JP2005528880A - BEHAB cleavage and suppression of primary central nervous system (CNS) tumors - Google Patents

Abstract

The present invention relates to primary CNS tumors, useful compositions for reducing tumor volume, extending the length of survival in mammals with primary CNS tumors, and thereby providing treatment for primary CNS tumors, and Provide a method. The present invention also relates to methods of identifying compounds for reducing tumor volume and extending animal survival, and thus it relates to the treatment of primary CNS tumors.

Description

Cross reference to related applications. S. C. In accordance with §119 (e), priority is given to US Provisional Patent Application No. 60 / 306,046 filed on July 17, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT The present invention is supported in part by funds obtained from the US Government (National Institutes of Health Numbers EY06511 and NS35228), and therefore the US Government has certain rights in the present invention. Can do.

  Glioma and primary brain tumor are notorious for their difficulty in control and treatment. Unlike secondary tumors or non-glial tumors that have metastasized to the brain, gliomas exhibit a unique invasion ability characterized by the lack of a well-defined boundary between cancer tissue and a healthy brain. Conventional therapies, including surgery, chemotherapy and radiation therapy, are often only effective or ineffective treatments that are partially effective due to glioma invasiveness. Therefore, the prognosis of patients suffering from glioma is always severe.

  Not only is the prognosis often negative, but the diagnosis of glioma is a difficult and expensive process as well. Brain tumor detection requires costly imaging equipment that is not readily available at all medical facilities, and confirmation of imaging results usually requires surgical sampling of invasive and dangerous tumors. To do.

  The nature of the extracellular matrix (ECM) in the brain may play a role in the ability of cancer cells to invade. During normal development, the composition of the brain's extracellular matrix changes dramatically. Cell growth, migration, neuronal and glue outgrowth and angiogenesis in the developing brain occur in a soluble matrix that allows cell movement. However, in the mature and developed brain, cell motility is significantly reduced, and mature cells are stabilized in cell interaction (Non-Patent Document 1).

  The mechanism used by malignant tumors to invade surrounding tissues in the developing brain is significantly different. For example, some malignant cells can produce their own ECM molecules, such as hyaluronic acid (HA), thereby changing the balance and structure of the adjacent environment (Non-patent document 2, Non-patent document 3, Non-patent document) Document 4, Non-Patent Document 5, Non-Patent Document 6). Furthermore, tumor cells can alter their interaction with ECM molecules by changing their cellular receptor composition, for example by up-regulating HA binding molecules RHAMM and CD44 (Non-Patent Document 7, Non-Patent Document 7, Patent Document 8, Non-Patent Document 9, Non-Patent Document 10). Furthermore, a malignant cell can destroy the normal matrix which exists by producing a proteolytic enzyme and digesting surrounding ECM (nonpatent literature 11, nonpatent literature 12, nonpatent literature 13). The mechanisms that facilitate the invasion of tumor cells into adjacent tissues are diverse, but it is probably a malignant that alters the composition of the surrounding matrix, including the production and digestion of matrix molecules that best characterize the invasive phenotype The ability of the tumor.

  The role of proteases in the invasion process has been shown in almost all histological types of tumors (Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13) and is strongly in the invasiveness of high-grade gliomas (Non-patent document 14, Non-patent document 15, Non-patent document 16, Non-patent document 17, Non-patent document 18, Non-patent document 19, Non-patent document 20). Many of the fact that matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, are highly upregulated in gliomas and that inhibition of these proteases can reduce glioma invasiveness. Documents show (Non-patent document 21, Non-patent document 22, Non-patent document 23, Non-patent document 24, Non-patent document 25, Non-patent document 26, Non-patent document 16, Non-patent document 17, Non-patent document 18 ). However, clinical attempts to use MMP inhibitors for the treatment of primary tumors have been disappointing due to severe and harmful side effects (Non-patent Document 27).

  One brain ECM molecule that plays an important role in glioma invasion and tumor progression is the brain-enriched hyaluronan binding (BEHAB) protein (Hockfield et al., 1997, patent). Reference 1). BEHAB is a member of the proteoglycan tandem repeat family of proteins, and exists as both a GPI-anchored protein having a hyaluronan-binding domain and a secreted protein (Non-patent document 28, Non-patent document 29, Non-patent document) 30). Recent studies have shown that BEHAB is a brain-specific protein that is down-regulated as development proceeds in the human brain (Non-Patent Document 31). Importantly, BEHAB is up-regulated by about 700% in almost all adult human glioma samples tested to date (Non-patent Document 32, Non-patent Document 31).

BEHAB cleavage plays a prominent role in glioma progression. Matthews et al. (33) showed that BEHAB was cleaved between Glu ^{395 and} Ser ³⁹⁶ by metalloproteinases into 50 kDa and 90 kDa fragments, but not by MMP. Instead, BEHAB is cleaved by metalloproteinases, particularly ADAMTS4, which have members of disintegrin and the thrombospondin motif (ADAMTS) group of metalloproteinases.

Unfortunately, because of adverse side effects, normal metalloproteinase inhibitors have been found to be ineffective methods for the treatment of glioma. Diagnosis and treatment of primary central nervous system (CNS) tumors with the inherent risks of both diagnosis and treatment of glioma using conventional methods and relatively new treatment management failures such as metalloproteinase inhibitors A method for both has long been required. The present invention satisfies this need.
Hockfield, Semin. Dev. Biol. 1990, 1: 55-63. Delpech et al. Written, International Journal of Cancer, 1997, 72: 942-948. Kosaki et al. Author. Cancer Research, 1999, 59: 1141-1145. By Turley. Cancer and Metastasis Reviews, 1992, 11: 21-30 Tzanakakis et al. Author. Biochimie, 1997, 79: 323-332. By Zetter. Seminars in Cancer Biology, 1993, 4: 219-229 Goldbrunner et al. Author. Acta Neurochirgica, 1999, 141: 295-305 By Hall and Turley, Journal of Neuro-Oncology, 1995, 26: 221-229. Hall et al. Written, Cell, 1995, 82: 19-28. Merzak et al. Written, Cancer Research, 1994, 54: 3988-3992. Furcht et al. Written, Laboratory Investigation, 1994, 70: 781-783. Mignatti and Rifkin, Physiological Reviews, 1993, 73: 161-195 Stettler-Stevenson et al. Written by FASEB Journal, 1993, 7: 1434-1441. DeClerrck et al. Written, Cancer Research, 1991, 51: 2151-2157. Mohanam et al. Author, Journal of Neuro-Oncology, 1994, 22: 153-160. Nakagawa et al. Author, Journal of Neurosurgery, 1994, 81: 69-77. Rao et al. Author, Cancer Research, 1993. 53: 2208-2211 Rao et al. Author, Journal of Neuro-Oncology, 1994, 18: 129-138. Vaithillingham et al. Author, Journal of Neurosurgery, 1992, 77: 595-600. Yamamoto et al. Written, Journal of Neuro-Oncology, 22: 139-151 Deryugina et al. Author, Journal of Cell Science, 1997, 110: 2473-2482. Forsyth et al. Written, British Journal of Cancer, 1999, 79: 1828-1835. Hamasauna et al. Written, International Journal of Cancer, 1999, 82: 274-281. Rao et al. Written, Clinical and Experimental Metastasis, 1996, 14: 12-18. Sawaya et al. Written, Clinical and Experimental Metastasis, 1996, 14: 35-42. Uhm et al. Written, Clinical and Experimental Metastasis, 1996, 14: 421-433. By Heath and Grochow, Drugs, 2000, 59: 1043-1055 US Pat. No. 5,635,370 Jawski et al. Author, J.H. Cell Biol. 1994, 125: 495-509. Yamada et al. Author, J.H. Biol. Chem. 1994, 269: 10119-10126. Seidenbecher et al. Author, J.H. Biol. Chem. 1995, 270: 27206-27212. Gary et al. Author, Gene, 2000, 256: 139-147. Jawski et al. Written, Cancer Research, 1996, 56: 2293-2298. Matthews et al. Author, J.H. Biol. Chem. , 2000, 275: 22695-22703

SUMMARY OF THE INVENTION The present invention includes an isolated nucleic acid encoding a mammalian mutant BEHAB molecule or fragment thereof. In one aspect, the isolated nucleic acid has 99.7% identity to the nucleic acid sequence shown in SEQ ID NO: 4. In other aspects, the isolated nucleic acid comprises the nucleic acid sequence shown in SEQ ID NO: 4.

  In yet another aspect, the invention includes an isolated nucleic acid encoding a mammalian mutant BEHAB molecule, wherein the amino acid sequence of the mammalian mutant BEHAB molecule comprises the amino acid sequence shown in SEQ ID NO: 3.

  Isolated polypeptides comprising a mammalian mutant BEHAB molecule are also included in the present invention. In one aspect, the mammalian mutant BEHAB molecule has 99.6% identity to the amino acid sequence set forth in SEQ ID NO: 3.

In one embodiment, the isolated nucleic acid of the invention further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto. Preferably, the tag polypeptide is a myc tag polypeptide, glutathione-S-transferase tag polypeptide, green fluorescent protein tag polypeptide, myc-pyruvate kinase tag polypeptide, His ₆ tag polypeptide, influenza virus hemagglutinin tag poly The peptide is selected from the group consisting of a flag tag polypeptide and a maltose binding protein tag polypeptide.

  In another aspect, the isolated nucleic acid of the invention further comprises a nucleic acid that specifies a promoter / regulatory sequence operably linked thereto.

  Furthermore, the present invention includes a vector containing the isolated nucleic acid of the present invention. In one embodiment, the vector further comprises a nucleic acid defining a promoter / regulatory sequence operably linked thereto.

  Also included are recombinant cells that contain an isolated nucleic acid of the invention or that contain a vector of the invention.

  The invention further includes an isolated nucleic acid that is complementary to an isolated nucleic acid encoding a mammalian BEHAB molecule or fragment thereof, wherein the complementary nucleic acid is in an antisense orientation. In one aspect, the nucleic acid is complementary to the nucleic acid sequence shown in SEQ ID NO: 5. In one embodiment, recombinant cells and vectors comprising mammalian BEHAB molecules or fragments thereof are provided, wherein the complementary nucleic acids are in the antisense orientation.

  The invention also includes a method for treating a primary CNS tumor in a mammal. The method includes administering to the mammal an effective amount of a BEHAB cleavage inhibitor, thereby treating the primary CNS tumor in the mammal. In one aspect, the BEHAB cleavage inhibitor is selected from the group consisting of antibodies, proteins, protease inhibitors, and peptidomimetics. In one embodiment, the antibody specifically binds BEHAB or a fragment thereof. In other embodiments, the protein comprises the amino acid sequence shown in SEQ ID NO: 3, or a fragment thereof.

  Further included in the present invention are methods for treating primary CNS tumors in mammals. The method includes administering to the mammal an effective amount of a BEHAB expression inhibitor, thereby treating the primary CNS tumor in the mammal. In one aspect, the BEHAB expression inhibitor is selected from the group consisting of ribozymes and antisense nucleic acids. In one embodiment, the antisense nucleic acid is complementary to an isolated nucleic acid encoding a BEHAB molecule or fragment thereof. In other embodiments, the ribozyme is complementary to an isolated nucleic acid encoding a BEHAB molecule or fragment thereof.

  Further included are methods of treating primary CNS tumors in mammals. The method includes administering to the mammal an effective amount of an inhibitor of BEHAB cleavage product activity, thereby treating the primary CNS tumor in the mammal. In one aspect, the inhibitor of BEHAB cleavage product activity is an antibody. In one embodiment, the antibody specifically binds to a BEHAB cleavage product.

  The invention further includes a method of diagnosing a primary CNS tumor in a mammal. The method obtains a biological sample from a first mammal, assesses the amount of BEHAB nucleic acid molecule in the biological sample, and determines the amount of BEHAB nucleic acid molecule in the biological sample otherwise identical. Compared to the amount of BEHAB nucleic acid molecule in a biological sample obtained from a second mammal, wherein the amount of BEHAB nucleic acid molecule in the biological sample from the second mammal is otherwise identical; A higher amount of BEHAB nucleic acid molecule in a biological sample from a higher first mammal is an indication that the first mammal is suffering from a primary CNS tumor, thereby causing a primary CNS tumor in a mammal Including diagnosis.

  In one aspect, the biological sample is selected from the group consisting of blood, neural tissue, cerebrospinal fluid, urine, saliva and brain tissue.

  Further included are methods of assessing the effectiveness of treatment for primary CNS tumors in mammals. The method assesses the amount of BEHAB molecules in a mammal before, during or after treatment for the primary CNS tumor in the mammal, where the amount of BEHAB molecules in the mammal prior to treatment for the primary CNS tumor is determined. Efficacy of treatment for primary CNS tumors in mammals when higher or lower amounts of BEHAB molecules in a mammal during or after treatment for a primary CNS tumor compared to the amount of BEHAB molecules Including assessing the efficacy of treatment for primary CNS tumors in mammals, thereby providing an index of sex.

  In one aspect, the treatment for the primary CNS tumor is selected from the group consisting of chemotherapy, radiation therapy and surgery.

  Further provided are methods for identifying compounds that affect the expression of BEHAB molecules in cells. The method comprises contacting a cell with a test compound and comparing the level of BEHAB molecule expression in the cell to a level of BEHAB molecule expression in a cell that is otherwise identical to the test compound, wherein the test compound is A higher or lower level of BEHAB molecule expression in cells contacted with the test compound compared to the level of BEHAB molecule expression in cells otherwise identical to that is not in contact with the test compound Identifying compounds that are indicative of affecting expression and thereby affecting the expression of BEHAB molecules in the cell.

  Also included are compounds identified by the methods described above.

  The present invention further provides methods for identifying compounds that reduce the expression of BEHAB molecules in cells. The method comprises contacting a cell with a test compound and comparing the level of BEHAB molecule expression in the cell to a level of BEHAB molecule expression in a cell that is otherwise identical to the test compound, wherein the test compound is A higher or lower level of BEHAB molecule expression in cells contacted with the test compound compared to the level of BEHAB molecule expression in cells otherwise identical to that is not in contact with the test compound It includes identifying compounds that are indicative of decreasing expression, thereby reducing the expression of BEHAB molecules in the cell.

  Further provided is a compound identified by the above method.

  The invention also includes a method for identifying a compound that inhibits BEHAB cleavage in a cell. The method involves contacting a cell with a test compound and comparing the level of BEHAB cleavage in the cell to the level of BEHAB cleavage in a cell that is otherwise identical to the test compound, wherein the cell is contacted with the test compound. A higher or lower level of BEHAB cleavage in cells in contact with the test compound compared to the level of BEHAB cleavage in otherwise identical cells indicates that the test compound inhibits BEHAB cleavage in the cell. Identifying compounds that serve as indicators and thereby inhibit BEHAB cleavage in cells.

  Further provided are methods for identifying compounds that inhibit BEHAB cleavage in a cell-free system. The method comprises contacting a cell-free system with a test compound and comparing the level of BEHAB cleavage in the cell-free system with a level of BEHAB cleavage in a cell-free system that is otherwise identical not in contact with the test compound; A higher or lower level of BEHAB cleavage in the cell-free system in contact with the test compound compared to the level of BEHAB cleavage in the cell-free system that is otherwise identical here but not in contact with the test compound. Identifying compounds that are indicative of inhibiting BEHAB cleavage in a cell-free system, thereby inhibiting BEHAB cleavage in a cell-free system.

  A kit for the treatment of primary CNS tumors is also included. The kit includes a BEHAB cleavage inhibitory amount of a composition comprising an isolated nucleic acid molecule encoding a mutant BEHAB molecule or fragment thereof and a pharmaceutically acceptable carrier, the kit further comprising an applicator and use thereof Includes instruction manual.

  Further included is a kit for the treatment of primary CNS tumors comprising an isolated mutant BEHAB polypeptide or fragment thereof and a BEHAB cleavage inhibiting amount of a composition comprising a pharmaceutically acceptable carrier, the kit comprising: In addition, the applicator and instructions for its use are included.

  Further included is a kit for the treatment of primary CNS tumors comprising a BEHAB cleavage inhibiting amount of a composition comprising an antibody that specifically binds to a mammalian BEHAB molecule or fragment thereof and a pharmaceutically acceptable carrier. The kit further includes an applicator and instructions for use thereof.

  A kit comprising a BEHAB molecule expression-inhibiting amount of a composition comprising an isolated nucleic acid complementary to the isolated nucleic acid encoding a mammalian BEHAB molecule or several fragments thereof, and a pharmaceutically acceptable carrier; The complementary nucleic acid also provides a kit for the treatment of primary CNS tumors in the antisense orientation, the kit further comprising an applicator and instructions for use thereof.

  Other kits for the treatment of primary CNS tumors are also included. The kit includes a BEHAB cleavage product inhibitory amount of a composition comprising an antibody that specifically binds to the BEHAB cleavage product or fragment thereof and a pharmaceutically acceptable carrier, and the kit further includes an applicator and instructions for use thereof. including.

BRIEF DESCRIPTION OF THE DRAWINGS For purposes of illustration of the invention, certain aspects of the invention are depicted in the drawings. However, the invention is not limited to the exact arrangement and means of the embodiments depicted in the drawings.

  FIG. 1, including FIGS. 1A-1C, shows Western blot analysis of media from cells stably transfected (FIG. 1C) or transiently (FIGS. 1A and 1B) using full length and / or mutant BEHAB. I'm drawing a statue. FIG. 1A depicts a Western blot of media from cells transiently transfected with full length (FL) and mutant (NVY) BEHAB. Both cell lines show strong staining of the full length protein of 145 kDa. In contrast, no cleavage product was detected with the B50 antibody in the NVY mutant, but a 50 kDa cleavage product was detected in the medium of cells transfected with FL. FIG. 1B depicts a Western blot of media from cells transiently co-transfected with 2 μg FL construct and 0, 1, 2, or 4 μg NVY mutant construct. Transfection of the NVY mutant increased the amount of full length BEHAB, but had no effect on normal BEHAB cleavage, as can be seen using the B50 antibody. FIG. 1C depicts a Western blot of medium from cells stably transfected with full length (FL) and mutant (NVY) BEHAB. The expression of BEHAB was examined using B6 and B50 antibodies in CNS-1-FL and CNS-1-NVY cells. As with transient transfection, cells stably transfected with CNS-1-FL produced and cut BEHAB, but were stably transfected with CNS-1-NVY. Cells produced full-length protein but did not cleave it.

