WO2003093787A2

WO2003093787A2 - Antiproliferative protein from hypericum perforatum and nucleic acids encoding the same

Info

Publication number: WO2003093787A2
Application number: PCT/US2003/013154
Authority: WO
Inventors: Kamel Khalili; Nune Darbinian Sarkissian
Original assignee: Temple Univ School of Medicine
Current assignee: Temple Univ School of Medicine
Priority date: 2002-04-30
Filing date: 2003-04-28
Publication date: 2003-11-13
Anticipated expiration: 2004-10-30
Also published as: AU2003245247A1; AU2003245247A8; WO2003093787A3

Abstract

A protein named CHP-10 has been isolated from Hypericum perforatum callus culture. This protein comprises a unique 20 amino acid sequence, has an apparent molecular weight of approximately 39 kDa by SDS-PAGE, and inhibits proliferation of abnormally proliferating cells from cancer or non-cancerous proliferative disorders. Methods of using CHP-10, or fragments, derivatives, homologs and analogs of CHP-10, to inhibit the proliferation of abnormally proliferating cells are also provided.

Description

ANTIPROLIFERATIVE PROTEIN FROM HYPERICUM PERFORATUM AND NUCLEIC ACIDS ENCODING THE SAME

Field of the Invention

This invention relates to the field of antiproliferative compounds and their use, in particular inhibiting proliferation of cells with a protein isolated from Hypericum perforatum.

Background of the Invention

The abnormal proliferation of cells is a hallmark of many disease states- including cancer and non-cancerous proliferative disorders. The cancers are characterized by cells which have escaped the normal controls on growth and position, leading to pathologic conditions such as tumor growth and metastasis. Cells involved in non-cancerous proliferative disorders have escaped normal growth controls, but do not metastasize. The non-cancerous proliferative disorders thus form benign tumors, fibroses, and other non-malignant growths. In order to combat disease states characterized by the abnormal proliferation of cells, physicians employ therapeutic compounds that have antiproliferative or cytotoxic effects. Many such compounds have been identified and isolated from natural sources; for example, the anti-cancer drug taxol was isolated from the bark of the yew tree. St. John's wort (Hypericum perforatum) is a medicinal plant known for its therapeutic benefits. This plant is easily cultivated, and extracts of H. perforatum are currently used to treat cancers and clinical depression, control bacterial and viral infections, and aid in wound healing. The therapeutically active compounds in H perforatum extracts are hypericin, hyperforin and certain flavenols (e.g., quercetin and hyperoside). These compounds have also been isolated from cultured, rather than cultivated, H perforatum tissues. To date, no therapeutically active protein has been identified in extracts from cultured or cultivated H. perforatum tissues.

Anti-tumor activity has been demonstrated for purified hypericin, but this effect requires activation of the compound by visible light. However, an anti-tumor compound such as hypericin is not desirable, since it is difficult to expose all but the most superficial tumors to the light needed for photoactivation. Furthermore, because hypericin is sensitive to visible light, the compound is unstable and difficult to store. A antiproliferative compound isolated from H. perforatum, whose activity does not depend on external stimuli, and which can be readily extracted or synthesized in commercially relevant quantities, would be useful in the treatment of diseases characterized by the abnormal proliferation of cells.

Summary of the Invention

A novel protein with antiproliferative activity, called CΗP-10, has been isolated from Hypericum perforatum. A partial amino acid sequence has been obtained for the protein, and is given in SEQ ID NO: 1. The invention thus provides a substantially purified CΗP-10 protein isolated from H. perforatum, characterized in that the protein comprises SEQ ID NO: 1, has an apparent molecular weight of approximately 39 kDa by SDS- PAGE, and inhibits proliferation of cultured T98G glioblastoma cells. The invention further provides biologically active fragments, derivatives, homologs or analogs of CΗP-10.

The invention also provides nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 1, complementary sequences, and fragments and homologs thereof.

The invention also provides antibodies that bind to specific epitopes on CΗP-10, and to specific epitopes on derivatives, homologs, analogs, or antigenic fragments of CΗP-10. The antibodies can be monoclonal or polyclonal, or can be an antibody fragment that is capable of specifically binding to a CΗP-10 epitope.

The invention further provides a hybridoma that produces a monoclonal antibody which specifically binds the compounds of the invention.

The invention also provides a method of treating a subject having cells deriving from a cancer or a non-cancerous proliferative disorder, comprising administering to the subject an effective amount of CΗP-10, or a biologically active fragment, derivative, homolog or analog of CHP-10, such that proliferation of the cells is inhibited.

The invention fiirther provides a pharmaceutical formulation for treating a cancer or a non-cancerous proliferative disorder comprising CHP-10, or a biologically active fragment, derivative, homolog or analog of CHP-10.

Amino Acid Abbreviations

The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule:

A Alanine Ala

C Cysteine Cys

D Aspartic Acid Asp

E Glutamic Acid Glu

F Phenylalanine Phe

G Glycine Gly

H Histidine His

I Isoleucine He

K Lysine Lys

L Leucine Leu

M Methionine Met

N Asparagine Asn

P Proline Pro

Q Glutamine Gin

R Arginine Arg

S Serine Ser

T Threonine Thr

V Naline Nal w Tryptophan Trp

Y Tyrosine Tyr Definitions

"Antibody" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered or removed from the natural state through the actions of a human being. For example, a nucleic acid sequence or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid sequence or protein may exist in substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

The expression "amino acid" as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. "Standard amino acid" means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, "synthetic amino acid" also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half life without adversely affecting their biological activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention.

Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

As used herein, "protecting group" with respect to a terminal amino group of a peptide means any of the various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert- butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.

As used herein, "protecting group" with respect to a terminal carboxy group of a peptide means any of various carboxyl-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.

