. Further examples will be known to those of skill 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.
  • BPGF proteins or peptides from cells whether native or recombinant.
  • one method involves applying dialyzed bone fibroblast conditioned medium to a heparin sepharose affinity column, which was previously equilibrated in 10 mM Tris-HCl buffer containing 0.1 mM
  • 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 ID NO:l.
  • 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 a 15 to about 50, or more preferably, to about 30 amino acid long sequence from SEQ ID NO:l.
  • 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.
  • immunologically effective amount 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) .
  • B cell response an antibody response
  • T cell response cytotoxic immune 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) .
  • 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) .
  • CTL cytotoxic T lymphocyte
  • Immunoformulations of this invention may comprise whole growth factor proteins or antigenic peptide fragments from these proteins.
  • 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 incorporates 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.
  • 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)
  • 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)
  • Hopp as enabled in U.S. Patent 4,554,101, incorporated herein by reference, that 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 incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
  • 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 include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like.
  • 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 ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, will be labeled with a detectable label and the test components, that 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.
  • BPGF blood pressure
  • a detectable label such as, e.g., biotin or an enzymatic, radioactive or fluorogenic label
  • the known antibody would be immobilized, e.g., by attaching 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 or by using a chromogenic substrate in connection with an enzymatic label or by 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.
  • 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.
  • 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 a nuclear magnetic spin resonance label.
  • a detectable label such as a radioactive, fluorogenic or a 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.
  • 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 uses in antigen or antibody purification, as appropriate.
  • immunodetection methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, such as a biological sample from a patient, and 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) .
  • a sample suspected of containing such a protein, peptide or antibody such as a biological sample from a patient
  • 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
  • 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.
  • 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.
  • 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.
  • a label or marker such as a radioactive, florigenic, biological or enzymatic label
  • enzyme tags such as alkaline phosphatase, horseradish peroxidase and glucose oxidase being suitable.
  • the antigen e.g., BPGF
  • antibody e.g., anti-peptide antibodies
  • the antigen e.g., BPGF
  • antibody e.g., anti-peptide antibodies
  • the antigen or antibody 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.
  • 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.
  • sample suspected of containing BPGF proteins, peptides or antibodies sought to be detected 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.
  • non- clinical samples such as in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.
  • kits that may be employed to detect the presence of BPGF factor proteins, peptides and/or antibodies in a sample.
  • 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.
  • 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, e.g., injection or blow-molded plastic containers into 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 RNAs has been through the construction of recombinant vectors which will express antisense RNA once the vector is introduced into the cell.
  • antisense RNA technology has been in connection with attempts to affect the expression of specific genes. For example, Delauney, et al . have reported the use antisense transcripts to inhibit gene expression in transgenic plants (Delauney, et al. , 1988) . 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.
  • inhibition or suppression of BPGF-1 gene expression is desired and antisense molecules will be employed.
  • antisense molecules By preparing a construct that encodes an RNA molecule that is in 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.
  • FIG. 1 Histomorphological and immunohistochemical characterization of fibroblast-induced LNCaP chimeric tumors.
  • Hematoxylin and eosin-stained sections reveal differences between LNCaP/rUGM tumors in male (a) and female (b) hosts, the former a carcinosarcoma, the latter a pure sarcoma with no epithelial component.
  • LNCaP/MS tumors formed only in male hosts and histologically are vascular carcinomas with a minor mesenchymal component .
  • Immunohistochemical staining with monoclonal antibodies against PSA demonstrates intense and generalized staining of the epithelial cells in only male LNCaP/rUGM (d) and LNCaP/MS (f) tumors but not of the sarcomatoid LNCaP/rUGM tumors (e) in female mice.
  • LNCaP/3T3 tumors are sarcomas histologically similar to LNCaP/rUGM tumors in females and also stained negatively for PSA.
  • FIG. 2 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, 20 ⁇ g, was loaded and probed with a complementary DNA probe for PSA. (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) .
  • FIG. 3 Differences in serum PSA levels (ng/ml) in animals with various LNCaP chimeric tumors paralleled their differences in histomorphology.