  FIG. 2, including FIGS. 2A and 2B, depicts the effect of stable transfection on cell proliferation and cell death. CNS-1-FL, CNS-1-GFP and CNS-1-NVY stable cell lines were transformed into CNS-1-FL, CNS-1-GFP and CNS for cell proliferation (FIG. 2A) and cell death (FIG. 2B). The effect of -1-NVY transfection was compared to parental CNS-1 cells. FIG. 2A is a graph depicting cell growth over 7 days using the MTT assay. The data shows the change in absorbance. Transfection with CNS-1-FL, CNS-1-GFP and CNS-1-NVY had no effect on cell proliferation. FIG. 2B is a graph depicting cell death over 7 days as assessed using the LDH assay. Transfection with CNS-1-FL, CNS-1-GFP and CNS-1-NVY had no effect on cell death.

  FIG. 3, including FIGS. 3A-3D, depicts the effect of transfection with CNS-1-FL, CNS-1-GFP and CNS-1-NVY on tumor volume. Stably transfected cells were implanted intracranially in rats for 8 days, and the relative dimensions of the resulting tumors were evaluated histologically. FIG. 3A depicts an image of a representative tumor derived from CNS-1-GFP cells. FIG. 3B depicts an image of a representative tumor derived from CNS-1-FL cells. FIG. 3C depicts a representative tumor image derived from CNS-1-NVY cells. CNS-1-FL tumors are larger and more invasive than CNS-1-GFP and CNS-1-NVY tumors. FIG. 3D is a graph depicting tumor volume derived from CNS-1-FL, CNS-1-GFP and CNS-1-NVY cells. These data are quantified by image analysis and volume reconstruction, with individual data points indicated by asterisks.

  FIG. 4 is a graph depicting the impact on animal survival of tumors derived from cells stably transfected with CNS-1-FL, CNS-1-GFP and CNS-1-NVY. Animals with CNS-1-FL tumors survived significantly shorter than animals with CNS-1-GFP tumors or CNS-1-NVY tumors. Survival in animals with CNS-1-NVY tumors was not significantly different from survival in animals with CNS-1-GFP standard tumors.

DETAILED DESCRIPTION OF THE INVENTION Manipulation of the brain extracellular matrix (ECM) plays an important role in the progression and invasive phenotype of primary CNS tumors. One component of the ECM, brain-enriched hyaluronan binding (BEHAB) protein, is crucial in glioma cell motility and thus invasion. The data disclosed herein show for the first time that affecting BEHAB function and / or cleavage mediates a decrease in tumor size and an increase in survival time in animals with primary CNS tumors. Accordingly, the present invention includes, but is not limited to, glioma, oligodendroglioma, astrocytoma, gliosarcoma, glioblastoma multiforme, lymphoma and reactive gliosis following brain injury. Compositions and methods for the treatment of non-primary CNS tumors.

The invention also includes compositions and methods for the diagnosis of primary CNS tumors in mammals. That is, the data disclosed herein includes primary, including, but not limited to, glioma, oligodendroma, astrocytoma and gliosarcoma using the methods disclosed herein. FIG. 5 shows that CNS tumors can be diagnosed in mammals.
Definitions As used herein, each of the following terms has the meaning associated with it in this section.

  The articles “a” and “an” are used herein to refer to one or more (ie, at least one) of the grammatical objects of the article. For example, “an element” means one element or more than one element.

  “Amplification” refers to a means of copying a polynucleotide sequence and thus expanding it to a larger number of polynucleotide molecules, eg, by reverse transcription, polymerase chain reaction and ligase chain reaction.

As used herein, the term “antibody” refers to an immunoglobulin molecule that can specifically bind to a specific epitope on an antigen. The antibody can be an intact immunoglobulin derived from a natural source or from a recombinant source, and can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. The antibodies in the present invention can exist in various forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F (ab) ₂ as well as single chain antibodies and humanized antibodies (Harlow et al., Using). Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, 1999; Harlow et al., Authors: A Laboratory Manual, Cold. USA, 1988, 85: 5879-5883; Bird et al., Science, 1988, 242: 423-426).

  As used herein, the term “synthetic antibody” means an antibody made using recombinant DNA methods, eg, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody made by synthesis of a DNA molecule encoding an antibody, which DNA molecule expresses an antibody protein or an amino acid sequence defining an antibody, wherein the DNA or amino acid sequence is Obtained using synthetic DNA or amino acid sequencing methods available in the art and well known.

  “Antisense” specifically refers to the nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule encoding a protein or a sequence substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule that encodes a protein. The antisense sequence need not be complementary only to the coding portion of the coding strand of the DNA molecule. The antisense sequence can be complementary to a regulatory sequence defined on the coding strand of the protein-encoding DNA molecule, which regulatory sequence regulates the expression of the coding sequence.

  By the term “applicator”, as that term is used herein, a subcutaneous syringe for administering to a mammal a mutant BEHAB nucleic acid, protein and / or anti-BEHAB antibody and antisense BEHAB nucleic acid of the invention, It means any device including but not limited to pipettes and the like.

  “BEHAB”, “normal BEHAB” or “endogenous BEHAB” when used interchangeably herein refers to a brain-enriched hyaluronan binding molecule that exists in its natural state in a mammal. Others are known as Brebican.

  “Biological sample” as the term is used herein means a sample obtained from a mammal that can be used to assess the level of expression of BEHAB, the amount of BEHAB protein present, or both. . Such samples include, but are not limited to, blood samples, neural tissue samples, brain samples, and cerebrospinal fluid samples.

  “Cleavage” is used herein to refer to the dissociation of a peptide bond between two amino acids in a polypeptide, thereby separating the polypeptide containing the two amino acids into at least two fragments. It is done.

  As used herein, a “cleavage inhibitor” refers to a molecule that prevents cleavage of a polypeptide by titrating the protease responsible for cleavage, blocking the cleavage site, or otherwise rendering the cleavage site unrecognizable to the protease, Used to refer to a compound or composition.

  “Cleavage inhibiting amount” is used herein to refer to an effective amount of a cleavage inhibitor.

  “Cleavage product” is used herein to refer to a fragment of an initial polypeptide that results from cleavage into two or more fragments of the initial polypeptide. For example, cleavage products of the 145 kDa BEHAB protein include 90 kDa and 50 kDa fragments.

  By “complementary to part or all of a nucleic acid encoding BEHAB” is meant a nucleic acid sequence that does not encode a BEHAB protein. Rather, the sequence expressed in the cell is identical to the non-coding strand of the nucleic acid encoding the BEHAB protein and thus does not encode the BEHAB protein.

  As used herein, the terms “complementary” and “antisense” are not completely synonymous. “Antisense” specifically refers to a nucleic acid sequence in the non-coding strand of a double-stranded DNA molecule encoding a protein or a sequence that is substantially homologous to the non-coding strand. As used herein, “complementary” refers to the broad concept of subunit sequence complementarity between two nucleic acids, eg, two DNA molecules. If a nucleotide position in both molecules is occupied by a nucleotide that can normally base pair with each other, the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids when a substantial number (at least 50%) of corresponding positions in each molecule are occupied by nucleotides that can normally base pair with each other (eg A: T and G: C nucleotide pairs) Are complementary to each other. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. The antisense sequence need not be complementary only to the coding portion of the coding strand of the DNA molecule. The antisense sequence can be complementary to a regulatory sequence defined on the coding strand of the protein-encoding DNA molecule, which regulatory sequence regulates the expression of the coding sequence.

  The “coding region” of a gene consists of nucleotide residues in the coding strand of the gene and nucleotides in the non-coding strand of the gene, each of which is homologous to or complementary to the coding region of the mRNA molecule produced by transcription of the gene. Is.

  The “coding region” of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule that either match the anticodon region of the transfer RNA molecule during translation of the mRNA molecule or encode a stop codon. Thus, the coding region may include nucleotide residues that correspond to amino acid residues that are not present in the mature protein encoded by the mRNA molecule (eg, amino acid residues in a protein export signal sequence).

  “Coding” refers to a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, having a limited sequence of nucleotides (ie, rRNA, tRNA and mRNA) or other sequences having a limited amino acid sequence. It refers to the intrinsic properties that serve as templates for the synthesis of polymers and macromolecules in biological processes and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produce the protein in a cell or other biological system. Both the coding sequence whose nucleotide sequence is identical to the mRNA sequence and the non-coding strand normally used as a template for transcription of the gene or cDNA as a protein or other product of the gene or cDNA Can be said to code.

  A first region of an oligonucleotide if the two regions are adjacent to each other or if the two regions are separated by no more than about 1000 nucleotide residues, preferably no more than about 100 nucleotide residues Is “adjacent” to the second region of the oligonucleotide.

  As used herein, the term “fragment” as applied to nucleic acids is usually at least about 20 nucleotides in length, typically at least about 50 nucleotides, more typically from about 50 to About 100 nucleotides, preferably at least about 100 to about 500 nucleotides, more preferably at least about 500 nucleotides to about 1000 nucleotides, even more preferably at least about 1000 to about 1500, more preferably At least about 1500 nucleotides to about 2000 nucleotides, even more preferably at least about 2000 to about 2500, more preferably at least about 2500 nucleotides to about 2600 nucleotides, even more preferably at least about 26 0 can be ~ about 2650 pieces, and most preferably nucleic acid fragments it may be longer than about 2652 nucleotides in length.

  When applied to proteins, a BEHAB “fragment” is about 20 amino acids in length. More preferably, the BEHAB fragment is about 100 amino acids in length, more preferably at least about 200, more preferably at least about 300, more preferably at least about 400, even more preferably at least about 500, more Preferably about 600 and more preferably, more preferably at least about 700, even more preferably at least about 800, more preferably about 850, and more preferably at least about 884 amino acids.

  As used herein, “usage instructions” are publications, records, figures and other representational media that can be used to convey the usefulness of the composition of the invention for its stated use. including. For example, instructions for use of the kit of the present invention can be attached to a container containing the composition or transported with the container containing the composition. Alternatively, the instructional material and composition can be used in a co-operatively manner by the recipient, and the instructional material can be transported separately from the container.

  An “isolated nucleic acid” is a nucleic acid segment or fragment that is separated from a sequence that naturally flank it, such as a sequence that normally flank a fragment, such as in the genome in which it naturally resides. Refers to a DNA fragment removed from the sequence adjacent to the fragment. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, such as RNA or DNA or proteins that naturally accompany it in the cell. Thus, the term is used, for example, as a molecule (eg, PCR or restriction enzyme) introduced into a vector, autonomously replicating plasmid or virus, or introduced into prokaryotic or eukaryotic genomic DNA, or isolated independently of other sequences. Recombinant DNA present as digested cDNA or genomic or cDNA fragments. It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

  “Mutant BEHAB” is used herein to refer to a brain-enriched hyaluronan binding molecule whose amino acid sequence has been altered to inhibit cleavage by a protease.

  “Naturally present” as applied to a subject refers to the subject being able to be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a source in nature and that has not been altered by humans is naturally occurring.

  Within the scope of the present invention, the following abbreviations for nucleobases that are normally present are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

  Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

  By describing two polynucleotides as “operably linked”, a single-stranded or double-stranded nucleic acid moiety has a physiological effect on the other that at least one of the two polynucleotides characterizes it. It is meant to include two polynucleotides arranged within the nucleic acid moiety in such a way as to be possible. For example, a promoter operably linked to a coding region of a gene can promote transcription of the coding region.

  “Polynucleotide” means a single strand of nucleic acid or parallel and antiparallel strands. Thus, the polynucleotide can be a single-stranded or double-stranded nucleic acid.

  The term “nucleic acid” typically refers to large polynucleotides.

  The term “oligonucleotide” typically refers to short polynucleotides, generally no more than about 50 nucleotides. Where a nucleotide sequence is represented by a DNA sequence (ie A, T, G, C), it is understood that this also includes RNA sequences (ie A, U, G, C) where “U” has replaced “T”. It will be.

  Herein, the usual notation is used to describe the polynucleotide sequence: the left end of the single-stranded polynucleotide sequence is the 5′-end; the left-hand direction of the double-stranded polynucleotide sequence is 5 ′ -Called the direction.

  The direction of 5 'to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. A DNA strand having the same sequence as mRNA is called a “coding strand”; a sequence on the DNA strand that is located 5 ′ to the reference point on DNA is called an “upstream sequence”; A sequence on the DNA strand that is 3 ′ to the other is called a “downstream sequence”.

  “Part” of a polynucleotide means at least about 20 contiguous nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide can include all nucleotide residues of the polynucleotide.

  As used herein, a “primary CNS tumor” refers to neoplasia that originates in the brain in that cancer cells originate in other parts of the body and have not spread to the brain. Used. Examples of primary CNS tumors include glioma, fully differentiated astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, ventricular ependymoma, oligodendroma, ganglion cell tumor, Mixed glioma, brainstem glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, reactive gliosis, primitive neuroectodermal tumor, neurofibroma, lymphoma, blood vessel Includes but is not limited to tumors and lymphomas.

  “Treatment of primary CNS tumor” as used herein refers to a reduction in the severity of primary CNS tumor symptoms, including the volume of the tumor or the frequency or both of which the patient experiences or symptoms of the tumor, or the progression of tumor progression. It is used to refer to a situation in which the time or survival time is extended.

  “Primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, ie, in the presence of nucleotides, complementary polynucleotide templates and agents for polymerization such as DNA polymerase. A primer is typically single-stranded, but can be double-stranded. Primers are typically deoxyribonucleic acids, but a variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template that it is designated to hybridize with and serves as a site for initiation of synthesis, but need not reflect the exact sequence of the template. In such cases, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. The primer can be labeled, eg, with a chromogenic, radioactive or fluorescent moiety and used as a detectable moiety.

  A “probe” refers to a polynucleotide that can specifically hybridize to a specified sequence of another polynucleotide. The probe specifically hybridizes to the target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such cases, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. The probe can be labeled with, for example, a chromogenic, radioactive or fluorescent moiety and used as a detectable moiety.

  “Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. Amplified or assembled recombinant polynucleotides can be included in a suitable vector, and the vector can be used to transform a suitable host cell.

  Recombinant polynucleotides can also perform non-coding functions (eg, promoters, origins of replication, ribosome-binding sites, etc.).

  A host cell comprising a recombinant polynucleotide is referred to as a “recombinant host cell”. A gene that is expressed in a recombinant host cell in which the gene contains a recombinant polynucleotide produces a “recombinant polypeptide”.

  A “recombinant polypeptide” is one that is produced when a recombinant polynucleotide is expressed.

  “Polypeptides” are polymers of amino acid residues, related naturally occurring structural variants, and non-naturally occurring synthetic analogs linked via peptide bonds, related naturally occurring Refers to structural variants and their analogs that do not occur in nature. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

  The term “protein” typically refers to large polypeptides.

  The term “peptide” typically refers to short polypeptides.

  Herein, the usual notation is used to represent the polypeptide sequence: the left end of the polypeptide sequence is the amino-terminus; the right end of the polypeptide sequence is the carboxyl-terminus.

  As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence necessary for the expression of the product of a gene operably linked to a promoter / regulatory sequence. In some cases this sequence can be a core promoter sequence, in other cases this sequence can also include enhancer sequences and other regulatory elements necessary for expression of the gene product. The promoter / regulatory sequence can be, for example, one that expresses the gene product in a tissue specific manner.

  As used herein, the term “specifically binds” refers to an antibody that recognizes and binds to an epitope of BEHAB protein but does not substantially recognize or bind to other molecules in the sample. Means.

  A “therapeutic” treatment is a treatment given to patients who show signs of pathology in order to reduce or eliminate those symptoms.

  A “therapeutically effective amount” of a compound is an amount of the compound sufficient to provide a beneficial effect to the patient to whom the compound is administered.

  As used herein, “transgene” means an exogenous nucleic acid sequence comprising a nucleic acid encoding a promoter / regulatory sequence operably linked to a nucleic acid encoding an amino acid sequence, the exogenous nucleic acid being Encoded by animals or cells.

  A “vector” is a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses. The term must also be considered to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-related viral vectors, retroviral vectors, and the like.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (eg naked or contained in liposomes) and viruses into which a recombinant polynucleotide has been introduced.
Description
I. Isolated nucleic acid
A. Sense Nucleic Acids The present invention includes an isolated nucleic acid encoding a mammalian mutant BEHAB molecule or fragment thereof, wherein the nucleic acid shares at least about 99.7% identity with a nucleic acid having the sequence of SEQ ID NO: 4. . The mammal is preferably a human. Preferably, the nucleic acid is about 99.8% homologous, more preferably, and most preferably about 99.9% homologous to SEQ ID NO: 4 disclosed herein. Even more preferably, the nucleic acid is SEQ ID NO: 4. An isolated nucleic acid of the present invention comprises an RNA or DNA sequence encoding a mutant BEHAB protein of the present invention as well as a nucleotide sequence when the nucleotide sequence is cell-free or associated with a cell. It should be considered to include its altered forms, including chemical alterations of DNA or RNA that make it more stable. Chemical alterations of the nucleotide can also be used to enhance the effectiveness of the nucleotide sequence being absorbed by the cell or that it is expressed in the cell. All combinations of nucleotide sequence alterations are encompassed by the present invention.

  The present invention should not be considered limited to the nucleic acid and amino acid sequences disclosed herein. In accordance with the present invention, the methods described herein in the experimental details section for the formation of other mammalian mutant BEHAB nucleic acids encoding mutant BEHAB polynucleotides disclosed herein (eg, It is well known in the art that other nucleic acids encoding mutant BEHAB proteins can be obtained by following site-directed mutagenesis, frameshift mutation, etc.) as well as methods well known or untapped in the art. Will be readily apparent to those skilled in the art.

  Further, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. Formation of mutant BEHAB derivatives or mutant forms using recombinant DNA methods well known in the art, such as the method described in the book (Current Protocols in Molecular Biology, Green & Wiley, New York, 1997). In addition, a plurality of other methods can be used.

  Methods for introducing amino acid changes in a protein or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in Sambrook et al. (Id., 1989); Ausubel et al. It is described in the book (Id., 1997).

The invention includes a nucleic acid encoding a mammalian mutant BEHAB, wherein a nucleic acid encoding a tag polypeptide is covalently linked thereto. That is, the present invention encompasses chimeric nucleic acids in which the nucleic acid sequence encoding the tag polypeptide is covalently linked to the nucleic acid encoding the mutant BEHAB polypeptide. Such tag polypeptides are well known in the art, eg, green fluorescent protein (GFP), myc, myc-pyruvate kinase (myc-PK), His ₆ , maltose binding protein (MBP), influenza virus hemagglutination Element tag polypeptides, flag tag polypeptides (FLAG) and glutathione-S-transferase (GST) tag polypeptides are included. However, the present invention should in no way be construed as limited to nucleic acids encoding the tag polypeptides listed above. Rather, any nucleic acid sequence encoding a polypeptide that can act in a manner substantially similar to these tag polypeptides should be considered to be included in the present invention.

  Using a nucleic acid comprising a nucleic acid encoding a tag polypeptide, placing the mutant BEHAB in a cell, tissue and / or whole organism (eg, a mammalian embryo), detecting the mutant BEHAB secreted from the cell; and The role or roles of mutant BEHAB in the cell can be studied. Furthermore, the addition of a tag polypeptide facilitates the isolation and purification of “tagged” proteins, allowing the proteins of the invention to be easily produced and purified.

