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Title:  Bone morphogenetic protein-2 in the treatment and diagnosis of cancer
United States Patent: 
7,473,561
Issued: 
January 6, 2009

Inventors:
 Langenfeld; John (Flemington, NJ)
Assignee:
  University of Medicine and Dentistry of New Jersey (Somerset, NJ)
Appl. No.:
 11/435,852
Filed:
 May 17, 2006


 

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Abstract

The present invention pertains to the use of BMP-2, which is overexpressed in most common cancers, as 1) a target for cancer treatment therapies and 2) a means to diagnose cancer. The therapeutic component of this invention involves administering to a patient a composition that inhibits bone morphogenetic-2 activity. Such inhibition may be accomplished by ligands or antibodies that bind to BMP-2 or BMP-2 receptors. It may also be achieved by preventing the processing of pro-BMP-2, or blocking transcription or replication of BMP-2 DNA or translation of BMP-2 mRNA. The diagnostic component of the invention involves measuring the BMP-2 level in biological samples from both a patient and a subject and comparing those levels. Elevated levels of BMP-2 in the patient compared to the non-cancerous subject indicate cancer.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is related to the discovery that bone morphogenetic protein-2 (BMP-2) is overexpressed in many common human cancers and is linked to cancer invasion and growth. Further, inhibiting BMP-2 activity reduces the size of cancerous tumors in nude mice and down regulates the expression of VEGF and sonic hedgehog in lung cancer cell lines. Thus, the present invention pertains to the use of BMP-2 as a target for cancer treatment therapies and a means to diagnose cancer. Specifically, the therapeutic component of this invention involves administering to a patient a composition that inhibits bone morphogenetic protein-2 activity. The diagnostic component of the invention involves measuring the BMP-2 level in biological samples from both a patient and a non-cancerous subject and comparing those levels. Elevated levels indicate increased probability of cancer in the patient.

A primary aspect of the present invention is to provide a method for the treatment of cancer by administering to a patient a therapeutically effective amount of a BMP-2 activity inhibitor. Some cancers that may be treated by this method are carcinomas, including, but not limited to, lung cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, lung cancer, ovarian cancer, thyroid cancer, endometrial cancer, omental cancer, testicular cancer, and liver cancer. In a preferred embodiment of this invention, the patient is human.

The BMP-2-inhibitor of this invention may be a polypeptide that binds specifically to bone morphogenetic protein-2 itself, a polypeptide that binds specifically to a BMP-2 receptor, or an antibody that binds specifically to BMP-2 or a portion of BMP-2. The BMP-2 inhibitor may also be an antisense oligonucleotide that binds to a BMP-2 nucleic acid sequence or some portion thereof.

This invention features several particular polypeptides that are BMP-2 inhibitors. Preferred embodiments of this invention feature known antagonists to BMP-2, such as noggin, chordin, cerberus 1 homolog, gremlin, and DAN (2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid). Noggin is particularly preferred. Another aspect of this invention relates to the use of fragments of noggin, chordin, cerberus 1 homolog, gremlin, and DAN as BMP-2 inhibitors.

Another embodiment of this invention provides a method for treating cancer by administering to a patient a therapeutically effective amount of an expression vector encoding a BMP-2 inhibitor, such as a polypeptide that binds BMP-2 or an antisense oligonucleotide that binds to the nucleic acid for BMP-2. Another aspect of this invention includes the expression vector described above in which the nucleic acid sequence for BMP-2 is operably linked to a selective promoter. One preferred selective promoter encompassed by this invention is carcinoembryonic antigen promoter.

This invention also encompasses a kit that includes packaging material, a BMP-2 activity inhibitor, and instructions that indicate that the compound can be used for treating cancer in a patient. One type of cancer that may be treated is carcinoma. Particular carcinomas encompassed by this invention are lung cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, ovarian cancer, thyroid cancer, endometrial cancer, omental cancer, testicular cancer, and liver cancer.

The diagnostic component of this invention includes a method for diagnosing cancer in a patient by obtaining a biological sample from a patient and measuring the level of BMP-2 in the biological sample, with an elevated level of BMP-2 indicating an increased likelihood of cancer in the patient.

Any assay available to measure BMP-2 levels is encompassed by this invention. Particularly preferred are immunoassays. Some examples of immunoassays included in this invention are Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, immunoprecipitation, in situ immunohistochemistry, and immunofluorescence. ELISA is most particularly preferred.