  • 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.
  • FIG. 4 LNCaP cells are androgen sensitive in vi tro .
  • LNCaP cells were stimulated in vi tro 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.
  • FIG. 5 Effect of defined growth factors on LNCaP cell growth in vi tro.
  • the growth of LNCaP cells are stimulated in vitro by bFGF in a concentration-dependent manner (a) , producing a 180% increase in cell number over 9 days.
  • Both TGF ⁇ and EGF had no significant effect on LNCaP growth in vi tro using concentrations from 0.1 to 50 ng/ml.
  • a 50% reduction in LNCaP cell growth was produced by 0.1 ng/ml TGF3 (b) . Points, averages of 6 replicated determinations from 3 separate studies; bars, SE ranging from 3-9%.
  • FIG. 6 Stimulation of LNCaP cell growth in vi tro by prostate- and bone-derived conditioned media.
  • 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.
  • 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%.
  • FIG. 7 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 ⁇ g/ml collagen IV for 12 hours followed by EGF, bFGF, or stromal conditioned media.
  • LNCaP cells, 2 x 10 6 were inoculated s.c. with treated Gelfoam, except at some control sites, where ECGF-treated Gelfoam was injected alone to detect angiogenesis.
  • 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 factors 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 r s of the molecular weight standards are indicated to the left.
  • FIG. 12 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.
  • FIG. 13A Stimulation of Rat Prostate NbE-1 Cell Growth by Conditioned Medium from BPGF-1 Transfected COS-1 Cells.
  • NbE-1 cells were plated at approximately 2000 cells per well in 96-well plate. Medium conditioned by COS-1 cells transfected with wither BPGF-1 or vector alone (open square) was added to the cells. Cells were assayed for proliferation after 3, 5, 7 days of growth by crystal violet assay. The data plotted represent the mean of quadruplicate wells +/- SD.
  • FIG. 13B Supernatants conditioned by BPGF-1 Transfected COS-1 Cells Stimulate the Anchorage-Independent Growth of NbE-1 Cells.
  • Supernatants from BPGF-1 transfected COS-1 cells or vector-transfected cells were concentrated 10-fold for use in this study. Cell input at the initiation of the study was approximately 2000 cells per well. Each bar represents the mean number +/- SD of OD from quadruplicate wells.
  • FIG. 14 Northern Blot Analyses of BPGF-1
  • RNA (10 ⁇ g) isolated from MS cells was subjected to Northern blot. The filter was hybridized with the BPGF-1 cDNA. Position of 28 S and 18 S RNA are shown at the right.
  • FIG. 15 Southern Blot Analysis of DNA from Human Prostate PC-3 Cells Using the Entire BPGF-1 As a Probe.
  • the probe was radiolabeled using random priming hybridization and washing of the blot was done at 65°C, and the final washing stringency was 0.1 X SSC, 0.1% SDS.
  • FIG. 16 The Effect of BPGF-1 Gene Expression in LNCaP Cells Treated with Varieties of Growth Factors.
  • Confluent LNCaP cells were kept in serum-free T medium. After 24 h, fresh medium was added and some cultures were supplemented with growth factors, as indicated above. After 24 h of culture, the cells were collected and cellular RNA prepared. The RNAs were subjected to Northern Blot analysis.
  • FIG. 17 Immunoblot Analysis of Recombinant BPGF-1 Expression in E.coli.
  • the entire cDNA of BPGF-1 was ligated to pTricHis B, and expressed in Bacteria. Cell lysis was subjected to PAGE-SDS and blot to the membranes. Lane 1, uninduced by IPTG. Lane 2, induced by IPTG. The molecular marker is indicated on the left.
  • FIG. 18 Identification of BPGF-1 Protein Using Anti-Peptide Antibodies. Ten ⁇ g of MSI fraction was loaded in the gel. The molecular weight marker was shown on the right.