B. Antisense Nucleic Acids In certain circumstances it may be desirable to inhibit the expression of BEHAB, and therefore the present invention includes compositions useful for the inhibition of BEHAB expression. Thus, the invention features an isolated nucleic acid that is complementary to part or all of a nucleic acid encoding a mammalian BEHAB molecule, which nucleic acid is in an antisense orientation with respect to transcription. Preferably, the antisense nucleic acid is complementary to a nucleic acid having at least about 99.7% homology with SEQ ID NO: 5 or a fragment thereof. Preferably, the nucleic acid is about 99.8% homologous to a nucleic acid complementary to part or all of a nucleic acid encoding mammalian BEHAB having the sequence of SEQ ID NO: 5 or a fragment thereof, most preferably about 99.9%. Homologous, it is in an antisense orientation with respect to transcription. Most preferably, the nucleic acid is complementary to part or all of a nucleic acid that is SEQ ID NO: 5 or a fragment thereof. Such antisense nucleic acids serve to inhibit the expression, function or both of BEHAB molecules.

Alternatively, antisense molecules of the invention can be made synthetically and then given to cells. Antisense oligomers of about 10 to about 30, more preferably about 15 nucleotides are preferred because they are easily synthesized and introduced into target cells. Synthetic antisense molecules encompassed by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (the disclosure of which is incorporated by reference). See Cohen, Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL, 1989; Tullis, U.S. Pat. No. 5,023,243, which is incorporated herein in its entirety. Wanna)
II. Isolated polypeptides The present invention also includes isolated polypeptides comprising mammalian mutant BEHAB molecules. Preferably, the isolated polypeptide comprising a mammalian mutant BEHAB molecule is at least about 99.6% homologous to a polypeptide having the amino acid sequence of SEQ ID NO: 3, or some fragment thereof. Preferably, the isolated polypeptide is about 99.7% homologous to SEQ ID NO: 3 or some fragment thereof, more preferably about 99.8% homologous, more preferably, and most preferably About 99.9% homology. Most preferably, the isolated polypeptide comprising a mutated BEHAB molecule is SEQ ID NO: 3.

The present invention also provides analogs of proteins or peptides comprising a mutated BEHAB molecule disclosed herein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences, or by changes that do not affect the sequence, or both. For example, conservative amino acid changes can be made that alter the primary sequence of a protein or peptide but do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups:
Glycine, alanine;
Valine, isoleucine, leucine;
Aspartic acid, glutamic acid;
Asparagine, glutamine;
Serine, threonine;
Lysine, arginine;
Phenylalanine, tyrosine.

  Modifications (which usually do not change the primary sequence) include in vivo or in vitro chemical derivatization of polypeptides such as acetylation or carboxylation. By modification of glycosylation, for example by altering the glycosylation pattern of the polypeptide during synthesis and processing of the polypeptide, or in further processing steps; for example, polyglycation to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. Also included are modifications made by exposing the peptide. Also included are sequences having phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine or phosphothreonine.

  Polypeptides modified using conventional molecular biology methods to improve the resistance of polypeptides to proteolysis or to optimize solubility or to make them more suitable as therapeutic agents Is also included. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the present invention are not limited to the products of any particular example method listed herein.

  The present invention should also be considered to include “derivatives” and “variants” of the peptide of the present invention (or the DNA encoding it), and the derivatives and variants of the peptide (or DNA) obtained are Although not identical to the sequences listed in the specification, the peptides appear to have the same biological properties as the peptides disclosed herein in that they have the biological / biochemical properties of the mutant BEHAB peptides of the invention. Or a mutated BEHAB peptide that is altered in one or more amino acids (or altered in one or more base pairs when referring to the nucleotide sequence encoding the peptide).

  The biological properties of the mutated BEHAB protein should be considered to include the ability of the peptide to be secreted from the cell or immobilized via GPI-binding, the ability to be cleaved by proteases, etc. Not limited to.

Based on the disclosure presented herein, the skilled person will have the biological activity of the mutant BEHAB expressed in the brain tissue, detected in the brain tissue, secreted from the cell, fixed to the cell It will be understood that this includes, but is not limited to, the ability of a molecule or compound not to be cleaved by a protease. “Activity of mutant BEHAB” includes the effect of mutant BEHAB circulating in the ECM or cerebrospinal fluid or produced locally in the brain. The biological activity of the mutant BEHAB mediates or is associated with, among other things, tumor progression, tumor invasiveness, tumor volume and size, animal survival, and the like.
III. In other related aspects of vectors , the invention preferably relates to a mammalian mutant BEHAB operably linked to a nucleic acid comprising a promoter / regulatory sequence so that the nucleic acid can direct the expression of the protein encoded by the nucleic acid. Isolated nucleic acid encoding. Thus, the present invention is disclosed in, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. Introduction of exogenous DNA into cells with concomitant expression of exogenous DNA in expression vectors and cells, such as those described in the book (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1997) Includes a method for

  Expression of a mutant BEHAB, alone or fused to a detectable tag polypeptide, in cells that normally express normal BEHAB serves to drive the expression of proteins with or without the tag. This can be done in the cell into which the vector is introduced by forming a plasmid, virus or other type of vector that contains the desired nucleic acid operably linked to a promoter / regulatory sequence. A number of promoter / regulatory sequences useful for driving constitutive expression of genes are available in the art, for example the cytomegalovirus immediate promoter enhancer sequence, SV40 early, both used in the experiments disclosed herein. Examples include, but are not limited to, promoters and Rous sarcoma virus promoters. Furthermore, inducible and tissue-specific expression of a nucleic acid encoding mutant BEHAB can induce a nucleic acid encoding mutant BEHAB with or without a tag or regulation of tissue-specific promoter / regulatory sequences. It can be done by putting it down. Examples of tissue specific or inducible promoter / regulatory sequences useful for this purpose include, but are not limited to, the MMTV LTR inducible promoter and the SV40 late enhancer / promoter. Furthermore, promoters well known in the art that are induced in response to inducers such as metals, glucocorticoids and the like are also encompassed by the invention. Thus, it will be appreciated that the present invention encompasses the use of any promoter / regulatory sequence that is known or unknown and that can drive the expression of the desired protein operably linked to the promoter / regulatory sequence. I will.

  Expression of mutant BEHAB using a vector allows the isolation of large amounts of recombinantly produced protein. Further, where normal BEHAB expression causes a disease, disorder or condition associated with such expression, expression of the mutant BEHAB driven by a promoter / regulatory sequence includes treatment of the gene to which the mutant BEHAB is given. There can be useful therapeutics that are not limited to that. Administration of mutant BEHAB may be useful for primary CNS tumors for diseases, disorders or conditions associated with increased levels of expression, amounts of protein or cleavage of protein or its cleavage products and / or increased levels of activity Glioma, fully differentiated astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, ventricular ependymoma, oligodendroma, ganglion celloma, mixed glioma, brain stem Glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, primitive neuroectodermal tumor, neurofibroma, vascular tumor, lymphoma, etc. Absent.

  Thus, not only does the present invention include methods of inhibiting normal BEHAB expression, translation, cleavage and / or activity, but the present invention also includes methods associated with a decrease in BEHAB expression, protein content, cleavage and / or activity, This is because a reduction in BEHAB expression, cleavage and / or activity or an increase in mutant BEHAB expression and / or activity may be useful in providing effective therapeutics.

  The selection of a particular plasmid vector or other DNA vector is not a limiting factor in the present invention, and a wide variety of vectors are well known in the art. Furthermore, it is well within the skill of the artisan to select specific promoter / regulatory sequences and operably link those promoter / regulatory sequences to the DNA sequence encoding the desired polypeptide. Such methods are well known in the art and are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1997).

  Thus, the present invention includes a vector containing an isolated nucleic acid encoding a mammalian mutant BEHAB. Introduction of a desired nucleic acid into a vector and selection of the vector are well known in the art, see, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1997).

  The invention also includes cells, viruses, proviruses, etc. containing such vectors. Methods for the formation of cells containing vectors and / or exogenous nucleic acids are well known in the art. For example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1997).

Nucleic acids encoding mutant BEHAB can be cloned into a variety of plasmid vectors. However, the present invention should not be regarded as limited to plasmids or any particular vector. Rather, the present invention should be considered to include too many vectors that are readily available and / or well known in the art.
IV. Recombinant cells The present invention comprises, among other things, an isolated nucleic acid encoding a mutant BEHAB, an antisense nucleic acid complementary thereto, a nucleic acid encoding an antibody that specifically binds to BEHAB or its cleavage product, and the like. Contains cells. In one aspect, a recombinant cell can be transiently transfected with a plasmid that encodes a portion of a nucleic acid encoding a mutant BEHAB. The nucleic acid need not be integrated into the cell genome nor expressed in the cell. Furthermore, the cells can be prokaryotic or eukaryotic cells, and the invention should not be considered limited to any particular cell line or cell type. Such cells include, but are not limited to, neurons, neurons, brain cells, glioma-derived cell lines, glial cell lines, non-glial cell lines, stem cell lines and the like.

  Furthermore, it is important to note that the purpose of transgene-containing cells, ie recombinant cells, should not be considered limited to the development of intracranial tumors. Rather, the present invention relates to any cell into which a nucleic acid encoding a mammalian mutant BEHAB has been introduced, including, but not limited to, prokaryotic cells and eukaryotic cells including an isolated nucleic acid encoding a mammalian mutant BEHAB. Should be considered to include types.

  The present invention includes a eukaryotic cell, wherein the transgene of the present invention is introduced therein and the protein encoded by the desired gene is then expressed, where it was not previously present in the cell. A benefit is obtained if it is expressed or has not been expressed, or if it is expressed at a different level or under different circumstances than before the transgene was introduced this time. Such benefits include a system in which the expression of the desired gene can be studied in vitro in the laboratory or in the mammal in which the cell resides, using the cell containing the introduced gene as a research, diagnostic and therapeutic tool. It can be provided that a system capable of creating a mammalian model useful for the development of new diagnostic and therapeutic tools for selected disease states in mammals and selected disease states can be included.

  Give a mutant BEHAB to a cell, tissue or whole mammal if a higher amount of the mutant BEHAB can be useful for treating or reducing a condition associated with a disease, disorder or normal BEHAB expression, cleavage and / or activity In order to do this, cells expressing an isolated nucleic acid encoding a mutant BEHAB can be used. Such diseases, disorders or conditions include primary CNS tumors, gliomas, fully differentiated astrocytomas, anaplastic astrocytomas, glioblastoma multiforme, ependymoma, ventricular ependymoma, oligodendrocytes Tumor, ganglion cell, mixed glioma, brainstem glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, primordial neuroectodermal tumor, neurofibroma, Examples include, but are not limited to, hemangiomas and lymphomas. Accordingly, the present invention reduces normal BEHAB expression, translation, cleavage and / or activity where increased mutant BEHAB expression, protein content and / or activity may be useful in the treatment or reduction of a disease, disorder or condition. Including cells expressing mutant BEHAB to allow or prevent.

  Based on the disclosure presented herein, the ordinary skilled artisan will be able to replace the two portions of the nucleic acid to which the “knock-in” or “knock-out” vector of the present invention is to be replaced or deleted, respectively. It will be appreciated that it contains at least two sequences homologous to. Two sequences are homologous to the sequences flanking the gene; that is, one sequence is homologous to a region in or near the 5 'portion of the coding sequence of normal BEHAB and the other sequences are first Further downstream from that of One of ordinary skill in the art will appreciate that, based on the disclosure presented herein, the present invention is not limited to any particular flanking nucleic acid sequence. Rather, the targeting vector removes, for example, some or all of normal BEHAB from or into the mammalian genome (ie, a “knock-out” vector) or a nucleic acid encoding a mutant BEHAB or fragment thereof. Can be included (ie, a “knock-in” vector). The critical feature of the targeting vector is the opposite, in the case of a “knock-out” vector, a homologous set of two sequences located towards the 5 ′ and 3 ′ ends of the normal BEHAB open reading frame (ORF). It contains sufficient parts to allow deletion / insertion by recombination to take place so that all or part of the normal BEHAB-encoding nucleic acid is deleted from a position on the mammalian chromosome. is there.

  The design of transgenes and knock-in and knock-out targeting vectors is well known in the art and is described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1997) and the like. The upstream and downstream portions adjacent to or within the BEHAB coding region to be used in the targeting vector are based on known methods and include the nucleic acid and amino acid sequences of mammalian BEHAB and mutant BEHAB. Can be readily selected according to the description disclosed herein based on the disclosure presented in the document. Having these sequences, one of ordinary skill in the art will be able to construct the transgene and knock-out vectors of the present invention.

  Those skilled in the art will recognize that, based on the present disclosure, cells containing reduced amounts of normal BEHAB protein, decreased levels of BEHAB and / or BEHAB cleavage product activity, or both may be inhibitors of BEHAB expression (eg, anti-HAHAB expression). It will be appreciated that cells including, but not limited to, cells that express sense or ribozyme molecules, synthetic antibodies or intrabodies).

  Methods and compositions useful for maintaining mammalian cells in culture are well known in the art, where mammalian cells include those obtained from mammals including but not limited to mice, rats, humans, etc. can get.

  To study the effect of qualitative and quantitative changes in the amount of BEHAB on tumor progression and invasiveness, the recombinant cells of the invention can be used. This is because BEHAB is secreted and has a hyaluronan binding domain, indicating that BEHAB is included in the function, composition or activity of ECM. In addition, recombinant cells can be used for the production of mutant BEHAB for use in therapeutic and / or diagnostic purposes. That is, using a recombinant cell that expresses a mutant BEHAB, a large amount of purification that can be administered for the treatment or alleviation of a disease, disorder or condition associated with or caused by BEHAB expression, activity and / or cleavage. Isolated mutant BEHAB can be produced.

  Alternatively, recombinant cells expressing the mutated BEHAB can be administered in vitro and in vivo, where administering the recombinant cells thereby administers the protein to cells, tissues and / or mammals. That is. In addition, recombinant cells are useful for the discovery of BEHAB receptors and BEHAB signaling pathways.

Recombinant cells of the present invention in which the cells have been engineered so that the cells do not express BEHAB or express mutant BEHAB lacking cleavability can be transformed into mammalian cells (eg, neuronal cells, brain cells, etc.) ) Or syngeneic matched donor cells have been engineered recombinantly as described elsewhere herein (eg, inserting an antisense nucleic acid or knock-out vector, thereby allowing BEHAB expression levels and / or (Alternatively, by reducing the amount of protein in the recombinant cell), it can also be used in in vitro and in vivo cell therapy where the recombinant cell is administered to the recipient mammal. In this way, recombinant cells expressing BEHAB in reduced amounts are administered to a mammal whose own cells express an increased amount of BEHAB, thereby improving as disclosed elsewhere herein. Any disease, disorder or condition associated with or mediated by the observed BEHAB expression can be treated or alleviated.
V. Also included are antibodies that specifically bind to the antibody BEHAB, a BEHAB cleavage product or fragment thereof.

  Based on the disclosure presented herein, one skilled in the art will recognize that antibodies that specifically bind to BEHAB and / or BEHAB cleavage products are BEHAB proteins or immunogenic portions thereof, preferably other It will be understood that it binds to the cleavage site discussed in section. In one embodiment, the antibody is directed to a mammalian BEHAB comprising the amino acid sequence, SEQ ID NO: 6.

  Polyclonal antibody formation involves inoculating a desired animal with an antigen, for example, Harlow et al. Using standard antibody production methods such as those described in the authors (Antibodies, A Laboratory Manual, Cold Spring Harbor, NY, 1988) and isolating antibodies that specifically bind to the antigen therefrom. It is. Such methods include maltose binding protein or glutathione (GSH) tag polypeptide moieties and / or other moieties that make BEHAB protein immunogenic (eg, keyhole limpet hemocyanin, BEHAB conjugated to KLH). Immunizing an animal with a chimeric protein containing portions of the protein and portions containing murine and / or human BEHAB amino acid residues, respectively. A chimeric protein is produced by cloning a suitable nucleic acid encoding BEHAB (eg, SEQ ID NO: 5) into a plasmid vector suitable for this purpose, such as, but not limited to, pMAL-2 or pCMX. Other methods for the production of antibodies that specifically bind to BEHAB and portions thereof are described in Matthews et al. (J. Biol. Chem., 2000, 275: 22695-22703).

  However, the present invention should not be considered limited to only polyclonal antibodies that bind to full-length BEHAB. Rather, the present invention should be considered to include other antibodies to mammalian BEHAB or portions thereof as defined elsewhere herein. Furthermore, the present invention is particularly useful for immunohistological staining of tissues that bind to BEHAB and are capable of locating BEHAB in tissues, and transiently using nucleic acids encoding at least a portion of BEHAB. Alternatively, it should be considered to include antibodies capable of binding to BEHAB present in immunofluorescence microscopy of stably transfected cells.

  Those skilled in the art will be able to specifically bind an antibody to any part of the protein and form a specific antibody with respect to it using the full-length protein, based on the disclosure presented herein. You will understand. However, the present invention is not limited to using full length proteins as immunogens. Rather, the present invention includes the use of an immunogenic portion of a protein for the production of antibodies that specifically bind to mammalian BEHAB. That is, the present invention includes immunizing an animal with an immunogenic portion or antigenic determinant of BEHAB protein, such as an epitope containing a cleavage site or a new antigenic site made by proteolytic cleavage.

  By immunizing animals such as but not limited to rabbits or mice with BEHAB protein or part thereof, or protein containing at least part of BEHAB or part containing, for example, a suitable BEHAB amino acid residue Antibodies can be produced by immunizing an animal with a fusion protein containing a tag polypeptide moiety that includes a maltose-binding protein tag polypeptide moiety covalently linked to. Those skilled in the art will appreciate that, based on the disclosure presented herein, relatively small fragments of these proteins can also be used to produce antibodies that specifically bind to BEHAB. .

  Those skilled in the art will recognize, based on the disclosure presented herein, isolated BEHAB polypeptides for the formation of antibodies to epitopes that include a cleavage site of BEHAB or an epitope present on the cleavage product of BEHAB. It will be appreciated that various parts of can be used. With detailed analysis to locate the various epitopes and cleavage products of BEHAB sequences and proteins, one skilled in the art, based on the disclosure presented herein, is well-known or undeveloped methods in the art. Will be used to understand how antibodies can be obtained that are specific for various portions of a mammalian BEHAB polypeptide.

  Thus, for those skilled in the art, antibodies that neutralize and / or inhibit BEHAB activity (eg, by inhibiting the required BEHAB cleavage product receptor / ligand interaction or BEHAB cleavage) based on the disclosure presented herein. It will be appreciated that the antibody can recognize BEHAB or BEHAB cleavage products.