"Derivative" includes any purposefully generated peptide which in its entirety, or in part, comprises a substantially similar amino acid sequence to CHP-10 and has CHP-10 biological activity. Derivatives of CHP-10 may be characterized by single or multiple amino acid substitutions, deletions, additions, or replacements. These derivatives may include (a) derivatives in which one or more amino acid residues of SEQ ID NO:l are substituted with conservative or non-conservative amino acids; (b) derivatives in which one or more amino acids are added to SEQ ID NO:l; (c) derivatives in which one or more of the amino acids of SEQ ID NO:l includes a substituent group; (d) derivatives in which SEQ ID NO:l or a portion thereof is fused to another peptide (e.g., serum albumin or protein transduction domain); (e) derivatives in which one or more nonstandard amino acid residues (i.e., those other than the 20 standard L-amino acids found in naturally occurring proteins) are incorporated or substituted into SEQ ID NO:l; and (f) derivatives in which one or more nonamino acid linking groups are incorporated into or replace a portion of SEQ ID NO:l. A "homolog" of CHP-10 includes any nonpurposely generated peptide which in its entirety, or in part, comprises a substantially similar amino acid sequence to SEQ ID NO:l and has CHP-10 biological activity. Homologs may include paralogs, orthologs, and naturally occurring alleles or variants of CHP- 10. An "analog" of CHP-10 includes any non-peptide molecule comprising a structure that mimics the physico-chemical and spatial characteristics of CHP- 10, and has CHP-10 biological activity.

"Biologically active," with respect to CHP-10, or fragments, derivatives, homologs and analogs of CHP-10 means the ability of the compound to inhibit proliferation of T98G glioblastoma cells, or exhibiting immunogenic characteristics of a CHP-10 epitope.

The ability of the compound to inhibit proliferation of T98G glioblastoma cells can be determined according to the in vitro cell viability assays given in Example 2 below. A compound which "exhibits immunogenic characteristics of a CHP-

10 epitope" means that the compound 1) elicits a specific humoral or cellular immune response in a mammal to an epitope of CHP-10. As used herein, an "epitope" is a distinct structural area of an immunogen that can combine with an antibody or T-lymphocyte receptor. Reactivity to CHP-10 epitopes may be determined by known immunological techniques, such as immunoprecipitations and Western blot analyses as described above and in the Examples. By way of illustration, a compound exhibiting immunogenic characteristics of a CHP-10 epitope will, on injection into a mouse, cause that mouse to develop antibodies that will react with CHP-10 as detected, for example, by Western blot or enzyme-linked immunosorbent assay.

By "libraries" is meant pools and subpools of compounds, for example fragments, derivatives, homologs, analogs, or pro-analogs of CHP-10. "Peptide" and "protein" are used interchangeably, and refer to a compound comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds (e.g., peptide isosteres). No limitation is placed on the maximum number of amino acids which may comprise a protein or peptide. The amino acids comprising the peptides or proteins described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred. The amino acid comprising the peptides or proteins described herein may also be modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide. Also, a given peptide may contain many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold F, Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors," Meth. Enzymol. (1990) 182: 626-646 and Rattan et al. (1992), "Protein Synthesis: Posttranslational Modifications and Aging," Ann NYAcadSci 663: 48-62. "Nariant" as the term is used herein, is a nucleic acid sequence or peptide that differs from a reference nucleic acid sequence or peptide respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Νon-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. As used herein, a peptide or a portion of a peptide which has a

"substantially similar amino acid sequence" to a reference peptide means the peptide, or a portion thereof, has an amino acid sequence identity or similarity to the reference peptide of greater than about 70%. Preferably, the sequence identity is greater than about 75%, more preferably greater than about 80%, particularly preferably greater than about 90%, and more particularly preferably greater than about 95%, and most preferably greater than about 98%. Amino acid sequence similarity or identity may be computed by using the BLASTP and TBLASTΝ programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm; BLASTP and TBLASTΝ settings to be used in such computations are indicated in Table 1 below. Amino acid sequence identity is reported under "Identities" by the BLASTP and TBLASTΝ programs. Amino acid sequence similarity is reported under "Positives" by the BLASTP and TBLASTΝ programs. Techniques for computing amino acid sequence similarity or identity are well known to those skilled in the art, and the use of the BLAST algorithm is described in Altschul et al. (1990), J Mol. Biol. 215: 403- 410 and Altschul et al. (1997), Nucleic Acids Res. 25: 3389-3402, the disclosures of which are herein incorporated by reference in their entirety. BLASTP and TBLASTN programs utilizing the BLAST 2.0.14 algorithm and may be accessed at http://www.ncbi.nlm.nih.gov/.

Table 1 - Settings to be used for the computation of amino acid sequence similarity or identity with BLASTP and TBLASTN programs utilizing the BLAST 2.0.14 algorithm.

*The SEG program is described by Wootton and Federhen (1993), Comput. Chem. 17: 149-163.

"Substantially similar nucleic acid sequence" means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur. Preferably, the substantially similar nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least 70%, Preferably, the sequence identity is greater than about 75%, more preferably greater than about 80%, particularly preferably greater than about 90%, and more particularly preferably greater than about 95%, and most preferably greater than about 98%. Substantial similarity of nucleic acid sequences may be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50 °C; preferably in 7% (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 1XSSC, 0.1% SDS at 50 °C, more preferably 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 0.5.XSSC, 0.1% SDS at 50 °C; and most preferably in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 0.1XSSC, 0.1% SDS at 65 °C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, but are not limited to: GCS program package (Devereux et al. (1984), Nucl. Acids Res. 12: 387), and the BLASTN or FASTA programs (Altschul et al. (1990), supra). The default settings provided with these programs are adequate for determining substantial similarity of nucleic acid sequences for purposes of the present invention.

"Substantially purified" refers to a peptide or nucleic acid sequence which is substantially homogenous in character due to the removal of other compounds (e.g., other peptides, nucleic acids, carbohydrates, lipids) or other cells originally present. "Substantially purified" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or formulation into a pharmaceutically acceptable preparation.

"Synthetic mutant" includes any purposefully generated mutant or variant protein derived from CHP-10, in particular from the amino acid sequence of SEQ ID NO:l. Such mutants may be purposefully generated by, for example, chemical mutagenesis, polymerase chain reaction (PCR) based approaches, or primer-based mutagenesis strategies well known to those skilled in the art.

Brief Description of the Figures

FIG. 1A is a photograph showing an SDS-PAGE of CHP-10 purified from a culture of Hypericum perforatum callus tissue. Lane 1 is a molecular weight marker; Lane 2 is the purified CHP-10, showing an apparent molecular weight of approximately 39 kDa, and Lane 3 is a protease digestion of the material from Lane 2, showing complete degradation of CHP-10. FIG. IB is a photograph showing an SDS-PAGE of CHP-10 purified from 8 separate H. perforatum callus cultures. Lane 1 is a molecular weight marker; Lane 2 is a whole cell extract from the flowers of cultivated H. perforatum plants; Lanes 3-

10 are whole cell extracts from H. perforatum callus tissue, showing CΗP-10 protein with an apparent molecular weight of approximately 39 kDa. FIG.2 is a plot showing the number of viable U87MG astrocytoma cells vs. hours treated with 12.5, 25, 50 or 75 micrograms CΗP-10 protein extract.