Another aspect of this invention is a method for the diagnosis of cancer in a patient by detecting overexpression of BMP-2 in the patient by (i) quantifying in vivo or in vitro the presence of BMP-2 in a patient or a biological sample obtained from a patient, (ii) comparing the result obtained in step (i) to that of a normal, non-cancerous patient, and (iii) diagnosing the presence of cancer based on an increased level of BMP-2 in step (ii) relative to a normal, non-cancerous patient.

An additional aspect of the present invention demonstrates that BMP-2 stimulates lung tumorigenesis by stimulating angiogenesis of developing tumors. Furthermore, BMP-2 induced angiogenesis involves a direct activation of human endothelial cells. The present invention has discovered that vascular endothelial growth factor (VEGF), which is an active target in cancer therapy, stimulates BMP-2. Thus, BMP-2 enhances tumorigenesis by mediating an angiogenic response.

A therapeutic aspect of the invention comprises administering a therapeutically effective amount of a BMP-2 activity inhibitor to a patient to treat tumors or to treat the risk of developing tumors in a patient by decreasing vascular development and/or angiogenesis. Preferably, the BMP-2 activity inhibitor is administered in a therapeutically effective carrier and the administration is continued until the tumor or risk of the tumor is treated.

BMP-2 also mediates robust tube formation in both human aortic endothelial cells and human umbilical vein endothelial cells, which demonstrates its role in inducing endothelial differentiation. BMP-2 activates cancer cells to express Id, cyclin E, phosphorylation of Rb and Erk 1/2. BMP-2 also activates PI-3, MEK, and p38, all of which are involved in pathways involved in the transformation of cancers. Thus, another aspect of the present invention comprises a method of decreasing expression of Id, cyclin E, and phosphorylation of Rb and Erk 1/2 in cancer cells or precancerous cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the present discovery that the overexpression of bone morphogenetic protein-2 (BMP-2) is linked to cancer invasion and growth. BMP-2 is overexpressed in many common human cancers and regulates molecular pathways that are involved in the promotion of cancer. Inhibiting BMP-2 activity reduces the size of cancerous tumors in nude mice and down regulates the expression of VEGF and sonic hedgehog, both known cancer promoters, in lung cancer cell lines. Thus, the present invention is directed toward using BMP-2 as a target for cancer treatment therapies and as a means to diagnose cancer.

The therapeutic component of this invention involves administering to a patient a composition that inhibits bone morphogenetic protein-2 activity. Such inhibition may be accomplished by ligands or antibodies that bind to BMP-2 or BMP-2 receptors. It may also be achieved by preventing the processing of pro-BMP-2, or blocking transcription or replication of BMP-2 DNA or translation of BMP-2 mRNA. Delivery of such compositions may be systemic or tissue-targeted. Additionally, decreased vascularization and decreased tumor expression may be achieved by decreasing the expression of Id, cyclin E, and phosphorylation of Rb and Erk 1/2 and controlling the activity of other tumor enhancing genes.

The diagnostic component of the invention involves measuring the BMP-2 level in biological samples from a patient and determining whether that level is elevated from a normal level. A related aspect of the invention involves measuring the BMP-2 level in biological samples from both a patient and a non-cancerous subject and comparing those levels. Presumably, the levels of BMP-2 in the non-cancerous subject are physiologically normal levels. Preferably, the levels are taken from a study of non-cancerous subjects of a similar age and the level of BMP-2 is from a similar or the same organ, fluid or bodily location. Elevated levels of BMP-2 in the patient compared to the non-cancerous subject indicate cancer.

Although specific embodiments of the present invention will now be described, it should be understood that such embodiments are examples that are merely illustrative of a small number of the many possible specific embodiments that can represent applications of the principles of the present invention. Various modifications obvious to one skilled in the art to which the present invention pertains are within the spirit, scope and contemplation of the present invention as further defined in the appended claims.

BMP-2 as a Target in the Treatment of Cancer

The present invention is directed to the use of BMP-2 as a target in the treatment of cancer. Amino acids #283-396 of SEQ ID NO: 2 constitute the amino acid sequence of mature human BMP-2. Nucleotides #372-1514 of SEQ ID NO: 1 constitute the nucleotide coding sequence for human BMP-2. Any composition that specifically binds BMP-2 or a BMP-2 receptor, thereby antagonizing BMP-2 activity, blocks the processing of pro-BMP-2, and/or prevents the replication or transcription of BMP-2 DNA or the translation of BMP-2 mRNA could be used as a therapy to treat cancer.