  The present invention should not be considered limited to any particular immunogenic portion of any of the antibodies disclosed herein or the proteins of the present invention. Rather, the invention is defined elsewhere herein in BEHAB or a portion thereof, or to a protein that shares at least about% homology with a polypeptide having the amino acid sequence of SEQ ID NO: 6. Should be considered to include other antibodies. Preferably, the polypeptide is about 1% homologous to BEHAB (SEQ ID NO: 6), more preferably about 5% homologous, more preferably about 10% homologous, even more preferably about 20% homologous, more preferably about 30%. Homology, preferably about 40% homology, more preferably about 50% homology, even more preferably about 60% homology, more preferably about 70% homology, even more preferably about 80% homology, preferably about 90% homology, More preferably about 95% homology, even more preferably about 99% homology and most preferably about 99.9% homology.

  Those skilled in the art will recognize, based on the disclosure presented herein, one or more in the cellular process of antigens that can be used to locate and recognize related proteins in cells using antibodies. You will find that you can study the role of. Further, detect and / or measure the amount of protein present in a biological sample using well-known methods such as but not limited to Western blotting and enzyme-linked immunosorbent assay (ELISA) Antibodies can be used to do this. In addition, antibodies can be used to immunoprecipitate and / or immuno-affinity purify antibody cognate antigens using methods well known in the art. In addition, antibodies can be used to reduce the amount of BEHAB or BEHAB cleavage product in the cell, thereby inhibiting the effect or effects of BEHAB or BEHAB cleavage product in the cell. Thus, by administering the antibody to mammalian cells or tissues or to the mammal itself, the necessary BEHAB receptor / ligand interaction is thus inhibited and the effects of BEHAB cleavage are also inhibited. Those skilled in the art will appreciate that inhibition of BEHAB cleavage using anti-BEHAB antibodies can include, but is not limited to, reduction of tumor size, extension of survival, and the like.

  Those skilled in the art will recognize, based on the disclosure presented herein, that antibodies that specifically bind to BEHAB to inhibit BEHAB cleavage in the brain, oral, parenteral, intraventricular. It will be appreciated that administration includes intrathecal, intraparenchymal, or by multiple routes. Administration is via bioengineered polymer, direct injection, Omaya reservoir (subcutaneous implanted device used to deliver anticancer drugs to cerebrospinal fluid) or neurosurgery Delivery via other such means well known to those skilled in the art.

  The present invention includes polyclonal, monoclonal, synthetic antibodies and the like. Those skilled in the art will understand, based on the disclosure presented herein, that a critical feature of the antibodies of the present invention is that the antibodies specifically bind to BEHAB. That is, the antibody of the present invention recognizes BEHAB or a fragment thereof (eg, an immunogenic portion or antigenic determinant thereof) on a Western blot by immunostaining the cells, and immunizes BEHAB using standard methods well known in the art. Allow to settle.

  Monoclonal antibodies directed against the full length or peptide fragment of a protein or peptide can be obtained by any well-known monoclonal antibody production method, eg, Harlow et al. (Antibodies, A Laboratory Manual, Cold Spring Harbor, NY, 1988) and Tuszynski et al. It can be produced using the method described in the book (Blood, 1988, 72: 109-115). Large quantities of the desired peptide can also be synthesized using chemical synthesis methods. Alternatively, DNA encoding the desired peptide can be cloned and expressed from a suitable promoter sequence in cells suitable for the production of large amounts of peptide. Monoclonal antibodies directed against the peptide are formed from mice immunized with the peptide using standard methods referred to herein.

  Available in the art and described in, for example, Wright et al. (Critical Rev. Immunol., 1992, 12: 125-168) and the references described therein, and monoclonal antibodies obtained using the methods described herein. The encoding nucleic acid can be cloned and sequenced.

  Further, for example, Wright et al. (Critical Rev. Immunol., 1992, 12: 125-168) and references cited therein and Gu et al. The antibodies of the present invention can be “humanized” using the methods described in the book (Thrombosis and Hematocyst. 1997, 77: 755-759). The invention also includes the use of humanized antibodies that are specifically reactive with an epitope of BEHAB. Such an antibody can specifically bind to BEHAB or a fragment thereof. A humanized antibody of the invention has a human framework and is specifically reactive with BEHAB or a fragment thereof, typically but not limited to one or It has more complementarity determining regions (CDRs). Thus, for example, antibodies directed to BEHAB adapted to the human body are primary CNS tumors such as glioma, fully differentiated astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, ependymoma, Oligodendroma, ganglion cell, mixed glioma, brainstem glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, primitive neuroectodermal tumor, nerve It is useful in the treatment of fibroma, vascular tumor, lymphoma and the like.

  When adapting the antibody used in the present invention to the human body, Queen, et al. (US Pat. No. 6,180,370), Wright et al. (Critical Rev. Immunol., 1992, 12: 125-168) and references cited therein or Gu et al. Antibodies can be formed as described in (Thrombosis and Hematocyst. 1997, 77 (4): 755-759). Queen et al. In part, the method disclosed in US Pat. It is directed to design immunoglobulins adapted to the human body that are produced by expressing recombinant DNA sections encoding decision regions (CDRs). In general, the invention in the Queen patent has applicability directed to the design of immunoglobulins adapted to virtually any human body. Queen will include an expression control DNA sequence operably linked to an immunoglobulin coding sequence adapted to the human body, where the DNA section typically includes a naturally occurring or heterologous promoter region. Explains. The expression control sequence can be a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells, or the expression control sequence can transform or transfect prokaryotic host cells. Prokaryotic promoter system in a vector that can be. Once the promoter is introduced into a suitable host, the host is maintained under conditions suitable for high level expression of the introduced nucleotide sequence and, as desired, light chain, heavy chain, light / heavy adapted to the human body. Collection and purification of chain dimers or intact antibodies, binding fragments or other immunoglobulin forms can be followed (by Beyok, Cell of Immunoglobulin Synthesis, the contents of which are incorporated herein by reference). , Academic Press, New York, 1979).

  Human constant region (CDR) DNA sequences from a variety of human cells can be isolated according to well-known methods. Preferably, the human constant region DNA sequence is isolated from immortalized B-cells such as those described in International Publication No. 7/02671, the contents of which are incorporated herein by reference. CDRs useful in the production of antibodies of the invention can also be derived from DNA encoding a monoclonal antibody capable of binding to BEHAB. Such humanized antibodies can be formed using conventional methods in common mammalian sources capable of producing antibodies, including but not limited to mice, rats, rabbits or other vertebrates. Suitable cells for the constant region and framework DNA sequences, as well as host cells in which the antibody is expressed and secreted, can be obtained from multiple sources, such as the American Type Culture Collection, Manassas, VA.

  In addition to the humanized antibodies discussed above, other modifications to the original antibody sequence can be easily designed and produced using various recombinant DNA methods well known to those skilled in the art. it can. In addition, a variety of different human framework regions can be used alone or in combination as a basis for humanized antibodies directed against BEHAB. In general, gene alterations can be readily performed using a variety of well-known methods, such as site-directed mutagenesis (Gillman and Smith, Gene, 1979, 8: 81-97; Roberts et al., Nature). 1987, 328: 731-734).

  Alternatively, a phage antibody library can be generated. To make a phage antibody library, a cDNA library is first obtained from the desired protein to be expressed on the phage surface, eg, mRNA isolated from cells that express the desired antibody, eg, a hybridoma. A cDNA copy of the mRNA is made using reverse transcriptase. A cDNA defining an immunoglobulin fragment is obtained by PCR, and the resulting DNA is cloned into a suitable bacteriophage vector to create a bacteriophage DNA library containing DNA defining the immunoglobulin gene. Methods for making bacteriophage libraries containing heterologous DNA are well known in the art, see, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1989).

  Manipulate the bacteriophage encoding the desired antibody so that the protein appears on its surface in such a way that it can be used to bind to its corresponding binding protein, eg, the antigen to which the antibody is directed be able to. Thus, when a bacteriophage that expresses a particular antibody is incubated in the presence of a cell that expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophages that do not express antibodies will not bind to the cells. Such sorting techniques are well known in the art and are described, for example, in Wright et al. (Critical Rev. Immunol., 992, 12: 125-168).

  Methods such as those described above were developed for the production of human antibodies using M13 bacteriophage display (Burton et al., Adv. Immunol., 1994, 57: 191-280). ). In essence, a cDNA library is formed from mRNA obtained from a population of antibody-producing cells. mRNA encodes a translocated immunoglobulin gene, and thus the cDNA encodes the same. The amplified cDNA is cloned into an M13 expression vector to form a library of phage that express human Fab fragments on the surface. Phage that reveals the antibody in question is selected by antigen binding and grown in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to normal monoclonal antibody synthesis, this method immortalizes human immunoglobulin-encoding DNA rather than cells that express human immunoglobulin.

  The method just shown describes the formation of a phage encoding the Fab portion of the antibody molecule. However, the present invention should not be regarded as limited solely to the formation of phage encoding Fab antibodies. Rather, phage that encode single chain antibodies (scFv / phage antibody library) are also included in the present invention. Fab molecules contain the entire Ig light chain, ie they contain both the variable and constant regions of the light chain, but only the variable region of the heavy chain and the first constant region domain (CH1). Single chain antibody molecules comprise a single chain of protein containing the Ig Fv fragment. An Ig Fv fragment contains only the variable regions of the heavy and light chains of the antibody and does not have the constant regions contained therein. Marks et al. A phage library containing scFv DNA can be prepared according to the method described in the book (J. Mol. Biol. 1991, 222: 581-597). Selection of the phage so produced for isolation of the desired antibody is performed in a manner similar to that described for phage libraries containing Fab DNA.

The present invention should also be considered to include synthetic phage display libraries that can synthesize heavy and light chain variable regions so that they contain almost all possible specificities (by Barbas, Nature Medicine, 1995). Year, 1: 837-839; de Kruif et al., J. Mol. Biol. 1995, 248: 97-105).
VI. Method
A. Methods of treating primary CNS tumors The present invention is based, in part, on the novel discovery that BEHAB plays a significant role in the survival time of mammals with primary CNS tumor progression, invasiveness and brain tumors. As shown by the data disclosed herein, BEHAB cleavage empowers the progression of primary CNS tumors, inhibiting the cleavage and / or inhibiting the function of BEHAB and its cleavage products in primary CNS tumors in mammals. Can be used as a treatment for. In all cases, whether treating or diagnosing a primary CNS tumor, the most preferred mammal is a human.

  The invention includes a method of treatment of a primary CNS tumor in a mammal, preferably a human. This is because BEHAB cleavage and / or the function, biological activity and expression of BEHAB cleavage products are determined by the progression and invasiveness of the primary CNS tumor, as shown by the data disclosed elsewhere herein Because it is. Thus, as is apparent from the data presented herein, inhibition of BEHAB cleavage and / or inhibition of BEHAB cleavage product function, biological activity and expression may serve as a treatment for primary CNS tumors. it can. Those skilled in the art, based on the present disclosure, will be aware that inhibition of BEHAB cleavage may include glioma, fully differentiated astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, ventricle Ependymoma, oligodendroglioma, gangliocytoma, mixed glioma, brainstem glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, primordial neuroectoderm It will be appreciated that it provides important and novel therapeutics for the treatment of tumors, neurofibromas, vascular tumors, lymphomas, reactive gliosis and the like. Those skilled in the art will also understand that primary CNS tumors result from injury to the CNS, the brain and spinal cord. Tumors resulting from these injuries are well known in the art and are often referred to as reactive gliosis. Reactive gliosis is described in detail, for example, in Stret (Toxicol. Pathol., 2000, 28: 28-30). The present invention includes a method for the treatment of reactive gliosis in a mammal, preferably a human.

  An inhibitor of BEHAB cleavage is administered to the mammal, thereby reducing BEHAB cleavage and providing a therapeutic benefit. Those skilled in the art will appreciate that, based on the disclosure presented herein, BEHAB cleavage can be inhibited using a wide range of methods known or to be developed in the future. Thus, the present invention encompasses inhibiting BEHAB cleavage in mammals, thereby preventing primary CNS tumor progression and invasiveness. The present invention discloses methods for inhibition of BEHAB cleavage in mammals, such as blocking the cleavage site, titrating the protease responsible for cleavage, and expressing or administering an uncleavable BEHAB mutant. This, as demonstrated by the data disclosed herein, affecting BEHAB cleavage includes, but is not limited to, reduced tumor size and increased survival in mammals suffering from primary CNS tumors. Because it mediates not a wide variety of effects, thereby providing new and potent therapeutics for primary CNS tumors.

  In addition to providing the present disclosure and the data presented herein, the skilled person can also administer an inhibitor of BEHAB and / or its cleavage product function, biological activity and expression to the mammal to produce a primary CNS tumor. It will be appreciated that it provides beneficial therapeutics to mammals with The present invention includes methods for reducing or preventing the expression of BEHAB and the binding of cleavage products and / or their ligands. As shown by the data disclosed herein, increased amounts of BEHAB and increased biological activity of BEHAB cleavage products mediate increased progression of primary CNS tumors, resulting in decreased survival and greater Causes a tumor. Accordingly, methods for inhibition of BEHAB expression or BEHAB cleavage product expression and / or function are included in the present invention.

One skilled in the art will appreciate that inhibition of BEHAB cleavage includes blocking of the cleavage site, titration of the protease responsible for cleavage, and expression and / or administration of a non-cleavable BEHAB mutant. The present invention includes a method for inhibition of BEHAB cleavage by blocking the cleavage site on the protein. As disclosed herein, the cleavage site comprises the BEHAB protein Glu ^395- Ser ³⁹⁶ . Therefore, inaccessibility to this cleavage site for proteases can prevent the cleavage of BEHAB. Thus, the present invention includes a method for inhibition of BEHAB cleavage by blocking protease access to the cleavage site. For example, an antibody against a portion containing a cleavage site for a protein or a peptide or small molecule that interacts with the cleavage site will block the protease from approaching the protein, thereby inhibiting BEHAB cleavage. Those skilled in the art, with the disclosure and data disclosed herein, can specifically bind an antibody to a shorter peptide containing a cleavage site or a larger portion of a BEHAB protein, provided that the antibody binds to the cleavage site. You will see that it shuts off.

  Methods of forming antibodies to BEHAB are well known in the art (Matthews et al., J. Biol. Chem., 2000, 275: 22669-22703) and are disclosed elsewhere herein. ing. Furthermore, methods for the production of antibodies that specifically bind to an epitope of a protein are well known in the art and can be performed using standard methods disclosed herein and elsewhere. Harlow et al. (Antibodies: A Laboratory Manual, Cold Spring Harbor, NY, 1988).

  One skilled in the art will appreciate that the antibody can be administered as a protein, as a nucleic acid construct encoding the protein, or both. Numerous vectors and other compositions and methods disclosed elsewhere herein are well known for administering proteins or nucleic acid constructs encoding proteins to cells or tissues. Accordingly, the present invention includes a method for administering an antibody specific for BEHAB or a nucleic acid encoding an antibody (synthetic antibody) (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 198). Year); Ausubel et al. Author, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1997).

  Those skilled in the art will understand that, based on the disclosure presented herein, an antibody can be administered so that it blocks the cleavage site on BEHAB present in the mammal. The invention further encompasses administering an antibody or nucleic acid encoding the antibody that specifically binds to BEHAB, wherein the molecule further causes the antibody to bind to BEHAB and prevent its GPI-fixed expression or secretion. Contain intracellular retention sequences. Such antibodies, often referred to as “intrabodies” are well known in the art and are described, for example, in Marasco et al. (US Pat. No. 6,004,490) and Beerli et al. (Breast Cancer Research and Treatment, 1996, 38: 11-17). Thus, the present invention comprises a method comprising inhibiting BEHAB cleavage when BEHAB is present in a mammal, as well as inhibiting BEHAB present in its GPI-immobilized form or its secreted form on its cell membrane. Methods of inhibition of BEHAB cleavage and such methods known in the future are included.

  The present invention also encompasses a method for inhibiting BEHAB cleavage by inhibiting the protease responsible for BEHAB cleavage. This is because, as is evident from the data presented herein, BEHAB is cleaved by proteases at certain sites, but inhibition of BEHAB cleavage results in smaller tumor volumes and improved animal survival, among other things. is there. Accordingly, the present invention includes a method of inhibiting BEHAB cleavage by inhibiting a protease that cleaves BEHAB.

  Those skilled in the art will appreciate that inhibition of protease includes administering an effective amount of a protease inhibitor to the mammal. Such inhibitors include, but are not limited to, metalloproteinase tissue inhibitor 2, metalloproteinase tissue inhibitor 3, small molecules, and chemical compounds including antibodies that specifically bind to proteases that cleave BEHAB. I can't. Specific protease inhibitors are well known in the art and are described in, for example, Martel-Pelletier et al. (Best Pract. Res. Clin. Rheumatol., 2001, 15: 805-29). Those skilled in the art, in light of the present disclosure and the description herein, will readily understand how to administer a protease inhibitor to a mammal, and thus the present invention provides protease inhibition as a treatment for primary CNS tumors. Includes agents.

  The invention also encompasses a method for inhibition of BEHAB cleavage by titration of a protease responsible for BEHAB cleavage. This is because, as is apparent from the data presented herein, BEHAB is cleaved by proteases at certain sites, while mutant BEHABs of the invention are measured in both in vivo and in vitro assays, This is because it cannot be cleaved by a protease. Furthermore, proteases that cleave BEHAB are present in the body in limited amounts and in limited locations compared to other metalloproteinases. Thus, non-cleavable BEHAB can titrate the protease and therefore it cannot be used to cleave endogenous BEHAB. Those skilled in the art will know that titration of proteases includes providing substances that reduce the functional concentration of proteases available for cleavage of BEHAB in mammals, preferably humans. Let's go. Protease titration further includes providing a substance that the protease recognizes and binds, and reducing the number of proteases or protease active sites available for cleavage of BEHAB.

  As described more fully elsewhere herein, tumors expressing mutants, non-cleavable forms of BEHAB, can be found in the presence of endogenous BEHAB, especially in smaller tumor volumes and survival in animals. The improvement is produced. These data indicate that even if endogenous BEHAB is present in the cells, the presence of non-cleavable BEHAB is the primary CNS tumor model in an in vivo primary CNS tumor model accepted in the art. It shows that the progress is reduced. The data further show that when tumors expressing exogenous mutant BEHAB are compared to tumors expressing exogenous normal BEHAB, tumors expressing mutant BEHAB produce smaller and longer animal survival times. Yes. While not wishing to be bound by any particular theory, the data presented herein show that non-cleavable BEHAB mutants titrate the protease responsible for BEHAB cleavage and tumor progression as a result of reduced cleavage This indicates that a decrease in

  Based on the present disclosure and the data disclosed herein, the skilled person is able to suppress the progression of tumors by reducing the size of the tumor with a non-cleavable substrate for proteases and improve survival in animals suffering from primary CNS tumors You will understand that Accordingly, the present invention includes a method for the treatment of primary CNS tumors by titration of a protease that cleaves BEHAB.