  • FIG. 19 Morphological Changes Induced by BPGF-1 cDNA Expressed in PC-3 Cells. Micrographs of the PC-3 parental in high intensity (a) , and in low intensity (c) . PC-3 cell transfected with BPGF-1 cDNA in high intensity (b) , and in low intensity (d) . Cells were cultured in T-medium with 5% FCS, and photographed with inverted microscope.
  • the inventors screened a cDNA expression library with a polyclonal antibody specifically against MS-1 fraction and isolated the clones that encodes an 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 evidence by Southern blot analysis.
  • BPGF-1 tissue specific and expressed predominantly in bone, seminal vesicles, and prostate, with substantially higher levels in bone than in prostate (at least 50 times higher in bone than in prostate) , although it is also present in minute quantities in other tissues. It is likely that the expression of BPGF-1 in 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.
  • TGF-3 (Wilding et al . , 1989 b) , 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) .
  • the inventors show that expression of the full-length of the BPGF-1 cDNA stimulates the growth of human and rat prostatic carcinoma cells. It was also observed that expression of the full-length of the BPGF-1 cDNA in human prostate PC-3 cells line results in the change of the cell morphology. The morphology of the BP cell line (transfected with BPGF-1) is different from the parental PC-3 cell line (FIG. 19) . BP cells were more elongated, and piled up when they reached confluence. These results indicate that BPGF-1 is capable of functioning as a proliferation factor on human and rat prostate 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 lymph node derived prostate cancer cell line
  • LNCaP cells were chosen for several reasons.
  • LNCaP cells have previously been shown to be nontumorigenic when injected subcutaneously in athymic mice with less than 4 x 10 6 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 .
  • PSA prostate specific antigen
  • LNCaP cells are androgen-responsive both in vivo (Sonnenschein et al., 1989) and in vi tro (Schuurmans et al. , 1989) which provides scope for the sex-dependent differences in chimeric tumor growth to be assessed.
  • LNCaP can be consistently grown in vi tro (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 vi tro 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 the control growth observed (if any) in animals given either LNCaP cells, or the test composition, alone.
  • compositions other than intact cells the inventors have developed a modified version of the assay. This is based upon the adsorption 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.
  • results disclosed herein demonstrate that certain fibroblasts can induce LNCaP tumor growth in vivo in a cell-type specific and androgen-dependent manner.
  • 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 vi tro .
  • 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.
  • LNCaP and rUGM conditioned media produce bidirectional increases in growth in a paracrine-, but not autocrine-, mediated fashion.
  • LNCaP cells participated in chimeric tumor formation preferentially in males, demonstrating initial in vivo androgen-sensitive growth. These results, along with their in vi tro androgen sensitivity, further support the view that the initial growth of LNCaP cells in vivo may be androgen-responsive (Sonnenschein et al . , 1989) .
  • the LNCaP androgen- refractory cell lines, C 4 and C 5 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 as, to date, there have been no reports concerning this area of investigation. The identification of factor(s) produced by such refractory cells may impact on the development of new therapeutic approaches to address the problem of hormonally refractory prostate cancer cell growth.
  • the substantially purified fraction contained novel polypeptides with apparent molecular weights on SDS/PAGE of: 227, 223, 218, 157, 90, 80, 48, and 20kD. 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.
  • 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.
  • bFGF-like protein may be responsible, in part, for the growth factor activity which stimulates prostate cell growth in vivo and in vi tro .
  • LNCaP cells passage 29, were obtained from Dr. Gary Miller (University of Colorado, Denver, CO) and grown in RPMI 1640 (Irvine Scientific, Santa Anna, CA) with 10% fetal 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 fetal 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).
  • MS cells were maintained in T-medium (80% DMEM, 20% F12K [Irvine Scientific], 3 g/1 NaHC0 3 , 100 u/ml penicillin G, 100 ⁇ g/ml streptomycin, 5 ⁇ g/ml insulin, 13.6 pg/ml triiodothyronine, 5 ⁇ g/ml transferrin, 0.25 ⁇ g/ml biotin, and 25 ⁇ g/ml adenine) with 5% FBS; passages 29-33 were used.
  • a rat prostatic fibroblast line, NbF-1 was established from 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
  • NRK normal rat kidney
  • 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
  • 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.