Untreated U87MG astrocytoma cells are shown as controls.

FIG. 3A is a light micrograph showing untreated T98G glioblastoma cells at 42 hrs. in culture. FIGS. 3B, 3C and 3D are light micrographs showing the effect of 5 micrograms, 10 micrograms, and 30 micrograms CΗP-10, respectively, on cultured T98G glioblastoma cells at 42 hrs. in culture, after approximately 26 hrs. exposure to CΗP-10.

FIG. 4A is a light micrograph showing untreated T98G glioblastoma cells at 42 hrs. in culture. FIGS. 4B and 4C are light micrographs showing, respectively, the effect of 12.5 micrograms CΗP-10 protein extract from Lane 2 in Fig. 1A and the protease digestion from Lane 3 in Fig. 1 A on cultured T98G glioblastoma cells.

FIG. 5 is a plot showing the number of viable T98G glioblastoma cells vs. hours treated with 12.5 micrograms CΗP-10. Untreated T98G glioblastoma cells and T98G glioblastoma cells treated with protease digested CΗP-10 are shown as controls.

Detailed Description of the Invention A cytotoxic and anti-cytoproliferative protein named CΗP-10 has been isolated and substantially purified from cultured callus tissue of Hypericum perforatum (St. John's wort). CΗP-10 has an apparent molecular weight of approximately 39 kDa by SDS-PAGE, and the first twenty amino acids of the N-terminal end as determined by automated Edman degradation are:

DINGGGATLPQALYQTSGNL (SEQ ID NO: 1). CHP-10 can be readily identified by the structural and functional characteristics listed above.

A suitable technique for isolating CHP-10 from H. perforatum callus tissue is given in Example 1 below. The CHP-10 protein was not found in whole cell extracts of the flowers of cultivated H. perforatum. Without wishing to be bound by any theory, it is possible that CHP-10 is expressed only in cultured H perforatum tissue (see Fig. IB). CHP-10 can also be produced synthetically by any known means, including synthesis by biological systems and by chemical methods. Biological synthesis of peptides is well known in the art, and includes the transcription and translation of a synthetic gene encoding CHP-10 sequences, especially SEQ ID NO: 1. Nucleic acid sequences encoding SEQ ID NO: 1 can be synthesized based on standard codon usage tables. See, e.g., Fig. 9.1 on pg. 214 of Lewin B, Genes NL Oxford University Press, Inc., New York, 1997, which is incorporated herein by reference. For example, nucleic acids encoding SEQ ID NO: 1 can have the formula:

5'-GAY ATH AAY GGN GGN GGN GCN ACN YTN CCN CAR GCN YTN TAYCARACNNNNGGNGTNYTN-3' (SEQIDNO: 2), wherein Y is T or C; H is A, C or T;

N is A, G, T or C; and R is G or A

These nucleic acids can be subcloned into an appropriate plasmid expression vector for propagation and expression in an appropriate host. The invention thus provides synthetic nucleic acids which encode CHP-10 protein sequences, their complementary sequences, and fragments and homologs thereof. Preferred nucleic acid sequences are those of SEQ ID NO: 2.

Techniques used to construct nucleic acid sequences, construct plasmid expression vectors, transfect host cells, and express a nucleic acid sequence of interest are widely practiced in the art, and practitioners of ordinary skill are familiar with the standard resource materials which describe specific conditions and procedures. For example, general methods for the cloning and expression of recombinant molecules are described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratories, 1982; and in Ausubel, Current Protocols in Molecular Biology, Wiley and Sons, 1987, the disclosures of which are incorporated herein by reference.

CHP-10 produced from an expression vector can be obtained from the host cell by cell lysis, or by using heterologous signal sequences fused to the expressed protein which cause secretion of the protein into the surrounding medium. Preferably, the signal sequence is designed so that it may be removed by chemical or enzymatic cleavage, as is known in the art. The CHP-10 thus produced can then be purified in a manner similar to that utilized for isolation of CHP- 10 from H. perforatum callus tissue.

Chemical peptide synthesis techniques suitable for directly synthesizing CHP-10, including manual and automated techniques, are well-known to those of ordinary skill in the art. For example, CHP-10 or SEQ ID NO: 1 can be synthesized de novo using conventional solid phase synthesis methods. In such methods, the peptide chain is prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group; various coupling reagents e.g., dicyclohexylcarbodiimide or carbonyldimidazole; various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide; and the various cleavage reagents, e.g., trifluoroactetic acid (TFA), HC1 in dioxane, boron tris-(trifluoracetate) and cyanogen bromide; and reaction in solution with isolation and purification of intermediates are methods well-known to those of ordinary skill in the art. A preferred chemical peptide synthesis method follows conventional

Merrifield solid phase procedures well known to those skilled in the art. Additional information about solid phase synthesis procedures can be had by reference to Steward and Young, Solid Phase Peptide Synthesis, W.H. Freeman & Co., San Francisco, 1969; the review chapter by Merrifield in Advances in Enzvmology 32:221-296, (Nold FF, ed.), Interscience Publishers, New York, 1969; and Erickson and Merrifield (1990), The Proteins 2:61-64, the entire disclosures of which are incorporated herein by reference. Crude peptide preparations resulting from solid phase syntheses may be purified by methods well known in the art, such as preparative HPLC. The amino-terminus may be protected according to the methods described for example by Yang et al. , EEES Eett. 272:61-64 (1990), the entire disclosure of which is herein incorporated by reference.

CHP-10 can also comprise a label a (e.g., substances which are magnetic resonance active; radiodense; fluorescent; radioactive; detectable by ultrasound; detectable by visible, infrared or ultraviolet light) so that the CHP-10 can be detected. Suitable labels include, for example, fluorescein isothiocyanate (FITC); peptide chromophores such as phycoerythrin or phycocyanin and the like; bioluminescent peptides such as the luciferases originating from Photinus pyrali; fluorescent proteins originating from Renilla reniformi; and radionuclides such as ³²P, ³³P, ³⁵S, I¹²⁵ or ¹²³I. For example, the label may comprise an NH₂-terminal fluorescein isothiocyanate (FITC)-Gly-Gly-Gly-Gly motif that is conjugated to a protein transduction domain.