A compound that specifically binds to BMP-2 is any compound such as a polypeptide or an antibody that has a binding affinity for any naturally occurring isoform, splice variant, or polymorphism of BMP-2. As one of ordinary skill in the art will appreciate, such "specific" binding compounds may also bind to other closely related proteins that exhibit significant homology (such as greater than 90% identity, more preferably greater than 95% identity, and most preferably greater than 99% identity) with the amino acid sequence of BMP-2. Any compound that binds to BMP-2 sufficiently to suppress BMP-2 activity is contemplated by the present invention.

Similarly, a compound that specifically binds to a BMP receptor is any compound that has a binding affinity for any naturally occurring isoform, splice variant, or polymorphism of the BMP receptor. As one of ordinary skill in the art will appreciate, such "specific" binding compounds may also bind to other closely related proteins that exhibit significant homology (such as greater than 90% identity, more preferably greater than 95% identity, and most preferably greater than 99% identity) with the amino acid sequence of a BMP receptor. The present invention embodies polypeptides that specifically bind to BMP-2, thereby inhibiting its activity, or that specifically bind to BMP receptors, thereby inhibiting BMP-2 activity. Specific embodiments of such polypeptides are described below.

The present invention encompasses known antagonists of BMP-2 activity, including noggin (35) (U.S. Pat. No. 6,075,007, Economides, et al.), chordin (36) (U.S. Pat. No. 5,896,056), gremlin (GENBANK Accession No. AF 154054), cerberus 1 homolog (GENBANK Accession No. NM-005454), and DAN.

Recombinant mouse noggin from R & D Systems (Minneapolis, Minn.) was used in the inhibition experiments described in the Results section below. Mouse and human noggin share 98% homology. Therefore, this invention also relates to use of a polypeptide with the amino acid sequence of mature mouse noggin (amino acids #20-231 of SEQ ID NO: 6) and with the amino acid sequence of mature human noggin (amino acids #20-231 of SEQ ID NO: 4) as a BMP-2 activity inhibitor. The amino acid sequence for human chordin is SEQ ID NO: 8, for human gremlin is SEQ ID NO: 10, and for cerberus 1 homolog is SEQ ID NO; 12. The nucleotide coding sequence for human noggin is SEQ ID NO: 3, for mouse noggin is SEQ ID NO: 5, for human chordin is nucleotides #247-3114 of SEQ ID NO: 7, for human gremlin is nucleotides #130-684 of SEQ ID NO: 9, for human cerberus 1 homolog is SEQ ID NO: 11.

This invention also embodies polypeptide fragments of noggin, chordin, gremlin, cerberus 1 homolog, and DAN that bind BMP-2 and inhibit its activity. Such polypeptides may be tested for inhibitory efficiency by culturing cells transformed with progressively shorter portions of the nucleotide sequences encoding the above proteins, recovering and purifying from the various cultures the resulting polypeptide, and testing those polypeptides for their ability to inhibit BMP-2 activity.

This invention also includes genetically altered BMP receptor proteins that inhibit BMP-2 activity. For example, altered BMP receptors that inhibit the binding effects of BMP-2 are described in U.S. Pat. No. 6,291,206 (Wozney, et al.).

Also included by this invention are polypeptides that bind BMP receptors without activating them. (37, 38) Particularly preferred are ligands that will bind BMP IB receptors, which are a subtype of BMP receptors. Aberrant expression of the BMP IB receptor in many human cancer specimens has been noted, as discussed in the Results section below. (39) The coding sequence for BMP IB precursor is nucleotides #274-1782 of SEQ ID NO: 13. The amino acid sequence for BMP IB is amino acids #14-502 of SEQ ID NO: 14.

This invention also encompasses expression vectors that incorporate a nucleotide sequence encoding an inhibitor of BMP-2 activity operably linked to control sequences that promote and/or regulate expression of the nucleotide sequence. The preparation of such expression vectors, as well as the use of various control sequences, is well known to those of skill in the art and is described in many references, such as Sambrook, et al. (1989). Expression vectors can be derived from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses and from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Promoters can be prokaryotic, such as lacI, lacZ, T3, T7, gpt, lambda PR, PL, and trp, or eukaryotic, such as CMV immediate early, HSV thymidine kinase, early and late SV40, LTR's from retrovirus, and mouse metallothionein-1. Selective promoters such as those described in Nettlebeck, D. M., et al., "Gene therapy: designer promoters for tumour targeting" Trends Genet 16(4); 174-81 (2000) that are tissue-specific, tumor-selective, treatment-responsive, or tumor endothelium directed may also be used. For example, the promoter of the carcinoembryonic antigen (CEA) is expressed on many breast, lung, and colorectal cancers.