  Compounds used for titration of proteases that cleave BEHAB include, but are not limited to, peptides, proteins, mimetopes and peptide mimetics. As disclosed elsewhere herein, non-cleavable BEHAB (mutant BEHAB, SEQ ID NO: 3) is a mutation in the amino acid sequence around the cleavage site, specifically Glu-Ser-Glu- Contains the native BEHAB protein with a mutation of Ser-Arg-Gly to Glu-Ser-Glu-Asn-Val-Tyr (SEQ ID NO: 1 and SEQ ID NO: 2, respectively). Those skilled in the art will readily appreciate that peptides derived from full-length mutant BEHAB can exhibit the same protease titration as the full-length mutant BEHAB protein shown in SEQ ID NO: 3. Thereby, the present invention encompasses full-length mutant BEHAB proteins and truncated mutant BEHAB peptides with protease titration activity.

As used herein, amino acids are indicated by their full name, their corresponding three letter code, or their corresponding one letter code, as shown in the table below:
Full name 3 letter code 1 letter code Aspartic acid Asp D
Glutamate Glu E
Lysine Lys K
Arginine Arg R
Histidine His H
Tyrosine Tyr Y
Cysteine Cys C
Asparagine Asn N
Glutamine Gln Q
Serine Ser S
Threonine Thr T
Glycine Gly G
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine Ile I
Methionine Met M
Proline Pro P
Phenylalanine Phe F
Tryptophan Trp W
The peptides of the present invention are described in Stewart et al. (Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Illinois, 1984, 2nd Edition), and Bodanzky and Pandsky (The Pde It can be readily prepared by the standard well established solid phase peptide synthesis (SPPS) described. First, a suitably protected amino acid residue is coupled via its carboxyl group to a derivatized insoluble polymeric support, such as a cross-linked polystyrene or polyamide resin. “Suitably protected” refers to the presence of a protecting group on both the α-amino group and the side chain functional group of an amino acid. Side chain protecting groups are generally stable to solvents, reagents and reaction conditions used throughout the synthesis and can be removed under conditions that do not affect the final peptide product. Stepwise synthesis of oligopeptides is performed by removal of the N-protecting group from the first amino acid and coupling to the carboxyl terminus of the next amino acid in the desired peptide sequence. This amino acid is also properly protected. Activate and support the carboxyl of the incoming amino acid by formation of a reactive group, for example, the formation of an “active ester” group such as carbodiimide, symmetrical anhydride or hydroxybenzotriazole or pentafluorophenyl ester It can be reacted with the N-terminus of the amino acid bound to the body.

  Examples of solid phase peptide synthesis methods include the BOC method using tert-butyloxycarbonyl as the α-amino protecting group and the FMOC method using 9-fluorenylmethyloxycarbonyl for protecting the α-amino of amino acid residues Both of these methods are well known by those skilled in the art.

  The introduction of N- and / or C-blocking groups can also be carried out using the usual scheme for solid phase peptide synthesis. For example, for the introduction of a C-terminal blocking group, synthesis of the desired peptide is typically a chemically modified support resin such that cleavage from the resin yields a peptide having the desired C-terminal blocking group. Is used as a solid phase. For example, in order to obtain a peptide having a primary amino blocking group at the C-terminus, synthesis is performed using p-methylbenzhydrylamine (MBHA) resin, and treatment with hydrofluoric acid is desired when peptide synthesis is completed. The C-terminally amidated peptide is released. Similarly, the introduction of an N-methylamine blocking group at the C-terminus is performed using N-methylaminoethyl-derivatized DVB resin, which is treated with HF (hydrofluoric acid) to produce N-methylamidated C- Releases the peptide having a terminal end. Blocking the C-terminus by esterification can also be performed using conventional methods. This involves the use of a resin / blocking group combination that allows release of the side chain peptide from the resin and allows subsequent reaction with the desired alcohol for the generation of the ester functionality. FMOC protecting groups in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker can be used for this purpose, and cleavage from the support is accomplished by TFA in dichloromethane. The esterification of the appropriately activated carboxyl function, for example using DCC, can then be performed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.

  While the peptide to be synthesized is still bound to the resin, an N-terminal blocking group can be introduced, for example by treatment with a suitable anhydride and nitrile. For example, for the introduction of an acetyl blocking group at the N-terminus, the peptide coupled to the resin can be treated with 20% acetic anhydride in acetonitrile. The N-blocking peptide product can then be cleaved from the resin, deprotected and subsequently isolated.

  To ensure that the peptide obtained from chemical or biological synthesis is the desired peptide, an analysis of the peptide composition must be performed. Such amino acid composition analysis can be performed by determining the molecular weight of the peptide using high resolution mass spectrometry. Alternatively or in addition, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, separating the components of the mixture, identifying and quantifying using HPLC or an amino acid analyzer. . It is also possible to unambiguously determine the sequence of a peptide using a protein sequencer that sequentially degrades the peptide and identifies the amino acids in sequence.

Prior to its use, the peptide is purified to remove contaminants. In this regard, it will be appreciated that the peptides will be purified to meet standards set by appropriate regulatory agencies or for specific uses. In order to obtain the necessary level of purity, for example, C ₄ -, C 8 _- to more conventional purification methods, including reverse phase high pressure liquid chromatography (HPLC) using an alkylated silica column such as silica _- or C ₁₈ Any one can be used. To achieve purification, a gradient mobile phase with increasing organic content, for example acetonitrile in an aqueous buffer usually containing a small amount of trifluoroacetic acid, is generally used. Ion exchange chromatography can also be used to separate peptides based on their charge.

  Those skilled in the art will readily be able to administer a mutant BEHAB protein or peptide capable of titrating a protease that cleaves BEHAB to a mammal as an isolated nucleic acid encoding the mutant BEHAB protein or peptide. Will be. Methods for expressing a desired protein in a cell or mammal are well known in the art, and when combined with the present disclosure and the data herein, skilled artisans can mutate BEHAB protein or in a cell or mammal without undue experimentation. The peptide could be expressed.

  Those skilled in the art can introduce a vector or expression vector containing an isolated nucleic acid encoding the desired protein or peptide into the cell or mammal for expression of the protein or peptide in the cell or mammal. It will be appreciated that there are many ways to include. One skilled in the art will further appreciate that the vector can comprise an isolated nucleic acid of SEQ ID NO: 4 or some biologically active portion thereof.

  The invention also includes mimetopes of mutant BEHAB proteins and peptides of the invention. As used herein, a mimetope of a mutant BEHAB protein or peptide has the ability to titrate the activity of such mutant BEHAB protein or peptide (eg, a protease that cleaves BEHAB, thereby preventing native BEHAB cleavage). ), Often because mimetope has a structure that mimics a mutated BEHAB protein or peptide. However, it should be noted that as long as the mimetope functionally mimics a protein, the mimetope need not have a structure similar to the mutant BEHAB protein or peptide. Mimetopes are: peptides modified to reduce their ease of degradation; anti-idiotype and / or catalytic antibodies or fragments thereof; non-proteinaceous immunogenic portions of isolated proteins (eg, carbohydrate structures) Can be, but is not limited to, synthetic or natural organic or inorganic molecules including nucleic acids; and / or other peptidomimetic compounds. The mimetop of the present invention can be designed using the structure of the mutant BEHAB protein or peptide of the present invention as drawn by a computer. Affinity chromatography methods where random samples of molecules such as oligonucleotides, peptides or other organic molecules are made and such samples are used with corresponding binding partners (eg proteases or anti-BEHAB antibodies that cleave BEHAB) Mimetope can also be obtained by screening according to the above. Preferred mimetopes are peptidomimetic compounds that are structurally and / or functionally similar to the mutant BEHAB protein or peptide of the invention, in particular to the cleavage site of the mutant BEHAB protein. Methods for the formation of mimetopes and peptidomimetics are well known in the art and are described, for example, by Kazmierski (Peptidometrics Protocols (Method in Molecular Medicine Vol. 23), Humana Press, Toda 99 details). ing.

  The invention also includes a method for inhibiting the expression and / or activity of BEHAB in a mammal. Those skilled in the art will understand that with the present disclosure and the data disclosed herein, higher levels of BEHAB expression increase tumor size and reduce survival in mammals suffering from primary CNS tumors. I will. That is, the data presented elsewhere herein show that when a mammal with a primary CNS tumor that overexpresses BEHAB is compared to a mammal that expresses a normal amount of BEHAB or a mammal that expresses mutant BEHAB, It shows for the first time that it has a larger tumor volume and a shorter survival time. Thus, one skilled in the art will certainly know that the method of treatment of the primary CNS tumor involves inhibition of BEHAB expression.

  Inhibitors of BEHAB expression and / or activity are administered to the mammal, thereby reducing BEHAB and providing a therapeutic benefit. Those skilled in the art will appreciate that based on the disclosure presented herein, BEHAB can be inhibited using a variety of methods well known in the art or to be developed in the future. That is, the present invention encompasses inhibition of BEHAB expression, such as inhibition of transcription and / or translation. This is illustrated by the data disclosed elsewhere herein, including a variety of but not limited to decreased levels of BEHAB expression and / or activity including decreased tumor size and increased survival. This is because the effect was mediated. Thus, inhibition of BEHAB includes, but is not limited to, inhibition of translation and / or transcription of nucleic acids encoding proteins.

  Further, the routineer will understand based on the disclosure presented elsewhere herein that inhibition of BEHAB includes, but is not limited to, inhibition of the biological activity of the molecule. . This is because inhibition of BEHAB activity limits the progression of primary CNS tumors in that BEHAB is not cleaved by endogenous proteases, as the data disclosed elsewhere in this specification shows. These data indicate that inhibition of BEHAB activity provides a therapeutic benefit for the treatment of diseases such as but not limited to primary CNS tumors.

  The present invention encompasses the inhibition of BEHAB by inhibiting the expression of a nucleic acid encoding BEHAB. Methods for inhibition of gene expression are well known to those of ordinary skill in the art and include the use of ribozymes or antisense nucleic acid molecules.

  Antisense nucleic acid molecules are DNA or RNA molecules that are complementary to some portion of the mRNA molecule. When present in a cell, an antisense nucleic acid hybridizes to an existing mRNA molecule and inhibits translation into a gene product. Inhibition of gene expression using antisense nucleic acid molecules is well known in the art (Marcus-Sekura, Anal. Biochem. 1988, 172: 289), and methods for expressing antisense nucleic acid molecules in cells are also so. (Inoue, US Pat. No. 5,190,931, 1993).

  The present invention encompasses the inhibition of BEHAB expression using ribozymes. The use of ribozymes to inhibit gene expression is well known to those of ordinary skill in the art (Cech et al., J. Biol. Chem., 1992, 267: 17479-17482; Hampel et al. Biochemistry, 1989, 28: 4929-4933; Altman et al., US Pat. No. 5,168,053, 1992). Ribozymes are catalytic RNA molecules that have the ability to cleave other single-stranded RNA molecules. Ribozymes are known to be sequence specific and are therefore modified to recognize specific nucleotide sequences (Cech, J. Amer. Med. Assn., 1988, 260: 3030-3034) It can allow selective cleavage of mRNA molecules. With the knowledge that the nucleotide sequence of BEHAB is well known in the art (Hockfield et al., US Pat. No. 5,635,370, 1997), one of ordinary skill in the art has disclosed and An antisense polynucleotide or ribozyme can be synthesized without undue experimentation with reference to the subject matter herein.

  The skilled person will further appreciate that the cleavage of BEHAB mediates the progression of primary CNS tumors, with the disclosure and the data presented herein. While not wishing to be bound by any particular theory, BEHAB is normally expressed endogenously in low amounts, and does not necessarily cause a primary CNS tumor during normal expression, but it may also result in cleavage of BEHAB or even In particular, the product of a cleavage event can be theorized to mediate primary CNS tumor progression in mammals. Thereby, as will be appreciated by those skilled in the art, inhibition of BEHAB cleavage product activity can be used as a method of treatment for mammals suffering from primary CNS tumors.

  The invention also encompasses the use of a suitable antibody, protein or peptide, mimetope, peptidomimetic and / or isolated nucleic acid pharmaceutical composition for carrying out the methods of the invention, the composition being suitable Antibody, protein or peptide, mimetope, peptidomimetic and / or isolated nucleic acid and a pharmaceutically acceptable carrier.

  As used herein, the term “pharmaceutically acceptable carrier” uses it to combine a suitable antibody, protein or peptide, mimetope, peptidomimetic and / or isolated nucleic acid. Means a chemical composition that can be used and, after being combined, can be used to administer a suitable antibody, protein or peptide, mimetope, peptidomimetic and / or isolated nucleic acid to a mammal.

  Pharmaceutical compositions useful for the practice of the present invention can be administered to deliver dosages of 1 ng / kg / day to 100 mg / kg / day. In one aspect, the invention contemplates administration of a dosage that results in a concentration of a compound of the invention of 1 μM to 10 μM in a mammal.

  Pharmaceutical compositions useful in the methods of the invention can be administered systemically in oral solid preparations, eye drops, suppositories, aerosols, topical or other similar preparations. They can be administered directly into the CNS, into the envelope, into the ventricle, into the parenchyma, via direct injection, or via a bioengineered polymer. In addition to suitable antibodies, proteins or peptides, mimetopes, peptidomimetics and / or isolated nucleic acids, such pharmaceutical compositions enhance and facilitate pharmaceutically acceptable carriers and drug administration. Other ingredients known to do can be included. Other possible formulations, such as nanoparticles, liposomes, reclosed red blood cells and immunologically based systems can also be used to administer suitable hypericin derivatives according to the method of the invention.

  Compounds identified using any of the methods described herein can be prepared and administered to a mammal for the treatment of the diseases disclosed herein.

  The present invention encompasses the preparation and use of pharmaceutical compositions comprising as an active ingredient a compound useful for the treatment of the diseases disclosed herein. Such a pharmaceutical composition may consist solely of the active ingredient in a form suitable for administration to a patient, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable substances. A carrier that can be included, one or more additional components, or some combination thereof. As is well known in the art, the active ingredient can be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, for example in combination with a physiologically acceptable cation or anion. .

  As used herein, the term “pharmaceutically acceptable carrier” is used to combine an active ingredient with it and is used to administer the active ingredient to a patient after it has been combined. A chemical composition that can be made.

  As used herein, the term “physiologically acceptable” ester or salt is compatible with any other component of the pharmaceutical composition and is detrimental to the patient to whom the composition is to be administered. Means an ester or salt form of the active ingredient that is not.

  Formulations of the pharmaceutical compositions described herein can be prepared by any method known or later developed in the pharmacological arts. In general, such methods of preparation involve combining the active ingredient with a carrier or one or more other accessory ingredients, and then shaping the product into the desired single- or multi-dosage units, if necessary or desirable. Including packaging.

  While the descriptions of pharmaceutical compositions presented herein are primarily directed to pharmaceutical compositions suitable for medical administration to humans, those skilled in the art It will be appreciated that it is suitable for administration to all types of mammals. Modifications of the pharmaceutical composition suitable for human administration to make the composition suitable for administration to a variety of animals are well understood and the ordinary skilled veterinary scientist will do Even so, it would be possible to design and make such changes using only routine experimentation. Patients intended for administration of the pharmaceutical compositions of the invention include humans and other primates, commercially relevant mammals such as cattle, pigs, horses, sheep, cats and dogs, mice It is intended to include, but is not limited to, birds, including commercially suitable birds such as mammals, including birds (including rats and mice), chickens, ducks, butterflies and turkeys.

  Pharmaceutical compositions useful in the methods of the present invention are oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular, intrathecal, intraventricular, parenchymal. It can be prepared, packaged, or sold in a formulation suitable for internal or other routes of administration. Other formulations contemplated include ejected nanoparticles, liposomal formulations, reclosed red blood cells containing active ingredients, and immunologically based systems.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in bulk, as a single unit dosage or as multiple single unit dosages. As used herein, a “unit dosage” is an isolated amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dosage of active ingredient that will be administered to a patient or a convenient percentage of such dosage, for example one half or one third of such dosage.

  The relative amounts of the active ingredient, pharmaceutically acceptable carrier and additional ingredients in the pharmaceutical composition of the present invention are determined by the identity, size and condition of the patient being treated, as well as the composition being administered. It will vary depending on the power path. For example, the composition can contain 0.1% to 100% (w / w) of the active ingredient.

  In addition to the active ingredient, the pharmaceutical composition of the present invention may further comprise one or more additional pharmaceutically active agents. Additional agents specifically contemplated include antiemetics and scavengers such as cyanide and cyanate scavengers.

  Conventional methods can be used to make controlled-release or sustained release formulations of the pharmaceutical compositions of the invention.

  Formulations of the pharmaceutical composition of the present invention suitable for oral administration include, but are not limited to, tablets, hard or soft capsules, sachets, troches or lozenges each containing a predetermined amount of active ingredient Prepared in a solid dosage unit form, packaged or sold. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions or emulsions.

  As used herein, an “oily” liquid is one that contains carbon-containing liquid molecules and exhibits a lower polarity than water.

  A tablet containing the active ingredient may be prepared, for example, by compressing or molding the active ingredient, optionally with one or more additional ingredients. An active ingredient in a flowable form, such as a powder or granule preparation, optionally mixed with one or more binders, lubricants, excipients, surfactants and dispersants, Compressed tablets can be made by compression in suitable equipment. Molded tablets can be made by molding in a suitable apparatus a mixture of the active ingredient, a pharmaceutically acceptable carrier and at least a liquid sufficient to wet the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pre-gelatinized corn starch, polyvinyl pyrrolidone and hydroxypropyl methylcellulose. Known lubricants include but are not limited to magnesium stearate, stearic acid and talc.

  The tablets can be uncoated or they can be coated using known methods to delay disintegration in the patient's gastrointestinal tract and thereby provide sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate can be used for tablet coating. Further, the tablets are coated and controlled osmotically using, for example, the methods described in US Pat. Nos. 4,256,108; 4,160,452; and 4,265,874. Release tablets can be formed. The tablets may further comprise sweetening agents, flavoring agents, coloring agents, preservatives or some combination thereof in order to obtain a pharmaceutically pleasing and palatable preparation.

  Physiologically degradable compositions such as gelatin can be used to make hard capsules containing the active ingredient. Such hard capsules contain the active ingredient and can further contain additional ingredients including an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin.