Methods of modifying peptide sequences with labels are well known to those skilled in the art. For example, methods of conjugating fluorescent compounds such as fluorescein isothiocyanate to short peptides are described in Danen et al, Exp. Cell Res., 238:188-86 (1998), the entire disclosure of which is incorporated herein by reference.

The invention also provides biologically active fragments of CHP-10. Biologically active fragments according to the invention can be obtained, for example, by chemical or enzymatic fragmentation of larger natural or synthetic CHP-10 peptides, or by biological or chemical syntheses as described above. A preferred CHP-10 fragment is SΕQ ID NO: 1.

The invention also provides biologically active derivatives of CHP-10. The techniques for obtaining these derivatives are known to persons having ordinary skill in the art and include, for example, standard recombinant nucleic acid techniques, solid phase peptide synthesis techniques and chemical synthetic techniques as described above. Linking groups may also be used to join or replace portions of CHP-10, especially SΕQ ID NO: 1, and other peptides. Linking groups include, for example, cyclic compounds capable of connecting an amino-terminal portion and a carboxyl terminal portion of SEQ ID NO: 1. Techniques for generating derivatives are also described in U.S. patent 6,030,942 the entire disclosure of which is herein incorporated by reference (derivatives are designated "peptoids" in the 6,030,942 patent). CHP-10 derivatives may also incorporate labels such as are described above into their structure.

Examples of derivatives according to the present invention include, for example, synthetic variants of CHP-10. CHP-10 derivatives also include fusion peptides in which a portion of the fusion peptide has a substantially similar amino acid sequence to SEQ ID NO: 1. Such fusion peptides can be generated by techniques well-known in the art, for example by subcloning nucleic acid sequences encoding SEQ ID NO: 1 and a heterologous peptide sequence into the same expression vector, such that the SEQ ID NO: 1 and the heterologous sequence are expressed together in the same protein. The heterologous sequence may comprise a peptide leader sequence that directs entry of the expressed protein into a cell. Such leader sequences include "protein transduction domains" or "PTDs", which are discussed in more detail below.

The present invention also provides biologically active homologs of CHP-10. Homologs comprise a peptide sequence comprising a substantially similar amino acid sequence to SEQ ID NO: 1 that exhibit CHP-10 biological activity, and can be identified on this basis. CHP-10 homologs can also incorporate labels such as are described above into their structure.

The present invention also provides biologically active analogs of CHP- 10. Such analogs can, for example, be small organic molecules capable of inhibiting proliferation of cells from a cancer or a non-cancerous proliferative disorder. CHP-10 analogs may incorporate labels such as are described above into their structure.

CHP-10 analogs preferably comprise a structure, called a pharmacophore, that mimics the physico-chemical and spatial characteristics of CHP-10, especially of SEQ ID NO: 1. CHP-10 analogs can be identified by screening a library of pro-analogs designed by the retrosynthetic, target oriented, or diversity-oriented synthesis strategies described by Schreiber (2000), Science 287:1964-1969, the entire disclosure of which is herein incorporated by reference. Retrosynthetic strategies require the identification of key structural elements in a molecule. These elements are then incorporated into the structure of otherwise distinct pro-analogs generated by organic syntheses. U.S. Patent 6,030,942, in particular Example 4 therein, describes retrosynthetic methods for the design and selection of analogs based on key structural elements of a protein.

Key structural elements of CHP-10 can be identified, for example, by evaluating the various portions of CHP-10 for the ability to inhibit proliferation of cells by the assays of Examples 2 and 3. Alternatively, CHP-10 key structural elements can be determined using nuclear magnetic resonance (NMR), crystallographic, and/or computational methods which permit the electron density, electrostatic charges or molecular structure of certain portions of CHP-10 or fragments thereof to be mapped. Preferably, CHP-10 key structural elements comprise the primary, secondary and tertiary structure of the amino acid sequence of SEQ ID NO: 1.

Pools and subpools of pro-analogs can be generated by automated synthesis techniques performed in parallel, such that all synthesis and resynthesis can be performed in a matter of days. Once generated, pro-analog libraries can be screened for analogs; i.e. compounds exhibiting the ability to inhibit proliferation of cells by the assays of Examples 2 and 3 below.

CHP-10, and fragments, derivatives, homologs, and analogs of CHP-10, can be modified to enhance their entry into abnormally proliferating cells. For example, the compounds of the invention can be encapsulated in a liposome prior to being administered. The encapsulated compounds are delivered directly into the abnormally proliferating cells by fusion of the liposome to the cell membrane. Reagents and techniques for encapsulating the present compounds in liposomes are well-known in the art, and include, for example, the ProVectin™ Protein Delivery Reagent from Imgenex. In a preferred embodiment, the compounds of the invention are modified by associating the compounds with a peptide leader sequence known as a "protein transduction domain" or "PTD." These sequences direct entry of the compound into abnormally proliferating cells by a process known as "protein transduction." See Schwarze et al. (1999), Science 285: 1569 - 1572.

PTDs are well-known in the art, and may comprise any of the known PTD sequences including, for example, arginine-rich sequences such as a peptide of nine to eleven arginine residues optionally in combination with one to two lysines or glutamines as described in Guis et al. (1999), Cancer Res. 59: 2577-2580, the disclosure of which is herein incorporated by reference. Preferred are sequences of eleven arginine residues or the NH₂-terminal 11- amino acid protein transduction domain from the human immunodeficiency virus TAT protein (SEQ ID NO: 3). Other suitable leader sequences include, but are not limited to, other arginine-rich sequences; e.g., 9 to 10 arginines, or six or more arginines in combination with one or more lysines or glutamines. Such leader sequences are known in the art; see, e.g., Guis et al. (1999), supra. Preferably, the PTD is designed so that it is cleaved from the compound upon entry into the cell.

A PTD may be located anywhere on the compound that does not disrupt the compound's biological activity. For compounds of the invention comprising a peptide, the PTD is preferably located at the N-terminal end.