  • mice 6-8 wk old athymic nude mice (BALB/c strain, Charles River Laboratory, Wilmington, MA) of both sexes were co-inoculated subcutaneously with 1 x 10 6 LNCaP cells and 1 x 10 6 of one of the 6 fibroblast cell lines described above. Up to 5 x 10 6 LNCaP cells and 2 x 10 6 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 morphological 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.
  • Gelfoam preparation Upjohn, Kalamazoo, MI
  • type IV collagen Collaborative Research, Bedford, MA
  • ECGF endothelial cell derived growth factor
  • ECGF endothelial cell derived growth factor
  • MS conditioned media 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
  • 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 vi tro (see below) .
  • Gelfoam a solid gelatin sponge
  • 100 ug/ml collagen IV for 12 hours at 4°C
  • the Gelfoam was then minced using a polytron to allow subcutaneous inoculation via an 18 gauge needle.
  • 2 x 10 6 LNCaP cells were inoculated with Gelfoam and collagen IV, with or without ECGF. Tumor incidence and size was monitored as described above. 3. Histology and Immunohi ⁇ tochemistry.
  • 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) .
  • H&E hematoxylin and eosin
  • 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.
  • 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 32 P-labeled human Alu repetitive sequences probe (Oncor, Gaithersburg, MD) . 6. 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 ⁇ g/200 mg tissue as quantified spectrophotometrically using 40 ⁇ g RNA/A 260 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.
  • Bio-Rad Zetaprobe membrane
  • the membrane was prehybridized in the presence of 1 M NaCl, 10% dextran sulfate, 1% SDS, and 200 ⁇ g/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.
  • bFGF basic fibroblast growth factor
  • TGFce transforming growth factors alpha and beta
  • EGF epidermal growth factor
  • unlabeled R1881 200-fold of [ 3 H-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 ⁇ l aliquot was added to a scintillation vial and counted with a scintillation counter (Beckman Instruments, Inc., Houston, TX) .
  • mice co-inoculated with LNCaP cells and various types of fibroblasts were compared (Table I) .
  • the observation period for all injections was 3 months.
  • LNCaP and all fibroblast cell lines were found to be nontumorigenic
  • 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 mm 3 and 72 ⁇ 52 mm 3 , respectively.
  • Lung CCD16 and NRK fibroblasts did not induce chimeric tumor growth in either sex.
  • the histomorphology and relative content of LNCaP cells in the various fibroblast-induced tumors differed markedly, as characterized below.
  • Chimeric tumors were characterized histomorphologically, immunohistochemically, and biochemically.
  • a difference in histomorphology 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. la), while 89% (16/18) of the tumors in females were pure sarcomas (FIG. lb) .
  • 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. lc) ; 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 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 (FIG. Id; FIG. 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.
  • FIG. 2 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 histomorphologic 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) . 3. Serum PSA Levels.
  • bFGF stimulated LNCaP cell growth 180% in a concentration-dependent manner compared to cells grown in serum-free media alone (FIG. 5a) .
  • TGF / 3 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) .
  • 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.
  • FIG. 7b (FIG. 7b) illustrating that certain growth factors could maintain their biological activity when injected subcutaneously with this technique.
  • LNCaP tumors formed at 60%, 50% and 38% of inoculated sites, respectively. Tumor latency, growth rate and size was similar, and did not differ from that 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
  • Example 7 As shown in Example 1, section 7, 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 vi tro 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. Firstly, 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 lOmM Tris/HCl, lmM 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.
  • 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 vi tro 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 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) .
  • HGF HGF
  • EGF nerve growth factor
  • 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.
  • mAbs monoclonal antibodies
  • the inventors propose the partially-purified growth factor preparation as a starting material for this procedure for the following reasons. Firstly, the action of the conditioned media cannot be neutralized using a single commercially available antibody directed against any of the known growth factors. Secondly, 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.
  • mAbs against such polypeptides are therefore potentially attractive diagnostic, prognostic, imaging, and therapeutic agents for the treatment of prostate cancer in man.