  Physiologically degradable compositions such as gelatin can be used to make soft gelatin capsules containing the active ingredient. Such soft capsules contain the active ingredient, which can be mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

  A liquid formulation of the pharmaceutical composition of the present invention suitable for oral administration is prepared in liquid form or in the form of a dry product intended to be reconstituted with water or other suitable vehicle prior to use. Can be packaged or sold.

  Liquid suspensions can be prepared using conventional methods for suspending the active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin. Liquid suspensions further include, but are not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffering agents, salts, flavoring agents, coloring agents and sweetening agents. Species or more additional components can be included. The oily suspension may further contain a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fat, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include naturally occurring phosphatides such as lecithin, alkylene oxides and fatty acids, long chain fatty alcohols, fatty acids and partial esters derived from hexitol, or fatty acids and hexitols. Condensation products with partial esters derived from anhydrides (for example, polyoxyethylene stearate, heptadecaethyleneoxycatechol, polyoxyethylene sorbitol monooleate and polyoxyethylene sorbitan monooleate, respectively). Absent. Known emulsifiers include but are not limited to lecithin and gum arabic. Known preservatives include, but are not limited to, methyl, ethyl or n-propyl-para-hydroxybenzoate, ascorbic acid and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose and saccharin. Known thickening agents for oily suspensions include, for example, biswax, hard paraffin and cetyl alcohol.

  Solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same way as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. . Solutions of the pharmaceutical composition of the present invention can contain each of the ingredients described for the liquid suspension, and the suspending agent will not necessarily help dissolve the active ingredient in the solvent. Will be understood. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin.

  Known methods can be used to prepare powdered and granular formulations of the pharmaceutical preparations of the present invention. Such a formulation can be administered directly to a patient, for example for the formation of tablets, for filling capsules, or for preparing aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. Can be used. Each of these preparations can further contain one or more dispersing or wetting agents, suspending agents and preservatives. Additional excipients, such as fillers and sweetening, flavoring or coloring agents, can also be included in these formulations.

  The pharmaceutical composition of the present invention can also be prepared, packaged or sold in the form of an oil-in-water emulsion or a water-in-oil emulsion. The oily phase can be a vegetable oil, for example olive or arachis oil, a mineral oil, for example liquid paraffin or combinations of these. Such compositions further comprise one or more emulsifiers such as naturally occurring gums such as gum arabic or tragacanth, naturally occurring phosphatides such as soy or lecithin phosphatides, a combination of fatty acid and hexitol anhydride. Esters or partial esters derived from, for example, sorbitan monooleate and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions can also contain additional ingredients including, for example, sweetening or flavoring agents.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in a formulation suitable for rectal administration. Such compositions can be in the form of, for example, suppositories, retention enema preparations, and solutions for rectal or colonic irrigation.

  Non-irritating pharmaceutically acceptable excipients and active ingredients that are solid at normal room temperature (ie about 20 ° C.) and liquid at the patient's rectal temperature (ie about 37 ° C. in healthy humans) Can be used to make suppositories. Suitable pharmaceutically acceptable excipients include but are not limited to cocoa butter, polyethylene glycol and various glycerides. Suppository formulations can further include a variety of additional ingredients including, but not limited to, antioxidants and preservatives.

  The active ingredient can be combined with a pharmaceutically acceptable liquid carrier to form a retention enema preparation or solution for rectal or colonic irrigation. As is well known in the art, enema preparations can be administered and packaged therein using delivery devices adapted to the patient's rectal anatomy. Enema preparations can further include various additional ingredients including, but not limited to, antioxidants and preservatives.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in a formulation suitable for vaginal administration. Such compositions can be in the form of, for example, suppositories, impregnated or coated vaginal insertable materials such as tampons, irrigation preparations or gels or creams or solutions for vaginal lavage.

  Methods for impregnating or coating a material with a chemical composition are known in the art, such as depositing or bonding a chemical composition on a surface, during the synthesis of the material, into the structure of the material A method of introducing a chemical composition (ie as using a physiologically degradable material) and a method of absorbing an aqueous or oily solution or suspension into an absorbent material followed by drying or non-drying Including, but not limited to.

  Irrigation preparations or solutions for vaginal irrigation can be prepared by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, irrigation preparations can be administered and packaged therein using delivery devices adapted to the anatomy of the patient's vagina. The irrigation preparation can further include various additional ingredients including, but not limited to, antioxidants, antibiotics, antibacterials and preservatives.

  As used herein, “parenteral administration” of a pharmaceutical composition refers to the route of administration of a pharmaceutical composition characterized by physical infringement of a patient's tissue and wounds in the tissue. Including. Thus parenteral administration includes administration of the pharmaceutical composition by injection of the composition, application of the composition via a surgical incision, application of the composition via a tissue-permeable non-surgical wound, etc. Is not limited to these. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

  Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable preparations can be prepared, packaged or sold in unit dosage form, for example, in ampoules or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions in oily or aqueous vehicles, solutions, emulsions, pastes, and implantable sustained or biodegradable formulations. Such formulations can further comprise one or more additional ingredients including but not limited to suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, for parenteral administration of the reconstituted composition using a suitable vehicle (eg, sterile pyrogen-free water) The active ingredient is provided in a dry (ie powder or granule) form.

  The pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oleaginous suspension or solution. This suspension or solution may be prepared according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing, wetting or suspending agents described herein. Such sterile injectable preparations can be prepared using a non-toxic parenterally acceptable diluent or solvent, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation can include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, slightly soluble polymers or slightly soluble salts.

  Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or coatings and solutions or suspensions. It is not limited to these. Topically administrable formulations can contain, for example, from about 1% to about 10% (w / w) of the active ingredient, although the concentration of the active ingredient is at the solubility limit of the active ingredient in the solvent. Can be so expensive. Formulations for topical administration can further include one or more additional ingredients as described herein.

  The pharmaceutical composition of the present invention can be prepared, packaged or sold in a formulation suitable for pulmonary administration via the oral vestibule. Such formulations can comprise dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers, preferably about 1 to about 6 nanometers. Such compositions conveniently use a device containing a dry powder reservoir that can direct the flow of propellant to disperse the powder, or dissolve or suspend in low-boiling propellant in a closed vessel. It is in the form of a dry powder for administration using a self-propelling solvent / powder-dispensing container such as a device containing a turbid active ingredient. Preferably such powders include particles such that at least 98% by weight of the particles have a diameter greater than 0.5 nanometers and at least 95% by number of particles have a diameter less than 7 nanometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of particles have a diameter less than 6 nanometers. The dry powder composition preferably includes a solid fine powder diluent such as sugar and is conveniently provided in unit dosage form.

Low boiling propellants generally include liquid propellants having a boiling point of less than 65 ^° F. at atmospheric pressure. In general, the propellant can constitute 50 to 99.9% (w / w) of the composition, and the active ingredient can constitute 0.1 to 20% (w / w) of the composition. The propellant can further include additional components such as liquid non-ionic or solid anionic surfactants or solid diluents, preferably having the same size as the particles containing the active ingredients.

  The pharmaceutical compositions of the invention prepared for pulmonary delivery can also provide the active ingredient in the form of a solution, suspension or drop of delayed-release polymer. Such preparations can be prepared, packaged or sold as aqueous or dilute alcoholic solutions or suspensions containing the active ingredients and optionally sterile, conveniently using nebulization therapy or nebulization equipment. Can be administered. Such formulations further include one or more additional ingredients including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants or preservatives such as methyl hydroxybenzoate. Can be included. The drops provided by this route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

  The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the invention.

  Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such preparations are administered in the manner in which nasal breathing is used, i.e. by rapid inhalation through the passage of the nose from a container of powder held close to the nares.

  Other formulations are active ingredients that are incorporated into the delayed-release polymer. Such polymers are well known in the pharmaceutical art and are described in detail, for example, in US Pat. Nos. (4,728,512; 4,728,513; 5,084,287; 5,285,186). Has been.

  Formulations suitable for nasal administration can contain small amounts of active ingredient such as, for example, roughly 0.1% (w / w) to as much as 100% (w / w), and are further described herein. One or more of the additional components described can be included.

  The pharmaceutical composition of the present invention can be prepared, packaged or sold in a formulation suitable for buccal administration. Such preparations can be in the form of tablets or lozenges made using conventional methods, for example, containing 0.1-20% (w / w) of the active ingredient, the rest being by mouth Contains a dissolvable or degradable composition and optionally one or more additional ingredients as described herein. Alternatively, formulations suitable for buccal administration can include powders containing the active ingredients or solutions or suspensions which are aerosolized or sprayed. Such powdered, aerosolized or aerosolized formulations, when dispersed, preferably have an average particle size or droplet size in the range of about 0.1 to about 200 nanometers, One or more additional ingredients described in the document may be included.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in a formulation suitable for ocular administration. Such preparations may be in the form of, for example, eye drops including, for example, a 0.1-1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. it can. Such eye drops may further comprise buffering agents, salts or one or more other additional ingredients as described herein. Other useful ophthalmically administrable formulations include those containing the active ingredient in microcrystalline form or in a liposomal preparation.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in a formulation suitable for direct CNS administration. Such preparations are administered, for example, by an Ommaya reservoir, by intracapsular or intraventricular administration, by direct intraparenchymal injection, by delayed-release polymers or by other such methods well known in the pharmaceutical and neurological arts. Can be in the form of a liquid.

  As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granules Binding agents; lubricants; sweeteners; flavors; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oil vehicles and solvents; A dispersing or wetting agent; an emulsifier; a demulcent; a buffer; a salt; a thickener; a filler; an emulsifier; an antioxidant; an antibiotic; an antibacterial agent; a stabilizer; and a pharmaceutically acceptable polymer or Hydrophobic material. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art, for example, Genaro, ed,. Remington's Pharmaceutical Sciences, Mack Publishing Co. , Easton PA, 1985, which is incorporated herein by reference.

  Typically, dosages of the compounds of the invention that can be administered to a mammal, preferably a human, range from 1 μg to about 100 g per kilogram of animal body weight. The exact dosage to be administered will vary depending on a number of factors including, but not limited to, the type of animal being treated and the type of disease state, the age of the animal and the route of administration. Preferably, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of animal body weight. More preferably, the dosage will vary from about 10 mg to about 1 g per kilogram of animal body weight.

  The compound can be administered to the mammal as frequently as several times daily, or less frequently, for example once a day, once a week, once every two weeks, once a month, or even more Less frequently, it can be administered, for example, once every few months or once a year or less. The frequency of dosing will be readily apparent to the skilled person and will depend on several factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

B. The present invention further includes glioma, fully differentiated astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, ventricular ependymoma, oligodendroma, neuron Ganglion cell, mixed glioma, brain stem glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, primitive neuroectodermal tumor, neurofibroma, vascular tumor, Includes methods for diagnosis of primary CNS tumors associated with BEHAB, other central nervous system tumors and other neuropathological disorders, including but not limited to lymphomas and the like. This is because BEHAB overexpression is highly associated with progression and invasiveness, such as primary CNS tumors, as shown by the data disclosed elsewhere herein. Accordingly, the present invention includes a method for determining the level of BEHAB expression in a mammal as well as a method for diagnosis of a primary CNS tumor. Whether treating or diagnosing primary CNS tumors, the most preferred mammal in all cases referred to herein is a human.

  The invention includes a method of diagnosing a primary CNS tumor in a mammal. The method obtains a biological sample from a first mammal and assigns the BEHAB level (expression, amount, activity) in the sample to a primary CNS tumor that is otherwise identical to the first mammal. Comparing to the level of BEHAB in a sample obtained from a normal second mammal not suffering. The level of BEHAB in the sample from the first mammal is higher compared to the level of BEHAB in the sample obtained from the second mammal not suffering from the primary CNS tumor that is otherwise identical. Of mammals suffer from primary CNS tumors. This is because, as disclosed elsewhere herein, increased levels of BEHAB expression are associated with greater tumor volume and decreased survival among others.

  The invention further includes a method of diagnosing primary CNS tumor progression in a mammal. As will be appreciated by those skilled in the art with the present disclosure and the data herein, BEHAB cleavage mediates brain tumor progression, among others resulting in greater tumor volume and reduced survival time. Accordingly, the present invention includes a method of diagnosing brain tumor progression in a mammal. The method obtains a biological sample from a first mammal and is otherwise suffering from a primary CNS tumor in which the level of BEHAB cleavage in the sample is otherwise identical to the first mammal, or A normal second suffering from a primary CNS tumor that is not progressing as fast as the primary CNS tumor in the second mammal, as can be readily determined by those skilled in the art using standard neurological indicators. Comparing to the level of BEHAB cleavage in a sample obtained from the mammal. A higher level of BEHAB cleavage in a sample from a first mammal compared to a level of BEHAB cleavage in a sample obtained from a second mammal that is otherwise identical is a faster rate for the first mammal. It is an indicator of suffering from a primary CNS tumor that progresses in This is because, as disclosed elsewhere herein, increased levels of BEHAB cleavage are associated with, among other things, greater tumor volume and decreased survival.

  With the present disclosure and the data herein, those skilled in the art will know how to determine the level of BEHAB cleavage, including Western blotting, ELISA, and other immunology-detection assays well known in the art. You will find that is not limited to these.

  In one aspect, the biological sample is selected from the group consisting of a blood sample, a neurological tissue biopsy, a cerebrospinal fluid sample, urine, saliva, and the like.

  The invention includes a method of assessing the efficacy of treatment for a primary CNS tumor in a mammal. The method is mediated by an increase in BEHAB expression, or before, during and after the course of a particular treatment for a disease, disorder or condition associated therewith (such as a primary CNS tumor), and the amount of BEHAB expression, And / or assessing the level of activity. This is because, as described previously elsewhere herein, an increase in BEHAB expression, amount and / or activity is associated with or mediates greater tumor volume and decreased animal survival. Yes, it is a feature of improved mortality due to primary CNS tumors.

  Thus, assessing the effect of the course of treatment on BEHAB expression / amount / activity indicates the effectiveness of the treatment, and a lower level of BEHAB expression, amount or activity indicates that the treatment is successful.

  The course of treatment to be evaluated can include, but is not limited to, surgery, chemotherapy, radiation therapy, and / or multiple treatment modalities for the primary CNS tumor disclosed herein.

  The present invention includes probes and primers for the detection of BEHAB gene expression, amount or activity. Having provided the disclosure and data disclosed herein, one skilled in the art will appreciate that a probe is provided that can specifically hybridize to DNA or RNA of the BEHAB gene. For purposes of the present invention, if the probe hybridizes to the BEHAB gene under conditions of high or moderate stringency stringency, but does not significantly or detectably hybridize to an unrelated gene, they are “Can hybridize” to DNA or RNA, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1989). Preferably, the probe is subjected to high stringency conditions, eg, 5X SSPE at 65 ° C, 1X Denhardt solution, hybridization in 0.1% SDS and 0.2X SSC, 1X Denhardt solution at 65 ° C, 0.1% Hybridize to a suitable nucleotide sequence with at least one wash for removal of unhybridized probe in the presence of SDS. Except as otherwise stated herein, the probe sequence is designed to allow hybridization to the BEHAB gene but not to DNA or RNA sequences from other genes. Probes are used to hybridize to nucleic acids present in biological samples including, but not limited to, blood, cerebrospinal fluid, lymph or tissue isolated from a patient, for example. The hybridized probe is then detected, thereby indicating the presence of the desired cellular nucleic acid. One skilled in the art will appreciate that cellular nucleic acids can be subjected to amplification methods such as polymerase chain reaction (PCR) prior to hybridization. Alternatively, the BEHAB gene can be amplified and the amplified product can be subjected to DNA sequencing. The BEHAB gene can be detected by DNA sequence analysis or hybridized with a BEHAB-specific oligonucleotide probe under sufficient conditions and for a sufficient time to allow hybridization to a specific allele Can do. Typically, the hybridization buffer can contain tetramethylammonium chloride and the like, see Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1989).

  The nucleic acid probes of the present invention can consist of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogs (eg, peptide nucleic acids) or combinations thereof, and can be as short as about 12 nucleotides, Usually it is about 14-18 nucleotides in length and can be as large as the entire sequence of the BEHAB gene. The choice of probe size depends somewhat on the probe application and is well within the skill of the art.

Suitable probes can be constructed and labeled using methods well known in the art. For example it is possible to relatively short probe of 12 bases is formed by combining, labeled with ^{32 P} using of T ₄ polynucleotide kinase. Relatively long probes from about 75 bases to less than 1.5 kb are preferably in the presence of a labeled precursor such as, but not limited to, [α ³² P] dCTP, digoxigenin-dUTP or biotin-dATP. Formed by PCR amplification. Probes larger than 1.5 kb are generally most easily obtained by transfecting cells with a plasmid containing the relevant probe, growing the transfected cells in large quantities, and purifying the relevant sequences from the transfected cells. In Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1989).

Probes can be labeled with a variety of markers including, for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers. The use of ³² P is just one example for the marking or labeling of specific probes.

  The ability to detect the presence of BEHAB mRNA or DNA in a sample using a probe is a feature of this aspect of the invention. However, if there are only a limited number of relevant samples, it may be beneficial to amplify the relevant sequence so that it can be detected or obtained more easily.

  To amplify selected sequences, for example, RNA amplification (Lizardi et al., Bio / Technology 1988, 6: 1197-1202; kramer et al., Nature, 1989, 339: 401-402; et al., Clinical Chem., 1989, 35: 1826-1831; see US Pat. No. 4,786,600) and DNA amplification using ligase chain reaction (LCR) or PCR (US patents). Nos. 4,683,195; 4,683,202 and 4,800,159) (US Pat. Nos. 4,876,187, which describe an alternating detection / amplification system involving the use of cleavable linkages). No. 5,011,769)) or fully A variety of methods can be used, including other nucleic acid amplification methods that are within the level of ordinary skill in the art. For example, for PCR, the method can be modified as known in the art. PCR transcription can be enhanced by the introduction of a bacteriophage T7 RNA polymerase promoter sequence in one of the primary oligonucleotides, and the product immunoenzyme from the emitter enhanced with anti-RNA: DNA antibodies Detection can be performed (Blais, Appl. Environ. Microbiol. 1994, 60: 348-352). PCR can also be used in combination with reverse dot-blot hybridization (Iida et al, FEMS Microbiol. Lett. 1993, 114: 167-172). PCR products can be quantitatively analyzed by introduction of dUTP (Duplaa et al., Anal. Biochem., 1993, 212: 229-236), and samples are filtered for PCR-gene probe detection (Filter sampled) (Bej et al., Appl. Environ. Microbiol. 1991, 57: 3529-3534).