For introduction of a gene that encodes a protein that antagonizes BMP-2 activity, an expression vector vehicle that will facilitate delivery of the desired gene to the affected cells may be used. One way to facilitate delivery is by using an expression vector derived from virus. Examples of viral vectors that have been successfully used to deliver desired sequences to cells with high infection efficiency are adenoviral, retrovital, vaccinia viral, and adeno-associated viral vectors. Commonly used viral promoters for expression vectors are derived from polyoma, cytomegalovirus, Adenovirus, and Simian Virus 40 (SV40). It is also possible to use promoter or control sequences normally associated with the desired gene sequence, if such control sequences are compatible with the host cell systems.

Non-viral expression vector vehicles are also available. For instance, the expression vector could be associated with one or more lipids. As is known in the art of lipid-based gene delivery, such nucleic acid-lipid complexes can be in a variety of different forms depending generally on the nature of the lipid employed, the ratio of nucleic acid to lipid and/or other possible components, and the method by which the complex is formed. Examples of complexes include liposomes and micelles. Liposome-mediated gene transfer seems to have great potential for certain in vivo applications in animals. Studies have shown that intravenously injected liposomes are taken up essentially in the liver and the spleen, by the macrophages of the reticuloendothelial system. Using a catheter to introduce liposomes coupled to expression vectors to particular cellular sites has also been described. (Nabel, E. G., et al., Science 249:1285-1288 (1990))

Another possible expression vector vehicle consists of a cell receptor-specific ligand and a DNA-binding agent that would bind to the expression vector. (Nishikawa, M. et al., Gene Therapy 7:548-55 (2000)). Such a vehicle could also comprise a cell receptor-specific ligand and the nucleic acid-lipid complex described above. (Nicolau, C. et al., Methods Enzymol 149: 157-76 (1987))

In addition, the present invention embodies antibodies that specifically bind BMP-2 or BMP receptors, thereby inhibiting BMP-2 activity. When raising antibodies to BMP-2 or BMP receptors, the entire protein (either the precursor or the processed protein), or a portion thereof, may be utilized. Information useful in designing an antigen for the production of antibodies to BMP-2 may be deduced by those of skill in the art by homology analysis of SEQ ID NO: 2, especially amino acids #283-396 of SEQ ID NO: 2.

A recombinant human BMP-2 protein is commercially available from R & D Systems (Minneapolis, Minn.) and portions of the BMP-2 protein may be produced by a variety of methods. In order to raise antibodies to particular epitopes, peptides derived from the full BMP-2 sequence may be used. Custom-synthesized peptides in the range of 10-20 amino acids are available from a multitude of vendors, and can be ordered conjugated to KLH or BSA. Alternatively, peptides in excess of 30 amino acids may be synthesized by solid-phase methods, or may be recombinantly produced in a recombinant protein production system. In order to ensure proper protein glycosylation and processing an animal cell system (e.g., Sf9 or other insect cells, CHO or other mammalian cells) is preferred.

Selection of antibodies which alter the activity of BMP-2 may be accomplished in several ways. Antibodies that alter the binding of BMP-2 to a receptor may be detected by well-known binding inhibition assays. For instance, according to standard techniques, the binding of a labeled (e.g., fluorescently or enzyme-labeled) antibody to BMP-2, which has been immobilized in a microtiter well, is assayed for BMP-2 binding in both the presence and absence of the appropriate receptor. The decrease in binding will be indicative of a competitive inhibitor relationship between the antibody and the receptor. In addition, antibodies that are useful for altering the function of BMP-2 may be assayed in functional formats, such as the cell migration assays described in the Results and Examples sections.

This invention also embodies compositions that prevent the processing of inactive BMP-2 precursors. BMP precursors are proteolytically activated by proprotein convertases. For example, BMP-2 is cleaved by furin convertase from human leukocytes Furin inhibitors are known. (40) While the BMP-2 inhibitors discussed above adversely affect BMP-2 activity after it is expressed, it will be readily apparent to one of ordinary skill in the art that specific prevention of BMP-2 biosynthesis will achieve the same goals as more direct inhibition of its activity. Consequently, this invention also encompasses inhibition of BMP-2 biosynthesis as a method for treating cancer. Such inhibition may be achieved by selectively degrading mRNA encoding BMP-2 or BMP-4 or by interfering with transcription or translation of such mRNA. (41) As mentioned above, BMP-2 shares 92% homology with BMP-4.