Kits and methods for constructing fusion proteins comprising a protein of interest (e.g., CHP-10) and a PTD are known in the art; for example the TransNector™ system (Q-BIOgene), which employs a 16 amino acid peptide called "Penetratin™" corresponding to the Drosophila antennapedia DNA- binding domain; and the Voyager system (Invitrogen Life Technologies), which uses the 38 kDa NP22 protein from Herpes Simplex Virus- 1. The present invention also provides antibodies against CHP-10 and derivatives, homologs, analogs, or antigenic fragments of CHP-10. An antibody of the invention specifically binds an epitope of CHP-10, especially epitopes of SEQ ID NO: 1. The antibody can be a monoclonal antibody, a polyclonal antibody or an antibody fragment that is capable of binding antigen. The antibodies of the invention include chimeric, single chain, and humanized antibodies, as well as Fab fragments and the products of an Fab expression library. Polyclonal antibodies of the invention can be produced by immunizing an animal with substantially pure CHP-10 or an immxmogenic fragment thereof, using techniques well-known in the art. Antibody fragments, such as Fab antibody fragments, which retain some ability to selectively bind to the antigen of the antibody from which they are derived, can be made using well known methods in the art. Such methods are generally described in U.S. patent 5,876,997, the entire disclosure of which is incorporated herein by reference.

Monoclonal antibodies can be prepared using the method of Mishell, B.B. et al., Selected Methods In Cellular Immunology, (Freeman WH, ed.) San Francisco, 1980, the disclosure of which is herein incorporated by reference. Briefly, a peptide is used to immunize spleen cells of Balb/C mice. The immunized spleen cells are fused with myeloma cells. Fused cells containing spleen and myeloma cell characteristics are isolated by growth in HAT medium, a medium which kills both parental cells, but allows the fused products to survive and grow. Thus in one embodiment, the invention comprises a hybridoma that produces a monoclonal antibody which specifically binds to the present compounds.

Antibodies of the invention can be used to purify the compounds of the invention, using immunoafϊinity techniques which are well known by those of skill in the art.

The compounds of the invention can be used to treat a subject having abnormally proliferating cells deriving from a cancer or a non-cancerous proliferative disorder. The compounds of the invention are administered to the subject in an amount effective to inhibit the proliferation of the abnormally proliferating cells (the "effective amount"). The subject can be any animal, preferably a mammal, particularly preferably a human being. As used herein, to "inhibit the proliferation of an abnormally proliferating cell" means to kill the cell, or permanently or temporarily arrest the growth of the cell.

Inhibition of proliferation can be inferred if the number of abnormally proliferating cells in the subject remains constant or decreases after administration of the present compounds. The number of abnormally proliferating cells in a subject's body can be determined by direct measurement (e.g, calculating the concentration of leukemic cells in the blood or bone marrow) or by estimation from the size of a tissue mass. As used herein, a "tissue mass" is any localized collection of abnormally proliferating cells in a subject's body; for example a tumor, fibroid body, restenotic plaque, etc. The size of a tissue mass can be ascertained by direct visual observation or by diagnostic imaging methods such as X-ray, magnetic resonance imaging, ultrasound, and scintigriphy. Diagnostic imaging methods used to ascertain size of a tissue mass can be employed with or without contrast agents, as is known in the art. The size of a tissue mass can also be ascertained by physical means, such as palpation of the tissue mass or measurement of the tissue mass with a measuring instrument such as a caliper.

The compounds of the invention can inhibit proliferation of abnormally proliferating cells from cancer types of diverse histologic subtype and origin, such as those listed and described in the National Cancer Institute's "CancerNet" at: http://cancemet.nci.nih.gov/pdq/pdq_frea1-ment.shtml which is herein incorporated by reference in its entirety. For example, the present compounds can be used to inhibit the proliferation of primary or metastatic tumor or neoplastic cells from cancers of at least the following histologic subtypes: sarcoma (cancers of the connective and other tissue of mesodermal origin); melanoma (cancers deriving from pigmented melanocytes); carcinoma (cancers of epithelial origin); adenocarcinoma (cancers of glandular epithelial origin); cancers of neural origin (glioma/glioblastoma and astrocytoma); and hematological neoplasias, such as leukemias and lymphomas (e.g., acute lymphoblastic leukemia, chronic lymphocytic leukemia, and chronic myelocytic leukemia).

The present compounds can also be used to inhibit the proliferation of primary or metastatic tumor or neoplastic cells from cancers having their origin in at least the following organs or tissues, regardless of histologic subtype: breast; tissues of the male and female urogenital system (e.g. ureter, bladder, prostate, testis, ovary, cervix, uterus, vagina); lung; tissues of the gastrointestinal system (e.g., stomach, large and small intestine, colon, rectum); exocrine glands such as the pancreas and adrenals; tissues of the mouth and esophagus; brain and spinal cord; kidney (renal); pancreas; hepatobiliary system (e.g., liver, gall bladder); lymphatic system; smooth and striated muscle; bone and bone marrow; skin; and tissues of the eye. Furthermore, the present compounds can inhibit the proliferation of cells from cancers or tumors in any prognostic stage of development, as measured, for example, by the "Overall Stage Groupings" (also called "Roman Numeral") or the Tumor, Nodes, and Metastases (TNM) staging systems. Appropriate prognostic staging systems and stage descriptions for a given cancer are known in the art, for example as described in: http://cancernet.nci.nih.gov/pdq/pdq_treatment.shtml, supra. The compounds of the invention are also effective in inhibiting proliferation of cells from non-cancerous proliferative disorders, including: hemangiomatosis in newborn; secondary progressive multiple sclerosis; chronic progressive myelodegenerative disease; neurofibromatosis; ganglioneuro- matosis; keloid formation; Paget's Disease of the bone; fibrocystic disease (e.g., of the breast or uterus); sarcoidosis; Peronies' and Duputren's fibrosis, cirrhosis, atherosclerosis and vascular restenosis.

The compounds of the invention can be administered to a subject by any technique designed to expose abnormally proliferating cells in the subject's body to the compounds, such that the compounds are taken up by the cells. For example, the compounds of the invention can be administered by any enteral or parenteral route. Parenteral administration is preferred.