The present invention encompasses a method of detection of BEHAB overexpression in which PCR amplification is used for detection of BEHAB DNA, and thus a method of diagnosis of primary CNS tumors. The DNA samples were denatured at about 92 ^o ~ about 95 ° C. As an example, to produce a single-stranded DNA. The DNA sample can be cDNA generated from RNA. The specific primer is then annealed to single stranded DNA at about 37 ° C. to about 70 ° C., depending on the ratio of AT / GC in the primer and other factors well known in the art. The primer is extended at about 72 ° C. using, for example, Taq DNA polymerase or other thermostable DNA polymerase to generate the opposite strand in the template. These steps constitute one cycle that can be repeated to amplify the selected sequence. Nested PCR can be performed for higher specificity. In nested PCR, second amplification is performed using a second set of primers derived from sequences in the first amplification product. The entire BEHAB coding region can be amplified from, for example, cDNA, using a number of primers suitable for generating fragment lengths that are convenient in size for their sequencing. The number of primers required will be well known to those skilled in the art.

  The invention further includes methods in which LCR amplification is used for amplification. The LCR primer can be synthesized so that the 5 'base of the upstream primer hybridizes to a unique base pair in the desired gene and the BEHAB gene can be specifically detected.

  Within embodiments of the invention, probes can be used in an automated non-isotopic strategy, in which the target nucleic acid sequence is amplified by PCR and the desired product is then converted into, for example, a colorimetric oligonucleotide ligation assay (OLA). (Nickerson et al., Proc. Natl. Acad. Sci. USA, 1990, 81: 8923-8927).

  Primers for amplification of the chosen sequence should be chosen from sequences that are highly specific for BEHAB and that form a stable double helix with the target sequence. As is well known in the art, a primer must also be non-complementary with itself or another primer, particularly at the 3 'end, not form a dimer, and other regions of DNA. And should not form secondary structures or double helices. In general, primers of about 18 to about 20 nucleotides are preferred and can be easily synthesized using methods well known in the art. PCR products and other nucleic acid amplification products can be quantified using methods well known in the art (Duplaa et al., Anal. Biochem. 1993, 212: 229-236; Higuchi et al., Bio / Technology, 1993, 11: 1026-1030).

  With the present disclosure and the data disclosed herein, one skilled in the art will readily understand that diagnostics can be developed that can detect the overexpression of BEHAB nucleic acids in mammals. This is indicated by BEHAB in mammals when compared to mammals with normal endogenous BEHAB expression or mammals with overexpression of an uncleavable BEHAB mutant, as shown by data elsewhere in this specification. This is because increased expression results in larger tumors and shorter survival times. Thereby, determination of the level of BEHAB expression in mammals or cells can be used as a powerful and novel diagnostic method for the detection of primary CNS tumors among others.

C. Methods for Identifying Useful Compounds The present invention further includes methods for identifying compounds that affect the expression of BEHAB in cells. The method involves contacting a cell with a test compound and comparing the level of BEHAB expression in a cell so contacted with the level of BEHAB expression in a cell that is otherwise identical to that not in contact with the compound. including. If the level of BEHAB expression in cells contacted with the test compound is higher or lower compared to the level of BEHAB expression in cells otherwise identical that are not in contact with the test compound, this is It is an indicator that the compound affects the expression of BEHAB in cells.

  The present invention encompasses methods for identifying compounds that affect the expression of BEHAB. Based on the disclosure presented herein, one skilled in the art can assess the amount of BEHAB using a probe (eg, an antibody and / or nucleic acid probe that specifically binds to BEHAB). It will be appreciated that the method can identify compounds that selectively affect the expression of BEHAB. Such compounds are useful for inhibiting the expression of BEHAB. Those skilled in the art may find that such compounds are useful for the suppression of diseases, disorders or conditions mediated by and / or associated with increased expression of BEHAB, eg, increased amounts of BEHAB are primary It will be appreciated that in connection with CNS tumors, BEHAB expression is associated with increased tumor volume and decreased survival. Thus, based on the disclosure presented herein, one skilled in the art will appreciate that it may be useful for reducing the expression of BEHAB.

  Similarly, the invention includes a method of identifying a compound that decreases the expression of BEHAB in a cell. The method comprises contacting a cell with a test compound and comparing the level of BEHAB expression in the cell contacted with the compound to the level of BEHAB expression in a cell otherwise identical that is not in contact with the compound. Including. A lower level of BEHAB expression in cells contacted with the compound compared to the level in cells not contacted with the test compound is an indication that the test compound decreases BEHAB expression in the cells.

  The invention also includes a method of identifying a compound that reduces BEHAB cleavage in a cell. The method comprises contacting a cell with a test compound and comparing the level of BEHAB cleavage in the cell contacted with the compound to the level of BEHAB cleavage in a cell that is otherwise identical not in contact with the compound. A lower level of BEHAB cleavage in cells contacted with the compound as compared to the level in cells not contacted with the compound is an indication that the test compound reduces BEHAB cleavage in the cells.

  Compounds that reduce BEHAB cleavage in cells are useful because it has been shown herein that BEHAB cleavage is associated with primary CNS tumor progression and invasiveness. Furthermore, the data disclosed herein indicate that BEHAB cleavage mediates or is associated with greater tumor volume and reduced animal survival. Thus, methods for identifying compounds that reduce BEHAB cleavage can be used to treat a variety of diseases, including but not limited to primary CNS tumors.

  The skilled person will further appreciate that the present invention is not limited to methods of identifying compounds useful in cells or animals. That is, the present invention includes a method for identifying compounds useful in cell-free systems. As used herein, a cell-free system refers to an in vitro assay in which the components necessary for the reaction to occur are present but not associated with the cell. Such components can include cellular enzymes, transcription factors, proteins, nucleic acids, etc., but they are substantially free from cells. As disclosed by the data herein, BEHAB cleavage assays can be performed away from cells or animals, including the use of immunoprecipitation assays. Thereby, the present invention includes a method for the identification of compounds useful for treating primary CNS tumors in a cell-free system.

  Based on the disclosure presented herein, one of ordinary skill in the art will be able to determine the level of BEHAB expression in a cell by determining the level of expression of mRNA encoding BEHAB. Alternatively, by using immunological methods to assess BEHAB production and cleavage, as exemplified herein using Western blot analysis with anti-BEHAB antibodies, the expression and / or cleavage of BEHAB The level can be determined. In addition, nucleic acid based detection methods such as Northern blots and PCR assays can be used as well. In addition, the level of BEHAB activity and / or cleavage in cells can be assessed by determining various parameters that can be affected by BEHAB activity and / or cleavage, such as tumor volume, tumor invasiveness and animal survival. it can. Thus, based on the disclosure and implementation variations presented herein, those skilled in the art will recognize in their cells, including the methods disclosed herein and other methods that may be developed in the future. It will be appreciated that there are a number of methods well known in the art that can be used for the assessment of BEHAB activity and levels of cleavage.

  In addition, proteins that specifically bind to BEHAB or its cleavage products, such as receptors or other BEHAB-related proteins, can be identified using, for example, a yeast two-hybrid assay. Yeast two-hybrid assays are well known in the art and use well-proven methods such as those described by Bartel and Fields (The Yeast Two-Hybrid System, Oxford University Press, Cary, NC). Can be done. Thus, provided with the description presented herein, eg, all amino acid and nucleic acid sequences of BEHAB protein, those skilled in the art will recognize BEHAB or its like, but not limited to, BEHAB target or receptor protein. Proteins that specifically bind to the cleavage products can be easily identified.

  Those skilled in the art will understand, based on the disclosure presented herein, that the invention encompasses molecules identified using methods discussed elsewhere herein. Let's go. That is, using a molecule associated with BEHAB, such as but not limited to a BEHAB receptor protein or BEHAB target protein, a disease, disorder or condition mediated by BEHAB cleavage product interaction with a BEHAB-related protein, eg, primary CNS tumor, glioma, well-differentiated astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, ventricular ependymoma, oligodendroma, ganglion cell tumor, mixed glioma , Brainstem glioma, optic glioma, meningioma, pineal tumor, pituitary tumor, pituitary adenoma, primitive neuroectodermal tumor, neurofibroma, vascular tumor and lymphoma Can be developed. That is, those skilled in the art will recognize that BEHAB cleavage product receptor / ligand interactions and other BEHAB binding interactions, as more fully shown elsewhere in the discussion of antibodies that specifically bind BEHAB. It will be appreciated that BEHAB-related proteins can be used to develop therapeutics that inhibit BEHAB cleavage product activity in cells by inhibiting action.

BEHAB-related proteins identified by the methods disclosed above can be used directly to inhibit BEHAB interactions by contacting cells with BEHAB-related proteins or portions thereof, or using them Thus, antibodies and / or peptide mimetics that can inhibit BEHAB-related protein interactions with BEHAB can be developed, thereby inhibiting BEHAB function, activity and cleavage. Thus, BEHAB-related proteins including BEHAB receptor proteins or BEHAB cleavage product proteins are useful and are encompassed by the present invention.
VII. Kits The present invention provides a nucleic acid encoding a mutant BEHAB, a mutant BEHAB polypeptide, an antibody that specifically binds BEHAB, a nucleic acid complementary to a nucleic acid encoding BEHAB but in an antisense orientation, and a BEHAB cleavage product. A variety of kits are included, including compounds comprising a plurality of antibodies, applicators and instructional materials describing the use of the compounds to perform the methods of the invention. Although model kits are described below, other useful kit contents will be apparent to those skilled in the art in view of this disclosure. Each of these kits is contemplated within the present invention.

  In one aspect, the invention includes a kit for the treatment of primary CNS tumors. The kit is used in the same manner as disclosed herein with respect to the present invention. In short, a kit can be used to contact a cell with a nucleic acid encoding a mutant BEHAB molecule of the invention. In addition, the kit includes applicators and instructions for use of the kit. These instructions simply embody the embodiments presented herein.

  The kit further includes a pharmaceutically acceptable carrier. The composition is provided in a suitable amount as indicated elsewhere herein. Further, the route of administration and frequency of administration are as previously indicated elsewhere herein.

  In another aspect, the invention includes a kit for the treatment of primary CNS tumors. The kit is used in the same manner as disclosed herein with respect to the present invention. In short, a kit can be used to contact a cell with a mutant BEHAB polypeptide molecule of the invention. In addition, the kit includes applicators and instructions for use of the kit. These instructions simply embody the embodiments presented herein. The kit further includes a pharmaceutically acceptable carrier. The composition is provided in a suitable amount as indicated elsewhere herein. Further, the route of administration and frequency of administration are as previously indicated elsewhere herein.

  The present invention further includes kits for the treatment of primary CNS tumors. One skilled in the art will appreciate that the kit can be used in accordance with the methods set forth herein. The kit includes an antibody, small molecule or peptide that binds BEHAB or a fragment thereof, an applicator and instructions for use substantially similar to the examples presented herein. The kit further comprises a pharmaceutically acceptable carrier, the composition, route of administration and frequency of administration being as previously disclosed elsewhere herein.

  The present invention further includes a kit comprising an antisense nucleic acid complementary to a nucleic acid encoding a mammalian BEHAB molecule or fragment thereof. Such kits can be used in accordance with the methods of the invention to mediate a reduction in BEHAB expression. In addition, the kit includes applicator and instructions for use of the kit. These instructions simply embody the embodiments presented herein. The kit further includes a pharmaceutically acceptable carrier. Antisense nucleic acids and pharmaceutically acceptable carriers are provided in suitable amounts as indicated elsewhere herein. Further, the route of administration and frequency of administration are as previously indicated elsewhere herein.

  The present invention further includes kits for the treatment of primary CNS tumors. In short, the kit comprises an antibody, small molecule or peptide that specifically binds to a BEHAB cleavage product or fragment thereof and can be used according to the methods set forth elsewhere herein. The kit of the present invention further includes instructions for use similar to the methods shown herein for applicator and kit use. The kit also includes a pharmaceutically acceptable carrier, the composition of which, the route and frequency of administration, and the dosage are shown previously herein.

Experimental Examples The invention will now be described with reference to the following examples. These examples are presented for illustrative purposes only, and the invention should not be construed as limited to any of these examples, but rather all variations that will become apparent as a result of the description presented herein. Should be considered to encompass.

  The materials and methods used in the experiments presented in this example will now be described.

Site-directed mutagenesis : Full-length BEHAB cDNA was obtained from Zhang et al. It was cloned into the EcoR1 site of the eukaryotic expression vector pCDNA3 (Invitrogen, Carlsbad, Calif.) As described in (Journal of Neuroscience, 1998, 18: 2370-2376). The full length BEHAB expression vector was mutated using the QUIKCHANGE site-directed mutagenesis kit according to the manufacturer's recommendations (Stratagene, La Jolla, Calif.). Sequencing using the fluorescently labeled dideoxynucleotide chain termination method confirmed the introduction of suitable mutations.

Cell culture and transfection : CNS-1 cells (American Type Culture Collection, Manassas, Va.) Were grown and maintained in RPMI with 10% fetal calf serum (FCS). Cells were split every 4 days (1: 6 to 1:10) and re-plated. 75% confluent cells were plated in 60 mm tissue culture plates and transfected with 4 μg of the appropriate expression construct and Fugene 6 according to the manufacturer's recommendations (Roche, Indianapolis, IN). In brief, DNA and Fugene 6 were incubated with cells in their standard medium for 6 hours, after which the medium was removed and replaced with fresh medium overnight. The next day, cells were selected for transgene expression using G-418 (80 μg / μl, Invitrogen). Holding the cells in G-418-supplemented medium for two and a half weeks induced a stable pool of transfected cells. Following selection, a pool of stably transfected cells was maintained in media containing G-418 at a concentration of (40 μg / μl).

In vitro proliferation and cell death assay : The effect of transfection on cell proliferation and cell death was assessed. For both assays, cells were grown in 96-well tissue culture plates with an initial plate density of ⁴ × 10 ⁴ cells per well in 200 μl medium. Cell proliferation was measured by cell uptake of MTT (3,-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, MO). In short, 100 μl of media was removed and stored for cell death assay. To the remaining cells and medium, add 25 μl MTT (Dulbecco's phosphate buffered saline; 2 mg / ml in DPBS) and incubate for 3 hours at 37 ° C. in a humidified incubator (5% CO _{2 in} air). did. The product obtained by adding 100 μl of 40 mM hydrochloric acid in isopropanol was solubilized and the sample was incubated at 37 ° C. for 2 hours. Samples were analyzed in quadruplicate by measuring absorbance at 550 nM using a microplate reader.

100 μl of cell-free medium was used for the cell death assay. The medium was assayed for lactate dehydrogenase (LDH) activity using the Cytotoxicity Detection Kit according to the manufacturer's instructions (Roche, Indianapolis, IN). Briefly, the medium was incubated with lactate that was oxidized to pyruvate by LDH and reduced NAD ⁺ to NADH. In the presence of diaphorase, NADH catalyzes the conversion of the yellow tetrazolium salt to the red formazan salt. Samples were analyzed in quadruplicate by measuring absorbance at 450 nM using a microplate reader.

Animal studies : Female Lewis rats (Charles River Laboratories, Wilmington, Mass.) Were anesthetized (75 mg / kg ketamine, 5 mg / kg xylazine), and an incisor bar was positioned 3.0 mm below the ear extension. Placed in the apparatus (David Kopf Instruments, Tujunga, CA). Approximately 70% -80% confluent CNS-1 cells were harvested from 100 mm tissue culture plates using trypsin. Cells were washed once in DPBS and the concentration of cells was increased in phosphate buffered saline (PBS) supplemented with 1 μg / μl MgCl ₂ , 1 μg / μl CaCl ₂ and 0.1% glucose. Suspended at a concentration of 5 × 10 ⁴ cells per μl, except in the case of survival curves with inhibitors that were 1 × 10 ⁵ per μl. Using a 10 μl Hamilton syringe fitted with a 26 gauge beveled needle, 2.8 mm posterior to bregma, 2.2 mm lateral to midline and 5.0 mm ventral to dura mater Intrathalamic injection into the right thalamus was performed at the coordinates. A total volume of 3 μl of cell suspension was injected over 3 minutes and the needle was left in place for an additional minute before being slowly withdrawn.

  Animal health and survival were assessed every 6 hours. Animals that could not stand upright within 15 seconds of being laid down were considered to have reached the end of survival and were sacrificed. The date of sacrifice was recorded on the last day of survival.

Histology : Rats were deeply anesthetized and transcardially perfused with PBS followed by ice-cold 4% PBS-paraformaldehyde. 40 μM frozen sections were sliced on a cryostat and brains from animals implanted with transfected CNS-1 cells were sliced coronally. Every fifth section was stained with cresyl violet for assessment of tumor volume.

Image analysis and tumor volume assessment : Tissue sections were analyzed using NIH Image (http://rsb.info.nih.gov/nih-image/). The tumor area was manually sectioned every fifth section through the tumor by a researcher who hid the experimental conditions. Tumor volume was reconstructed using Calvaleri's estimator of morphology volume (Rosen and Harry, Journal of Neuroscience, 195, 19:35, 19:35).

Electrophoresis and Western analysis : Samples were electrophoresed on an 8% SDS-PAGE gel, and then proteins were transferred to nitrocellulose by electrophoresis (Laemmli et al., Nature, 1970, 227: 680-685; Towbin et al., Proc. Natl. Acad. Sci. USA, 1979, 76: 4350-4354). Blots were performed with rabbit affinity purified antisera (1: 10,000) or B50 (1: 5000) (Matthews et al., J. Biol. Chem., 2000, 275: 22695-22703), followed by Incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (1: 4000, Jackson ImmunoResearch Labs, West Grove, PA). Immunoreactive bands were visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.

Statistical analysis : The significance of comparison between animals transplanted with transfected cell lines was determined by one-way analysis of variance (ANOVA). Results from in vitro proliferation and cell death assays were analyzed using measured ANOVA repeated 4X7 times (cell line X days).

  The results of the experiment shown in this example will now be described.

Effect of NVY mutations on BEHAB cleavage : To investigate the role of BEHAB cleavage in primary CNS tumors, constructs were formed that block or inhibit BEHAB cleavage. The effect of mutations at the BEHAB Glu ^395- Ser ³⁹⁶ cleavage site on the cleavage of the full-length protein was examined. Previous studies on aggrecan with a cleavage site very similar to the BEHAB Glu ^395- Ser ³⁹⁶ site show that the ARG to NVY mutation of the 3 amino acids immediately downstream of the cleavage site completely blocks the cleavage. (Fosang et al., J. Biol. Chem., 2000, 275: 33027-33037). Therefore, site-directed mutagenesis was used to examine the impact of NVY mutations on BEHAB cleavage. This mutation changed the amino acid cleavage site from ³⁹³ Glu-Ser-Glu-Ser-Arg-Gly ³⁹⁸ to ³⁹³ Glu-Ser-Glu-Asn-Val-Tyr ³⁹⁸ (SEQ ID NO: 1 and SEQ ID NO: 2 respectively). . To assess the impact of NVY mutations on BEHAB cleavage, CNS-1 cells were transiently transfected with full-length BEHAB or a mutated BEHAB construct, and the resulting proteins were expressed as B6 and B50 antibodies (BEHAB, respectively). Analysis by Western blot using Matthews et al., J. Biol. Chem., 2000, 275: 22695-22703), which specifically binds to the C-terminal and neo-epitope generated by cleavage.