Inhibition of BMP-2 biosynthesis to treat for cancer could also be achieved through antisense therapy. Antisense therapy is the administration or in situ generation of oligonucleotides that specifically hybridize, under cellular conditions, with the cellular mRNA or genomic DNA encoding a BMP-2 protein or some portion of such cellular or genomic DNA, thereby inhibiting biosynthesis of the BMP-2 protein. Antisense therapy refers generally to the range of techniques known by one of ordinary skill in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.

Delivery of an antisense oligonucleotide of the present invention can occur in a variety of ways. For example, an antisense oligonucleotide can be delivered as an expression vector that produces RNA, which is complementary to at least a unique portion of the cellular mRNA encoding BMP-2. Such an expression vector could be delivered to cells by one of the expression vector vehicles described above. Alternatively, the antisense oligonucleotide could be generated ex vivo as an oligonucleotide probe which, when introduced to the cell, inhibits biosynthesis of BMP-2 proteins by hybridizing with the mRNA or genomic sequences encoding BMP-2. Such oligonucleotide probes could be modified oligonucleotides that are resistant to endogenous nucleases and therefore are stable in vivo. General methods to construct oligomers useful in antisense therapy are known in the art. (Van der krol, et al., Biotechniques 6:958-976 (1988); Stein, et al., Cancer Res. 48:2659-2668 (1988).

Dosage forms of the BMP-2 inhibitors of this invention include pharmaceutically acceptable carriers known to those of ordinary skill in the art. Pharmaceutically acceptable components are those that are suitable for use with mammals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. The carrier can be a solid or liquid. The type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspension reconstituted from non-effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners and melting agents. Parenteral and intravenous forms may also include isotonic salts and other materials to make them compatible with the type of injection or delivery system chosen.

For administration of an antibody to BMP-2, the pharmaceutically acceptable carrier will usually be an aqueous solution, such as normal saline or phosphate-buffered saline (PBS), Ringer's solution, lactate-Ringer's solution, or any isotonic physiologically acceptable solution for administration by the chosen means. In addition to additives for adjusting pH or tonicity, the antibody may be stabilized against aggregation and polymerization with amino acids and non-ionic detergents, polysorbate, and polyethylene glycol. Optionally, additional stabilizers may include various physiologically acceptable carbohydrates and salts. In addition, polyvinylpyrrolidone may be added in addition to the amino acid. Suitable therapeutic immunoglobulin solutions, which are stabilized for storage and administration to humans are described in U.S. Pat. No. 5,945,098. Other agents, such as human serum albumin (HSA), may be added to the pharmaceutical composition to stabilize the antibody conjugates.

The method of administration can be any suitable method that effectively alleviates the particular tumor and/or cancer being treated. Possible methods of administration are oral, rectal, parenteral, enteric, subcutaneous, transdermal, peritoneal, intratumoral, or intravenous.

Any suitable dosage of the compounds may be given in the method of the invention. Dosage levels and requirements are well recognized by those of ordinary skill in the art. As one of ordinary skill in the art will appreciate, an amount constituting an effective amount will vary depending on particular factors. For instance, specific dosage and treatment regimens will depend on facts such as the patient's general health profile, the type of cancer being treated, the severity and course of the patient's disorder, other therapeutics being administered to treat the cancer, and the judgment of the treating physician.

The present invention also provides kits for treating cancer using BMP-2 activity inhibitors. For example, such kits can comprise any one or more of the following materials: packaging material, at least one type of BMP-2 activity inhibitor, and instructions regarding dosage, method of administration, or the like for using the inhibitor to treat cancer.
 

Claim 1 of 1 Claim

1. A method for identifying a candidate agent for use in reducing vascularization in non-small cell lung tumors comprising: (a) contacting bone morphogenetic protein-2 (BMP-2) and BMP-2 receptor IB in the presence of a test agent under conditions suitable to permit the formation of a BMP-2/BMP-2 receptor complex, wherein said BMP-2 comprises the amino acid sequence of amino acids 283-396 of SEQ ID NO: 2 and wherein said BMP-2 receptor IB comprises the amino acid sequence of amino acids 14-502 of SEQ ID NO: 14; (b) determining the amount of the BMP-2/BMP-2 receptor complex formed in the presence of said test agent; and (c) determining that said test agent is a candidate agent for use in reducing vascularization in non-small cell lung tumors if the amount of the BMP-2/BMP-2 receptor complex formed in the presence of said test agent is lower than the amount of the BMP-2/BMP-2 receptor complex formed in the absence of said test agent, thereby identifying a candidate agent for use in reducing vascularization in non-small cell lung tumors.

 

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