  • 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 an appropriate buffer, such as Tris HCl, (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, for example, dialysis and lyophilization, or desalting using dry sephadex gels or sephadex gel exclusion column chromatography followed by lyophilization.
  • mice of approximately 3 months in age will be immunized mtraperitoneally (day 0) with 10 to 50 ⁇ g/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 (day 7 and 14) .
  • booster inoculation of antigens alone will be given.
  • the inventors contemplate that the novel booster method described below will be advantageously employed.
  • mice will be surgically opened to expose the spleen and a sterile solution of 5 to 20 ⁇ g of the growth factor antigens will be injected directly into the spleen. The mouse 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.
  • mice Five to 7 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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 in (Chan et al . , 1987) .
  • Immune splenocytes (10 8 cells) obtained from two hyperimmunized mice and 8-azaguanine-resistant SP2/0 mouse myeloma cells (10 7 cells) will be fused using 37% (v/v) polyethylene glycol 1500 (M. . 500-600 M.A.
  • 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 2 weeks by indirect ELISA.
  • HAT selection medium
  • 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/50 ⁇ l/well
  • 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.
  • the mAbs 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) .
  • FOG. 11 growth factor polypeptide
  • 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.
  • 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.
  • the inventors propose that the levels of these growth factors may correlate positively with prostate cancer progression.
  • 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 im unoblot 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.
  • 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 [ 131 I] or mAb-immunotoxins such as mAb-ricin A chain 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 a 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 then poured into a column and washed with a suitable wash buffer. An aqueous mixture including the growth factor polypeptides could then passed over the column under conditions to 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.
  • CNBr cyanogen bromide
  • CL4B cyanogen bromide
  • 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.
  • 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.
  • MS cells were maintained in T medium ( 80% DMEM, 20% F12K, 3 g/ml NaHC0 3 , 100 units/ml penicillin G, 100 ⁇ g/ml streptomycin, 5 ⁇ g/ml insulin, 13.6 pg/ml triiodothyronine, 5 ⁇ g/ml transferrin, 0.25 ⁇ g/ml biotin, and 25 ⁇ g/ml adenine) with 5% FBS; passages 29-33 were used.
  • T medium 80% DMEM, 20% F12K, 3 g/ml NaHC0 3 , 100 units/ml penicillin G, 100 ⁇ g/ml streptomycin, 5 ⁇ g/ml insulin, 13.6 pg/ml triiodothyronine, 5 ⁇ g/ml transferrin, 0.25 ⁇ g/ml biotin, and 25 ⁇ g/ml adenine
  • 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, 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.
  • 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 ⁇ g of MS poly(A) mRNA was used to construct MS cDNA expression library in the ⁇ ZAP II vector( Stratagene) .
  • Double-strands cDNA primed with a oligo(dT) 12 _ 18 was synthesized as described by the manufacture's protocols ( Pharmacia LKB Biotechnology) .
  • the final expression library contained a total of 2 x 10 6 clones, with more than 95% of the clones containing the cDNA inserts.
  • 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 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.
  • MS cDNA expression library was screened with the polyclonal antibody against MSI fraction. Screening of cDNA expression library was performed as described by the manufacture'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 MacVector ® . Northern Blot analyses
  • Total RNA was prepared from variety of human tissues and cells as described above. 20 ⁇ g 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 32 p random primer-labeled cDNA containing entire insert of BPGF-1 (1 X 10 6 dpm/ml hybridization buffer) . The membranes were incubated at 65°C overnight.
  • 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.
  • 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-HCl, 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 32 P 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
  • membranes were blocked with 5% powdered milk in TBS, 0.1% Tween 20 for 3 hours. Membranes then incubated with primary antibodies diluted in the blocking solution with 3% powder milk for 1 hour. After washing with TBS, 0.1% Tween 20, membranes were incubated in the blocking solution with 3% powdered milk for 1 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) .
  • BPGF-1 amino acid sequences 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.