Cells transiently transfected with both normal and mutated BEHAB produced the full-length product as shown using the B6 antibody (FIG. 1A). Normal full length BEHAB was cleaved at the ³⁹³ Glu-Ser-Glu-Ser-Arg-Gly ³⁹⁸ cleavage site as shown using the B50 antibody. In stark contrast, cells transfected with the NVY mutant construct produced large amounts of full-length protein, but no cleavage was detected (FIG. 1A). To determine if the NVHA mutant form of BEHAB could not be completely cleaved, samples were immunoprecipitated with B50 antibody to detect traces of such cleavage products. No reactive product was detected using this more sensitive assay, indicating that the NVY mutation completely inhibited cleavage of the full-length protein.

  The effect of NVY mutations on BEHAB cleavage was further investigated by co-transfecting CNS-1 cells with normal full length BEHAB and NVY constructs. These experiments were designed to determine whether the NVY mutant BEHAB can inhibit normal substrate cleavage. Cells were co-transfected with 2 μg of normal BEHAB expression construct and 0, 1, 2 or 4 μg of mutated BEHAB construct. The addition of the NVY mutant construct increased the amount of full length BEHAB, which had no effect on the ability of the protease to cleave normal BEHAB protein (FIG. 1B). While not wishing to be bound by any particular theory, these results suggest that the mutant form of BEHAB itself is not cleavable, but does not necessarily inhibit the protease.

Effect of NVY mutant on cell proliferation and cell death :
To evaluate the effects of BEHAB NVY cleavage site mutations on cell proliferation and cell death, a green fluorescent protein (GFP) expression vector (CNS-1-GFP), a normal BEHAB expression vector (CNS-1-FL) Alternatively, CNS-1 cells were stably transfected with an NVY mutant vector (CNS-1-NVY). A pool of cells stably transfected in G418 was selected and analyzed as described above. As observed using transient transfection, the pool of CNS-1-NVY cells produced full-length BEHAB but did not cleave it (FIG. 1C).

  Cell growth was analyzed in vitro for stably transfected cells using the MTT assay. The succinate-tetrazolium reductase system, an enzyme system of the mitochondrial respiratory chain, converted MTT to a dye, thereby providing a measure of the number of viable cells. Proliferation was assessed over 7 days. There was no difference in cell proliferation in any of the transfected cells (FIG. 2A). Furthermore, none of the transfected pools showed a difference from the untransfected parent CNS-1 cells.

  Cell death was assessed using the LDH assay. LDH is an enzyme that normally exists only in the cytoplasm. LDH activity is measured in the medium, where LDH is present only when it is released by dead or dying cells. There was a low level of cell death of about 2% to about 3% over the first 4 days in all cell lines. However, cell death thereafter increased slightly and there was no difference in percent cell death between any cell line at any time point (FIG. 2B). Thus, the mutated construct is not cleavable, but it has no obvious effect on cell growth or death in vitro.

NVY non-cleavable BEHAB does not affect the phenotype of CNS-1 tumors : previous studies show that CNS-1 cells transfected with normal full-length BEHAB form larger tumors than GFP transfected standards It is shown that. However, in 9L cells that neither produce nor cleave BEHAB, transfection with full-length BEHAB does not change the tumor phenotype (Zhang et al., J. of Neuroscience, 1998, 18: 2370-2376). ). To investigate the role of BEHAB cleavage in tumor progression, a pool of CNS-1 cells stably transfected with the above expression vector was grown as an intracranial graft. Eight days after tumor implantation, animals (n = 8) were perfused and their brains were processed for histology. Every fifth section was stained with cresyl violet, and the tumor area was estimated by image analysis. Volumes were reconstructed from these analyses. Consistent with previous results, CNS-1-FL tumors were larger and more invasive than CNS-GFP. CNS-1-NVY tumors were not significantly different in size from CNS-1-GFP tumors. Importantly, however, CNS-1-NVY tumors were significantly smaller than CNS-1-FL tumors (Table 1, FIG. 3). While not wishing to be bound by any particular theory, these results suggest that although BEHAB production alone is insufficient to enhance tumor progression, full-length protein cleavage plays a critical role in this process. It shows that it fulfills.

  To further investigate the effect of mutated BEHAB on tumor progression, the effect of mutated BEHAB construct on animal (n = 6) survival was examined. It was previously discovered that CNS-1-FL tumors reduce animal survival (Nutt et al., Cancer Res., 2001, 61: 7056-7059). Consistent with these results, animals transplanted with CNS-1-FL tumors reached an endpoint of survival on average 3 days earlier than animals transplanted with CNS-1-GFP tumors. However, CNS-1-NVY transplanted animals did not reach the end of survival earlier than standard. In fact, animals with CNS-1-NVY tumors survived slightly longer than animals transplanted with CNS-1-GFP, but with the number of animals in this study to date, the difference is significant. is not. CNS-1-NVY transplanted animals survived an average of 4 days longer than CNS-1-FL transplanted animals, with a 23% increase in survival (Table 1, FIG. 4).

Discussion Previous studies non -. 9 L gliosarcoma cell line grown as invasive tumors, indicating that the invasive are transfected with the 5 'fragment of BEHAB (Zhang et al al , J. Neuroscience, 1998, 18: 2370-2376). However, transfection of 9L cells with full length BEHAB does not change the tumor phenotype from wild type 9L cells. In stark contrast, CNS-1 cells transfected with full length BEHAB have improved tumor size and invasiveness. The difference in the phenotype of the different cell lines is explained by the difference in the ability of these two cell lines to cleave BEHAB. CNS-1 cells cleave the full length protein at the Glu ^395- Ser ³⁹⁶ cleavage site, forming 90 kDa and 50 kDa fragments. In contrast, 9L cells do not proteolytically process full-length proteins. Further previous studies have shown that BEHAB cleavage in CNS-1 cells is mediated by ADAMTS-4 (Matthews et al., J. Biol. Chem., 2000, 275: 22669-22703). The role of BEHAB cleavage in glioma progression was assessed using CNS-1 cells transfected with a mutated, uncleavable form of BEHAB. The effects of this construct on tumor progression and animal survival were studied and for the first time have demonstrated the role of BEHAB cleavage in glioma progression.

The mutation of the BEHAB / brevican cleavage site from ³⁹³ Glu-Ser-Glu-Ser-Arg-Gly ³⁹⁸ to ³⁹³ Glu-Ser-Glu-Asn-Val-Tyr ³⁹⁸ (SEQ ID NO: 1 and SEQ ID NO: 2 respectively) is The protein was made completely non-cleavable by CNS-1 cells. Importantly, this uncleavable mutant had an effect on rat CNS-1 tumors that was different from the normal full-length construct. Although CNS-1-FL tumors were larger than standard tumors and reduced animal survival time relative to animals with them, CNS-1-NVY tumors could not be phenotypically distinguished from standard tumors . These studies clearly demonstrate the critical role that both cleavage and processing play in BEHAB function in primary CNS tumors.

  CNS-1-NVY tumors were smaller than CNS-1-FL tumors, but were phenotypically similar to standard tumors. This result provides information regarding the mechanism of BEHAB cleavage-mediated effects in gliomas, especially in view of the fact that CNS-1 cells are induced to express endogenous BEHAB when grown in the brain. Thus, all three cell lines used in these experiments express normal amounts of BEHAB; CNS-1-FL tumors express exogenous BEHAB in a cleavable form in addition to endogenous BEHAB; CNS-1-NVY tumors express exogenous BEHAB in an uncleavable form in addition to endogenous BEHAB. Thus, if the NVY mutant acts as a dominant negative in CNS-1 cell tumors, it is expected that CNS-1-NVY tumors will be significantly smaller than standard tumors (CNS-1-GFP) due to disruption of the normal function of BEHAB It is not true. Rather, it appears that CNS-1-NVY tumors were instead transfected with molecules that do not produce changes in their phenotype compared to standard tumors, and the BEHAB cleavage product is not mediated by the full-length protein. It strongly suggests mediating interactions and / or functions. For example, if the BEHAB cleavage product simply solubilizes the matrix and allows cell movement, production of uncleavable BEHAB will interfere with this effect. However, tumors with or without uncleavable substrates appear to be very similar. These results suggest that the BEHAB cleavage product has a unique function that is not mediated by the full-length protein itself. The data evident herein show that BEHAB cleavage can power primary CNS tumor progression and that inhibition of BEHAB cleavage can reduce tumor progression. These studies indicate that inhibition of BEHAB cleavage can provide important new therapeutic strategies, and thus inhibition of BEHAB cleavage or cleavage product function will serve as an effective and novel method for the treatment of primary CNS tumors. It shows strong deafness.

  The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety.

  While the invention has been disclosed with reference to particular embodiments, other embodiments and variations of the invention can be devised by other skilled in the art without departing from the true spirit and scope of the invention. It is clear. It is intended that the appended claims be regarded as including all such aspects and equivalent variations.

Western blot of medium from cells transiently transfected with full length (FL) and mutant (NVY) BEHAB. Western blot of medium from cells transiently co-transfected with 2 μg FL construct and 0, 1, 2, or 4 μg NVY mutant construct. Western blot of medium from cells stably transfected with full length (FL) and mutant (NVY) BEHAB. Graph depicting cell growth over 7 days using MTT assay. Graph depicting cell death over 7 days as assessed using LDH assay. Representative tumor image derived from CNS-1-GFP cells. Representative tumor image derived from CNS-1-FL cells. Representative tumor image derived from CNS-1-NVY cells. Graph depicting tumor volume derived from CNS-1-FL, CNS-1-GFP and CNS-1-NVY cells. Graph depicting the effect on tumor survival of tumors derived from cells stably transfected with CNS-1-FL, CNS-1-GFP and CNS-1-NVY.

[Sequence Listing]

Claims (43)

  An isolated nucleic acid encoding a mammalian mutant BEHAB molecule or fragment thereof.   An isolated nucleic acid encoding a mammalian mutant BEHAB molecule, wherein the isolated nucleic acid has 99.7% identity to the nucleic acid sequence set forth in SEQ ID NO: 4.   3. The isolated nucleic acid of claim 2, wherein the isolated nucleic acid comprises the nucleic acid sequence shown in SEQ ID NO: 4.   An isolated nucleic acid encoding a mammalian mutant BEHAB molecule, wherein the amino acid sequence of the mammalian mutant BEHAB molecule comprises the amino acid sequence set forth in SEQ ID NO: 3.   An isolated polypeptide comprising a mammalian mutant BEHAB molecule.   An isolated polypeptide comprising a mammalian mutant BEHAB molecule, wherein the mammalian mutant BEHAB molecule has 99.6% identity to the amino acid sequence set forth in SEQ ID NO: 3.   2. The isolated nucleic acid of claim 1, wherein the nucleic acid further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto. The tag polypeptide is a myc tag polypeptide, a glutathione-S-transferase tag polypeptide, a green fluorescent protein tag polypeptide, a myc-pyruvate kinase tag polypeptide, a His ₆ tag polypeptide, an influenza virus hemagglutinin tag polypeptide, 8. The isolated nucleic acid of claim 7, selected from the group consisting of a flag tag polypeptide and a maltose binding protein tag polypeptide.   2. The isolated nucleic acid of claim 1, wherein the nucleic acid further comprises a nucleic acid defining a promoter / regulatory sequence operably linked thereto.   A vector comprising the isolated nucleic acid of claim 1.   11. The vector of claim 10, wherein the vector further comprises a nucleic acid defining a promoter / regulatory sequence operably linked thereto.   A recombinant cell comprising the isolated nucleic acid of claim 1.   A recombinant cell comprising the vector of claim 10.   An isolated nucleic acid in an antisense orientation that is complementary to an isolated nucleic acid encoding a mammalian BEHAB molecule or fragment thereof.   15. The isolated nucleic acid of claim 14, wherein the nucleic acid is complementary to the nucleic acid sequence shown in SEQ ID NO: 5.   A recombinant cell comprising the isolated nucleic acid of claim 14.   A vector comprising the isolated nucleic acid of claim 14.   A method for treating a primary CNS tumor in a mammal comprising administering to the mammal an effective amount of a BEHAB cleavage inhibitor, thereby treating the primary CNS tumor in said mammal.   19. The method of claim 18, wherein the BEHAB cleavage inhibitor is selected from the group consisting of antibodies, proteins, protease inhibitors, and peptidomimetics.   20. The method of claim 19, wherein the antibody specifically binds to BEHAB or a fragment thereof.   20. The method of claim 19, wherein the protein comprises the amino acid sequence shown in SEQ ID NO: 3 or a fragment thereof.   A method for treating a primary CNS tumor in a mammal comprising administering to the mammal an effective amount of a BEHAB expression inhibitor, thereby treating the primary CNS tumor in said mammal.   23. The method of claim 22, wherein the BEHAB expression inhibitor is selected from the group consisting of a ribozyme and an antisense nucleic acid.   24. The method of claim 23, wherein the antisense nucleic acid is complementary to an isolated nucleic acid encoding a BEHAB molecule or fragment thereof.   24. The method of claim 23, wherein the ribozyme is complementary to an isolated nucleic acid encoding a BEHAB molecule or fragment thereof.   A method of treating a primary CNS tumor in a mammal comprising administering to the mammal an effective amount of an inhibitor of BEHAB cleavage product activity, thereby treating the primary CNS tumor in said mammal.   27. The method of claim 26, wherein the inhibitor of BEHAB cleavage product activity is an antibody.   28. The method of claim 27, wherein the antibody specifically binds to a BEHAB cleavage product.   A biological sample is obtained from a first mammal, the amount of BEHAB nucleic acid molecules in the biological sample is evaluated, and the amount of BEHAB nucleic acid molecules in the biological sample is otherwise identical. Compared to the amount of BEHAB nucleic acid molecule in a biological sample obtained from two mammals, wherein the amount of BEHAB nucleic acid molecule in the biological sample from a second mammal that is otherwise identical The amount of BEHAB nucleic acid molecule in the biological sample from the first mammal that is higher in comparison is an indication that the first mammal is suffering from a primary CNS tumor, thereby causing a primary A method of diagnosing a primary CNS tumor in a mammal comprising diagnosing a sex CNS tumor.   30. The method of claim 29, wherein the biological sample is selected from the group consisting of blood, neural tissue, cerebrospinal fluid, urine, saliva and brain tissue.   The amount of BEHAB molecule in the mammal is assessed before, during or after treatment for the primary CNS tumor in the mammal, wherein the mammal in the mammal prior to treatment for the primary CNS tumor. A higher or lower amount of the BEHAB molecule in the mammal during or after the treatment for a primary CNS tumor compared to the amount of BEHAB molecule is greater than the amount of primary CNS tumor in the mammal. The efficacy of treatment for primary CNS tumors in mammals, comprising assessing the efficacy of the treatment for primary CNS tumors in the mammal Evaluation method.   32. The method of claim 31, wherein the treatment for the primary CNS tumor is selected from the group consisting of chemotherapy, radiation therapy and surgery.   Contacting a cell with a test compound and comparing the level of BEHAB molecule expression in the cell to the level of BEHAB molecule expression in a cell otherwise identical to the test compound, wherein the test compound and A higher or lower level of BEHAB molecule expression in the cell in contact with the test compound compared to the level of BEHAB molecule expression in the otherwise identical cell that is not contacted, A method for identifying a compound that affects the expression of a BEHAB molecule in a cell, comprising identifying a compound that is indicative of affecting the expression of the BEHAB molecule and thereby affects the expression of the BEHAB molecule in the cell.   34. A compound identified by the method of claim 33.   Contacting a cell with a test compound and comparing the level of BEHAB molecule expression in the cell to the level of BEHAB molecule expression in a cell otherwise identical to the test compound, wherein the test compound and A higher or lower level of BEHAB molecule expression in the cell in contact with the test compound compared to the level of BEHAB molecule expression in the otherwise identical cell that is not contacted, A method of identifying a compound that decreases the expression of a BEHAB molecule in a cell, comprising identifying a compound that is indicative of decreasing the expression of the BEHAB molecule, thereby decreasing the expression of the BEHAB molecule in the cell.   36. A compound identified by the method of claim 35.   Contacting the cell with a test compound and comparing the level of BEHAB cleavage in the cell to the level of BEHAB cleavage in a cell otherwise identical to the test compound, wherein the cell is contacted with the test compound; A higher or lower level of BEHAB cleavage in the cell in contact with the test compound compared to the level of BEHAB cleavage in the otherwise identical cell, the test compound inhibits BEHAB cleavage in the cell A method of identifying a compound that inhibits BEHAB cleavage in a cell, comprising identifying a compound that is indicative of, thereby inhibiting BEHAB cleavage in the cell.   Contacting a cell-free system with a test compound and comparing the level of BEHAB cleavage in the cell-free system with a level of BEHAB cleavage in a cell-free system that is otherwise identical to the test compound, wherein A higher or lower level of BEHAB cleavage in the cell-free system in contact with the test compound compared to the level of BEHAB cleavage in the cell-free system that is otherwise identical to the test compound; Of a compound that inhibits BEHAB cleavage in a cell-free system, comprising identifying a compound that inhibits BEHAB cleavage in a cell-free system, wherein the test compound is indicative of inhibiting BEHAB cleavage in the cell-free system Identification method.   A BEHAB cleavage inhibitory amount of a composition comprising an isolated nucleic acid molecule encoding a mutated BEHAB molecule or fragment thereof and a pharmaceutically acceptable carrier, further comprising an applicator and an instruction manual for use A kit for the treatment of primary CNS tumors.   A BEHAB cleavage inhibiting amount of a composition comprising an isolated mutant BEHAB polypeptide or fragment thereof and a pharmaceutically acceptable carrier, and further comprising an applicator and instructions for use. A kit for the treatment of primary CNS tumors.   A composition comprising an antibody that specifically binds to a mammalian BEHAB molecule or fragment thereof, and a pharmaceutically acceptable carrier, comprising a BEHAB cleavage inhibitory amount, further comprising an applicator and instructions for use. A kit for the treatment of primary CNS tumors.   A composition comprising an isolated nucleic acid complementary to an isolated nucleic acid encoding a mammalian BEHAB molecule or several fragments thereof, as well as a BEHAB molecule expression inhibiting amount of a composition comprising a pharmaceutically acceptable carrier. A kit for the treatment of primary CNS tumors, wherein the complementary nucleic acid is in an antisense orientation and further comprises an applicator and instructions for use. A BEHAB cleavage product inhibitory amount of a composition comprising an antibody that specifically binds to a BEHAB cleavage product or fragment thereof and a pharmaceutically acceptable carrier, further comprising an applicator and instructions for use. A kit for the treatment of a primary CNS tumor comprising.

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