Suitable parenteral administration methods include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, infra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g. peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); and direct application to the abnormally proliferating cells or to tissue comprising the abnormally proliferating cells, for example by a catheter or other placement device. It is preferred that subcutaneous injections or infusions be given in the area near the abnormally proliferating cells, particularly if the cells are on or near the skin.

The compounds of the invention can be injected in a single dose or in multiple doses. Infusion of the compounds of the invention can comprise a single sustained dose over a prolonged period of time or multiple infusions.

Direct injection into tissue comprising the abnormally proliferating cells is preferred.

An effective amount of the compounds of the invention can be based on the approximate weight of the tissue mass to be treated. The approximate weight of a tissue mass can be determined by calculating the approximate volume of the tissue mass, wherein one cubic centimeter of tissue mass volume is roughly equivalent to one gram.

An effective amount of the compounds of the invention based on the weight of a tissue mass can be at least about 10 μg compound/gram of tissue mass, and is preferably between about 10-1000 μg compound/gram of tissue mass. More preferably, the effective amount is at least about 60 μg compound/gram of tissue mass. Particularly preferably, the effective amount is at least about 100 μg compound/gram of tissue mass. It is preferred that effective amounts based on the weight of the tissue mass be injected directly into the tissue mass.

An effective amount of the compounds of the invention can also be based on the approximate or estimated body weight of the subject to be treated.

Preferably, such effective amounts are administered systemically; e.g. by intravascular injections and infusions, subcutaneous depositions or infusions, or intramuscular or intraperitoneal administrations.

For example, an effective amount of the compounds of the invention administered by single intravascular injection in humans (assuming a 60 kg subject) can range from about 5 - 3000 μg compound/kg of body weight, is preferably between about 700 - 1000 μg compound/kg of body weight, and is more preferably greater than about 1000 μg compound/kg of body weight.

An effective amount of the compounds of the invention used for multiple intravascular injections can be the same or lower than that used for single intravascular injections. For example, an effective amount for multiple intravascular injection in humans is preferably greater than about 250 μg compound/kg body weight, and is more preferably greater than about 500 μg compound/kg body weight. An effective amount of the compounds of the invention administered by single sustained intravascular infusion can be the same as that used for single and multiple intravascular injections, but may also be lower. For example, an effective amount for single sustained infusions in humans is preferably greater than about 90 μg compound kg body weight, and is more preferably greater than about 100 μg compound/kg body weight.

An effective amount of the compounds of the invention administered by multiple sustained intravascular infusions can be the same as that used for single and multiple injections and single sustained intravascular infusion, but may also be lower. For example, an effective amount for multiple sustained intravascular infusions in humans is preferably greater than about 35 μg/kg, and is more preferably greater than about 50 μg/kg.

An effective amount of the compounds of the invention administered by subcutaneous, intramuscular or intraperitoneal routes can be the same as that used for intravascular administration, but is preferably between about 200 and 1000 μg compound/kg body weight. More preferably, the effective amount is greater than 500 μg compound/kg of body weight.

An effective amount of the compounds of the invention can also be based on the approximate surface area of the subject to be treated. Effective amounts based on surface area are typically expressed in terms of μg compound/square meter of surface area (m²). It is preferred to base effective amounts on the surface area of a subject, because better inter-species comparisons can be made. Also, effective amounts based on surface area allow amounts to be determined for human adults and children without further adjustment. The assumptions underlying the inter-species and adult to child conversion of effective amounts based on surface area are found in EJ Freireich et al., (1966), Cancer Chemotherapy Reports 50: 219-244, the disclosure of which is herein incorporated by reference in its entirety.

Table 2 provides approximate surface area-to-weight ratios for various species. The surface area-to-weight ratio can be used to convert effective amounts based on body weight (expressed in μg/kg) to effective amounts based on surface area (expressed in μg/m²). The surface area-to-weight ratio is also used to calculate the conversion factors found in Table 3, which can be used to convert effective amounts expressed in terms of μg/kg from one species to another.

Table 2 - Surface Area to Weight Ratios of Various Species

Surface Area to

SpeciesBody Weight (kg) Surface Area (m²) Weight Ratio (kg/m²)

M Moouussee 0 0..0022 0.0066 3.0

R Raatt 0 0..1155 0.025 5.9

M Moonnkkeeyy 3 3 0.24 12

D Doogg 8 8 0.40 20

Human cchhiilldd 2200 0.80 25 adult 60 1.6 37

*Adapted from DeVita, VT, "Principles of Chemotherapy," pgs. 292-3, in Cancer: Principles and Practice of Oncology, (3^rd edit., DeVita VT, Hellman S, and Rosenberg SA, eds.), 1989, J. B. Lipincott Co., Phila., PA.

As shown in Table 2, to convert an effective amount based on body weight in μg/kg for any given species to the equivalent effective amount based on surface area (in μg/m²), multiply the effective amount based on body weight by the approximate surface area to weight ratio. For example, in the adult human 100 μg/kg is equivalent to 100 μg/kg X 37 kg/m² = 3700 μg/m².

Table 3 gives approximate factors for converting effective amounts expressed in terms of μg/kg from one species to an equivalent surface area effective amount expressed in the same units (μg/kg) for another species. For example, given a dose of 50 μg/kg in the mouse, the appropriate dose in man (assuming equivalency on the basis of μg/m²) is 50 μg/kg X 1/12 = 4.1 μg/kg. For the present invention, equivalency on the basis of μg/m² is assumed. Table 3 - Equivalent Surface Area Dosage Conversion Factors

Mouse Rat Monkey Dog Man

(20g) (150g) (3 kg) (8 kg) (60 kg)

Mouse 1 1/2 1/4 1/6 1/12

Rat 2 1 1/2 1/4 1/7

Monkey 4 2 1 3/5 1/3

Dog 6 4 5/3 1 1/2

Man 12 7 3 2 1 *Adapted from DeVita, VT, "Principles of Chemotherapy," pgs. 292-3, in Cancer: Principles and Practice of Oncology, (3^rd edit., DeVita VT, Hellman S, and Rosenberg SA, eds.), 1989, J. B. Lipincott Co., Phila., PA.

An effective amount of the compounds of the invention based on surface area is preferably admimstered systemically, as described above for effective amounts based on body weight. However, effective amounts based surface area can also be administered by peri- or infra-tissue mass injection or by direct application to the tissue mass.