  • TCM serum-free defined media supplement, Celex Co., Minnetonka, MN
  • the conditioned media was collected after 48 hours, and filtered through a 0.2 ⁇ M filter and then 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 O.Ol ⁇ M phenylmethylsulfonyl fluoride, water was changed every 24 hours. Samples were lyophilized and reconstituted to a 10-fold concentration with T medium, filtered, and diluted to the desired working concentration with T media containing 1% TCM.
  • PC-3 and NbE-1 cells were seeded in 96-well plate 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 was changed every other day. Cell number will be determined by crystal violet assay (Gillies, et al. , 1987) . The cells were grown in 96-well plates for another 3, 5, 7 days prior to fixation in 1% glutaraldehyde. The fixed Cells will be 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.
  • NbE-1 Cells were trypsinized and washed in PBS, and 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 grown 3 to 4 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 culture were supplement with variety of growth factors. The total cellular RNA was prepared 48 hours after treatment and subjected to Northern blot analysis.
  • 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.
  • Bone stromal cell conditioned medium contains factors that were able to stimulate human prostate tumor growth both in vi tro 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 a 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.
  • 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 vi tro soft agar colony formation (table 6) .
  • a polyclonal antibody specifically against MS-l 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 ⁇ g 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 ⁇ ZAPII 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.
  • 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) with a single, large open reading frame of 1620 nucleotides (SEQ ID N0:1) .
  • the BPGF-1 gene map is presented in FIG. 12.
  • the predicted BPGF-1 protein is 59-kD composed of 540 amino acids before post-translational modification.
  • the BPGF-1 contains two potential N-linked glycosylation sites.
  • a pi of 9 for the mature peptide is predicted from the cDNA sequence, ignoring any possible secondary ionic effects, such as contributions by the carbohydrate component.
  • the 3'UTR extended for an additional 858 bases, terminating with a poly(A) tail.
  • a conventional polyadenylation consensus sequence (AATAAA) was found 16 bases upstream of the poly(A) tail.
  • the BPGF-1 sequences were found to be unique in a search of the nucleic acid and protein databases (GenBank) , and they were not closely related to any known sequences.
  • BPGF-1 was not expressed (Table 7) .
  • BPGF-1 specific transcripts were substantially higher in bone than in prostate (>50 :1) .
  • Human prostate fibroblasts derived from the transitional and peripheral zones were examined, and the results showed that BPGF-1 was highly expressed in the transitional zone but weakly expressed in the peripheral zone.
  • HeLa Cervical cancer cells
  • MCF-7 Breast cancer cells
  • UPS7 (Ovarian cells) -
  • Southern blot analysis was performed on a human prostate cell line to determine the size of the gene.
  • Hybridization of BPGF-1 cDNA to human genomic DNA detected a single 15 kb BamHI fragment. Three hybridizing fragments were detected in Hindlll-digested DNA. Four hybridizing fragments were detected in EcoRI-digested DNA (FIG. 15) .
  • the cDNA contains no BamHI site, one EcoRI site, and two Hindlll sites.
  • BPGF-1 gene expression could be modulated by growth factors.
  • Northern blot analysis was performed and found that PDGF, EGF, HGF, KGF, and TGF-/3 could increase BPGF-1 gene expression in human prostate cancer cell line (LNCaP) .
  • DHT a classical prostate mitogen, was failed to induce BPGF-l gene expression (FIG. 16) .
  • the inventors have chemically synthesized 3 peptides that represent the internal amino acid sequences of the cloned BPGF-l cDNA. It has been shown that one of such polyclonal antibody obtained from the immunized mice recognized a protein band with an apparent molecular weight of 70 KDa (FIG. 18) . This protein could represent the product of BPGF-l.
  • a mammalian cell expression vector encoding an entire open reading frame of BPGF-l was constructed and expressed in COS-1 cells.
  • Conditioned medium from transfected COS-1 cells was collected and the activity was measured by whether the conditioned medium could support the growth of rat and human prostatic carcinoma cells.
  • the conditioned medium from transfected COS-1 cells contains potent stimulator for PC-3 and NbE-1 cell growth, while, transfection of COS-1 cells with vector alone yielded conditioned medium incapable of supporting PC-3 and NbE-1 cell growth.
  • transfecting the expression vector of BPGF-l to PC-3 cells resulted in an enhanced rate of cell growth and altered cell morphology, suggesting that BPGF-l may serve both as a mitogen and a morphogen.
  • the methods and compositions of the instant invention are useful for the growth and maintenance of prostatic carcinoma cells in culture, and further may be used in methods to assay the ability of selected cell or tissue types to undergo stimulation by BPGF-l.
  • compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the 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.
  • MAb human plasma-associated hepatitis
  • IGFs Insulin-like growth factors
  • IGF receptors IGF receptors
  • IGF-binding proteins IGF-binding proteins in primary cultures of prostate epithelial cells. J. Clin. Endocrinol. Metab. 73, 401-407.
  • TGF- ⁇ and ⁇ GF- ⁇ 2 transforming growth factor-/S secreted by cells in culture. J. Cell. Physiol. 138, 79-86.
  • AIDS-Kaposi's sarcoma-derived cells express cytokines with autocrine and paracrine growth effects. Science 243:223-226, 1989.
  • Fiorelli G., De Bellis, A., Longo, A., Pioli, P., Costantini, A., Giannini, S., Forti, G., and Serio, M. (1991) . Growth factors in the human prostate. J. Steroid. Biochem. Mol. Biol. 40,199-205.
  • Human transforming growth factor type b 2 production by a prostatic adenocarcinoma cell line, purification, and initial characterization. Biochemistry 26, 2406-2410.
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "DNA”
  • CCCCCAGCCT GTCCCCCACT GGAGCCTGCC AAGCTGGAGG AGATTGATGG ATTCTTTGCG 480
  • Val Asn Trp lie Gly Cys Gin Gly Ser Glu Pro His Phe Arg Gly Phe
  • Lys lie Ala Pro Thr Val Trp Lys Leu Ala Asp Arg Ser Lys lie Tyr 5 35 40 45
  • Lys lie Pro Tyr Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg Lys Glu 20 115 120 125
  • Gly Ala Val Leu Ala Lys Lys Val Asn Trp lie Gly Cys Gin Gly Ser 130 135 140
  • Val Trp Glu lie Gin Glu Asn Glu Leu Leu Gin 530 535

Abstract

On a recherché le rôle que jouent les facteurs de croissance spécifiques du stroma et l'interaction entre le stroma de l'hôte et les cellules tumorales dans le développement, la progression et la métastase du cancer de la prostate sur le squelette axial. On a identifié et cloné le facteur de croissance (BPGF-1) dérivé des os et de la prostate à partir d'une bibliothèque d'ADNc d'expression préparée à partir des cellules stromales osseuses humaines. L'ADNc du BPGF-1 cloné comprend 3171 nucléotides et un cadre de lecture ouvert unique de 1620 nucléotides. Le BPGF-1 code deux produits de transcription (3,3 et 2,5 kb) d'intensité approximativement égale. Les anticorps polyclonaux générés à partir des peptides synthétiques correspondant aux séquences nucléotidiques de l'ADNc du BPGF-1 cloné réagissent avec une protéine putative unique du BPGF-1 dont la masse moléculaire apparente est de 70kDa. L'analyse par transfert de Southern d'un ADN génomique humain a révélé qu'il y a une copie unique du gène du BPGF-1 ayant une taille de 15 kb dans les sites BamHI. L'ADNc du BPGF-1 code une protéine qui stimule la prolifération des cellules épithéliales prostatiques. Le gène du BPGF-1 est exprimé de manière prédominante dans les os, la prostate et les vésicules séminales, et présente une expression dans les os sensiblement supérieure à celle de la prostate et des vésicules séminales.
PCT/US1995/009261 1994-08-01 1995-07-20 Procedes et compositions s'appliquant a l'expression d'un facteur de croissance derive des os et de la prostate WO1996004379A1 (fr)

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US5914251A (en) * 1996-10-22 1999-06-22 Amgen Inc. Nucleic acid molecules encoding placental-derived growth factors
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