CHP-10, and fragment, derivatives and homologs thereof can also be administered to a subject by fransfection of the abnormally proliferating cells in the subject's body with a nucleic acid sequence encoding the compounds. Preferably, the nucleic acid sequence comprises a plasmid expression vector. Such plasmids may be generated by recombinant nucleic acid and molecular cloning techniques well-known in the art, as discussed above. Transfection methods for eukaryotic cells are well known in the art, and include, for example, direct injection of the nucleic acid into the nucleus or pronucleus; electroporation; liposome transfer; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; and transfection mediated by viral vectors. In a preferred method, the transfection is performed with a liposomal transfer compound, e.g., DOTAP (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate, Boehringer - Mannheim) or an equivalent, such as LIPOFECTIN. The amount of nucleic acid used is not critical to the practice of the invention; acceptable results may be achieved with 10 mM nucleic acid/10⁵ cells. A ratio of about 500 nanograms of plasmid vector in 3 micrograms of DOTAP per 10⁵ cells may be used.

Other suitable methods for the construction and propagation of plasmid vectors capable of expressing the present compounds, and techniques for transfecting such vectors into eukaryotic cells so that the compounds are expressed, are known in the art.

The present invention also provides pharmaceutical formulations for treating cancer or non-cancerous proliferative disorders, comprising CHP10, or biologically active fragments, derivatives, homologs or analogs of CHP-10. Pharmaceutical formulations of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use.

Pharmaceutical formulations of the invention can be prepared by mixing the present compounds with a physiologically acceptable carrier medium to form solutions, suspensions or dispersions. Preferred physiologically acceptable carrier media are water or normal saline.

Pharmaceutical formulations of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example,

DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions (e.g. 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical formulations of the invention can be prepared in a manner fully within the skill of the art.

The invention will be illustrated with the following non-limiting examples. Example 1 - Isolation and Characterization of CHP-10

Culture of H perforatum callus - Explants of H perforatum were cultured on 0.6% agar in Murashige-Skoog N32 medium containing 0.5-1.0 mg/1 of 2,4-dichlorophenoxyacetic (2,4-D), 3% sucrose, and all necessary hormones as described in Popov YG et al. (1999), "In vitro cultivation of plant tissues and organs: application aspects and prospects," Materials ofSci. Conf "Science to production," pp. 103-105, Yerevan and Popov YG et al. (2000), "Setting up the collection of isolated cultures of Armenian flora plants and its biotechnological potential," Reports of Internal Seminar "Conversion Potential of Armenia and ISTC Programs," part II, pp.116-119, Yerevan, the disclosures of which are herein incorporated by reference in their entirety. Callus tissue was collected, lyophilized, and stored until used.

CHP-10 Protein Extraction - Whole cell extract was prepared from lyophilized H. perforatum callus tissue by resuspension and homogenization in ice-cold TNN buffer (100 mM Tris, pH 8.0; 100 mM NaCl; 0.5% NP-40) plus protease inhibitors (ImM AEBSF; 40 uM Bestatin; 14 uM E-64; 22 uM Leupeptin; 15 uM Pepstatin; and 800 nM Aprotinin). After homogenization, the lysates were cleared by centrifugation at 14,000 rpm at 4°C. Whole cell extracts from the flowers of cultivated H perforatum plants were prepared as above for the callus tissue, as a control. The callus tissue and cultivated flower whole cell extracts were assayed for protein content by Bradford analysis (Bio-Rad) according to the manufacturer's instructions, and analyzed by SDS-PAGE (see below). Analysis of CHP-10 bv SDS-PAGE - The CHP-10 protein extract was resolved on 10% acrylamide gels in IX Laemmli running buffer (0.025 M Tris, 0.192 M Glycine and 0.1 % SDS) at 130 V for 1.5 h, according to the Laemmli Gel method as described in Ausubel FM et al, Current Protocols in Molecular Biology - Vol. II. Harvard Medical School, Massachusetts General Hospital, 2001, the disclosure of which is herein incorporated by reference. The SDS- PAGE reagents were obtained from Bio-Rad. Following electrophoresis, the gels were stained with Coomassie Blue according to Ausubel FM (2001), supra.

The results are given in Figs. 1 A and IB.

Lane 2 of Fig. 1A shows a protein with an apparent molecular weight of approximately 39 kDa isolated from an H perforatum callus, which was named CHP-10. Digestion of the H. perforatum extract with agarose-bound protease type XI-A (Sigma, St. Louis, MO; used according to the manufacturer's instructions) abolished the approximately 39 kDa band (see Fig. 1A, lane 3).

Fig. IB shows the approximately 39 kDa CHP-10 protein isolated from eight different H. perforatum callus cultures (see lanes 3-10). The CHP-10 protein was not present in whole cell extracts from the H. perforatum flowers (Fig. IB, lane 2).

Primary Amino Acid Sequence of CHP-10 - The CHP-10 protein isolated from H. perforatum callus cultures was subjected to automated Edman degradation, and the first twenty amino acids of the N-terminal end were determined. The sequence of the first twenty N-terminal amino acids from

CHP-10 is given in SEQ ID NO: 1.

Example 2 - Inhibition of Proliferation of U87MG Human Astrocytoma Cells with CHP-10

Culture of U87MG Cells - The U87MG human astrocytoma cell line was obtained from the ATCC (catalog no. HB-14), and was maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY) with 10% fetal bovine serum (Gibco BRL, Rockville, MD) and penicillin/streptomycin in a humidified incubator containing 5% CO₂.

Inhibition of U87MG Cell Proliferation - Approximately 5 x 10^"4 U87MG cells were plated per 60 mm dish in culture media containing fetal bovine serum, and grown for 16 hours. The cultured U87MG cells were treated with 12.5, 25, 50 or 75 micrograms CHP-10 protein extract prepared as in Example 1. Untreated U87MG cells were used as a control. At 0, 2.5 and 24 hrs. post-treatment, the treated and control cells were harvested and counted by phase microscopy using a hemacytometer. The experiment was performed on triplicate plates and 3 different samples were counted from each plate. Fig. 2 shows that CHP-10 protein extract inhibited the growth of the cultured U87MG cells in a dose dependent manner. For the cells treated with 75 micrograms of CHP-10 protein extract, no U87MG cells were left alive at 24 hrs. post- treatment.

Example 3 - Inhibition of Proliferation of T98G Glioblastoma Cells with CHP-10

Culture of T98G Glioblastoma Cells - The T98G human glioblastoma multiforme cell line was obtained from the ATCC (catalog no. CRL-1690), and was maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY) with 10% fetal bovine serum (Gibco BRL, Rockville, MD) and penicillin/streptomycin in a humidified incubator containing 5% CO₂.

Inhibition of T98G Cell Proliferation - Approximately 5 x 10^"4 T98G cells were plated per 60 mm dish in culture media containing fetal bovine serum, and grown for 16 hours.

In one experimental group, the cultured T98G cells were treated with 5, 10 or 30 micrograms of the CHP-10 protein extract prepared as in Example 1 for an additional 26 hours (total of 42 hours in culture). Untreated T98G cells were used as a control. Figs. 3A-3D show that 5 micrograms of CHP-10 extract significantly inhibits proliferation of the T98G cells, and that virtually 100% of the T98G cells were dead after treatment with 10 and 30 micrograms CHP-10 extract.

In a second experimental group, cultured T98G cells were treated with 12.5 micrograms CHP-10 protein extract and protein extract treated with protease prepared as in Example 1, for an additional 26 hours (42 hours total in culture). Untreated T98G cells were used as a control. Figs. 4A-4C show an almost complete inhibition of proliferation in the T98G cells freated with 12.5 micrograms CHP-10 protein exfract. This effect was abolished by prefreatment of the CHP-10 protein extract with protease, indicating that the antiproliferative activity is due to the CHP-10 protein, and not a non-proteinaceous factor co- purified with the CHP-10. In a third experimental group, cultured T98G cells were treated with 12.5 micrograms CHP-10 protein extract and protein extract treated with protease prepared as in Example 1. Untreated T98G cells were used as a control. At 0, 4-6 and 24 hrs. and at 4 days post-treatment, the treated and control cells were harvested and counted by phase microscopy using a hemacytometer. The experiment was performed on triplicate plates and 3 different samples were counted from each plate. Fig. 5 shows that no T98G cells treated with CHP-10 protein extract were alive after 4 days in culture. The cells freated with CHP-10 protein extract plus protease showed moderate growth, and the untreated confrol cells showed the expected exponential growth.

Taken together, the results of all three experimental groups indicate that CHP-10 protein inhibits proliferation of human glioblastoma cells in culture in a dose dependent manner.

All documents referred to herein are incorporated by reference. While the present invention has been described in connection with the preferred embodiments and the various figures, it is to be understood that other similar embodiments may be used or modifications and additions made to the described embodiments for perforating the same function of the present invention without deviating therefrom. Therefore, the present invention should not be limited to any single embodiment, but rather should be construed in breadth and scope in accordance with the recitation of the appended claims.

Claims

We claim:

1. An isolated CHP- 10 protein, characterized in that: i) the protein comprises SEQ ID NO: 1 ; ii) has an apparent molecular weight of 39 kDa by SDS-PAGE through a 10% polyacrylamide gel in IX Laemmli buffer at 130V for 1.5 hours; and iii) inhibits proliferation of cultured T98G glioblastoma cells.

2. The isolated CHP-10 protein of claim 1, wherein the protein is substantially purified from Hypericum perforatum callus culture.

3. A compound that inhibits proliferation of cultured T98G glioblastoma cells, comprising a fragment, derivative, homolog or analog of the CHP-10 protein of claim 1.

4. The compound of claim 3 wherein the compound comprises a fragment having the amino acid sequence of SEQ ID NO: 1.

5. An isolated nucleic acid encoding comprising:

(a) a sequence encoding SEQ ID NO: 1; or

(b) the complement of (a).

6. The isolated nucleic acid of claim 5, wherein (a) comprises a nucleic acid of the formula:

5'-GAYATH AAY GGN GGN GGN GCN ACN YTN CCN CAR GCN YTN TAYCARACNNNNGGNGTNYTN-3' (SEQ IDNO: 2), wherein Y is T or C;

H is A, C or T;

N is A, G, T or C; and

R is G or A.

7. An antibody that specifically binds to an epitope on the isolated CHP-10 protein of claim 1.

8. The antibody of claim 7 which is a polyclonal antibody.

9. The antibody of claim 7 which is a monoclonal antibody.

10. A hybridoma producing the monoclonal antibody of claim 9.

11. An antibody that specifically binds to an epitope SEQ ID NO: 1.

12. The antibody of claim 11 which is a polyclonal antibody.

13. The antibody of claim 11 which is a monoclonal antibody.

14. A hybridoma producing the monoclonal antibody of claim 13.

15. A method of treating a subject having cells deriving from a cancer or a non-cancerous proliferative disorder, comprising administering to the subject an effective amount of a compound comprising the isolated CHP-10 protein of claim 1, or a biologically active fragment, derivative, homolog or analog thereof, such that proliferation of the cells is inhibited.

16. The method of claim 15, wherein the cells derive from cancer.

17. The method of claim 16, wherein the cancer is selected from the group consisting of: sarcoma; melanoma; carcinoma; adenocarcinoma; glioma; glioblastoma; astrocytoma; leukemia; and lymphoma.

18. The method of claim 16, wherein the cancer has its origin in organs or tissues selected from the group consisting of: breast; tissues of the male and female urogenital system; lung; tissues of the gastrointestinal system; pancreas; adrenals; tissues of the mouth and esophagus; brain and spinal cord; kidney; liver; gall bladder; lymphatic system; smooth and striated muscle; bone and bone marrow; skin; and tissues of the eye.

19. The method of claim 15, wherein the cells derive from a non- cancerous proliferative disorder.

20. The method of claim 19, wherein the non-cancerous proliferative disorder is selected from the group consisting of: hemangiomatosis in newborn; secondary progressive multiple sclerosis; chronic progressive myelodegenerative disease; neurofibromatosis; ganglioneuromatosis; keloid formation; Paget's Disease of the bone; fibrocystic disease; sarcoidosis; Peronies fibrosis; Duputren's fibrosis, cirrhosis, atherosclerosis and vascular restenosis.

21. The method of claim 15, wherein the compound is administered by direct injection into a tissue comprising the cells deriving from a cancer or a non-cancerous proliferative disorder.

22. A pharmaceutical composition comprising the CHP-10 protein of claim 1, or a biologically active fragment derivative, homolog or analog thereof.