WO2011094671A2

WO2011094671A2 - N-terminally conjugated polypeptides for targeted therapy and diagnosis

Info

Publication number: WO2011094671A2
Application number: PCT/US2011/023140
Authority: WO
Inventors: Ahmad Safavy
Original assignee: UAB Research Foundation
Current assignee: UAB Research Foundation
Priority date: 2010-01-29
Filing date: 2011-01-31
Publication date: 2011-08-04
Anticipated expiration: 2012-07-29
Also published as: WO2011094671A3

Abstract

The present disclosure provides polypeptide conjugates comprising a polypeptide moiety and an agent, wherein the polypeptide moiety is linked to the agent through the -amino group of the polypeptide moiety. The present disclosure also provides methods for the manufacture of polypeptide conjugates comprising a polypeptide moiety and an agent, wherein the polypeptide moiety is linked to the agent through the -amino group of the polypeptide moiety. The methods disclosed provide for the selective attachment of the agent to the polypeptide moiety. Furthermore, the method of attachment utilizes mild conditions to link the polypeptide moiety and the agent, thereby maintaining the overall molecular structure of the polypeptide moiety so that is biological activity and/or binding activity is maintained. Such conjugates have many uses, for example in targeting specific cell types, treating disease, preventing disease, and diagnosing disease.

Description

N-TERMINALLY CONJUGATED POLYPEPTIDES FOR TARGETED THERAPY AND DIAGNOSIS

Inventor: Ahmad Safavy

RELATED APPLICATIONS

This application cites the benefit of US 61/299,783, filed on January 29, 2010, and which is currently pending, for priority purposes.

FIELD OF THE DISCLOSURE

The present disclosure is directed generally to the design, synthesis and use of polypeptide conjugates. The present disclosure is specifically directed to the design, synthesis and use of polypeptide conjugates wherein the polypeptide moiety is conjugated to an agent via the a-amino group of the N-terminal amino acid residue of the polypeptide.

BACKGROUND

Diagnostic and/or therapeutic targeting of agents is an area of importance and interest in medical field. The primary benefit of tissue targeting is the specific delivery of the diagnostic or therapeutic agent to the site of a disease, minimizing impact on tissues and organs not involved in the disease. The immediate benefits of this approach include more effective diagnostic and therapeutic outcomes and preventing and/or reducing the damage to healthy tissue, thereby increasing the quality of life for the subject.

Current methods of tissue-targeting generally utilize polypeptide conjugates, with the polypeptide serving as the targeting moiety. The manufacture of such polypeptide conjugates involves a chemical attachment of the agent to be delivered (for example, a drug) to the polypeptide in a way that the tissue-targeting quality of the polypeptide is preserved. In some embodiment, the linkage between the agent and the polypeptide is designed so that the agent is released in vivo once the localization of the conjugate to the tissue of interest is complete.

Preservation of the biological activity of polypeptides generally requires the preservation of the overall molecular structure of the polypeptide. The overall molecular structure of the polypeptide is dependent on the secondary and tertiary structures, which are a direct function of the primary structure. The primary structure of the polypeptide is determined by the specific sequence of the amino acids making up the polypeptide. The primary sequence determines how a polypeptide folds and that folding determines the secondary and tertiary structures as well as the final overall molecular structure. As interaction of the polypeptide with its target is dependent on the overall molecular structure, any disturbance of the overall molecular structure is likely to result in loss or reduction of the polypeptide's biological activity or the ability of the polypeptide to bind to its target. Many of the methods currently practiced in the art utilize reaction chemistry for the linkage between the polypeptide and the agent that damages the overall molecular structure of the polypeptide.

In addition, many current methods to manufacture such polypeptide conjugates are not specific in nature, resulting in an inability to control the location on the polypeptide where the agent is linked. Furthermore, in many cases it desirable to produce such a conjugate with a 1 : 1 molar ratio of agent to polypeptide. Again, many current methods to manufacture such polypeptide conjugates are not able to control the agen polypeptide ratio. As a result, such a conjugate containing multiple copies of the agent may not exhibit the desired activity.

As a result of the foregoing, the biological activity of the polypeptide or the ability of the polypeptide to bind its target are decreased or eliminated. Due to these issues, the conjugate may not show the desired activity or may not work for its intended purpose. The art is lacking polypeptide conjugates and methods of making the same where the agent is linked to the polypeptide in a controlled and defined manner and in a manner that preserves the biological activity and/or the ability of the polypeptide conjugate to bind its target efficiently. Furthermore, the art is lacking polypeptide conjugates where the agent and polypeptide are present in a 1 : 1 molar ratio.

Therefore, it is desirable to link an agent to the polypeptide specifically and in a manner that preserves the biological activity and/or the ability of the polypeptide conjugate to bind its target efficiently. Furthermore, it is desirable to link the agent to the polypeptide in a 1 : 1 molar ratio. The present disclosure addresses these shortcomings.

SUMMARY

A method of linking an agent to a polypeptide is provided, comprising: providing a polypeptide comprising an unprotected and reactive terminal a- amino group, and at least one non-terminal amino group, wherein all said non-terminal amino groups are unreactive; contacting the polypeptide with at least one of an agent or an agent coupled to a linker, said agent or agent coupled to a linker comprising exactly one unprotected aldehyde group; creating a covalent bond selectively between the terminal oamino group and the unprotected aldehyde group; and producing a conjugate molecule with a ratio of polypeptide to agent of 1 : 1.

A method of linking an agent to a polypeptide is provided, comprising: providing polypeptide comprising a terminal a-amino group having a pK2 value, and at least non-terminal amino group, each non-terminal amino group having a pKR value; mixing said polypeptide with at least one of an agent or an agent conjugated to a linker, said agent or agent conjugated to a linker comprising exactly one unprotected aldehyde group, to form a reaction mixture; maintaining the reaction mixture at a reaction pH, wherein the polypeptide selectively forms a covalent bond with the terminal G?-amino group through a reaction between the aldehyde group and the terminal oamino group thereby producing a conjugate molecule with a ratio of polypeptide to agent of 1 : 1.

Conjugates are provided that are the products of the processes described above.

A polypeptide conjugate is provided, said polypeptide conjugate having the structure A- Li-poly, wherein A is an agent, L is a linker, poly is a polypeptide moiety and 1 is 0 or 1, wherein the agent is linked to the polypeptide moiety, either directly or through L, to an alpha amino group of the polypeptide moiety and wherein the agent and polypeptide moiety are present at a ratio of polypeptide to agent of 1 : 1.

The polypeptide moiety of the conjugate may be, for example, Leiurus quinquestriatus chlorotoxin (CTX) polypeptide, or derivatives thereof. Other examples of the polypeptide moiety include related small polypeptide scorpion toxins, which share significant homology with CTX.

A pharmaceutical composition is provided comprising pharmaceutically acceptable derivatives of any of the conjugates disclosed and a pharmaceutically acceptable carrier.

A method of delivering an agent to a cell is provided, comprising contacting the cell with the conjugates disclosed. Also provided is a method for the treatment or diagnosis of a neoplastic disease, comprising identifying a subject in need of such treatment or diagnosis, and administering the pharmaceutical composition disclosed herein to the subject. A use of the conjugates is provided in the manufacture of a pharmaceutical composition for the treatment or diagnosis of a neoplastic disease. Also provided is an ex vitro method for diagnosing a neoplastic disease comprising contacting a sample from a subject in need of such diagnosis with the conjugates disclosed.

The methods, compositions, and uses of this disclosure have many applications, including the targeting, treatment, and diagnosis of particular cell types and disease states related to them, such as cancers that express matrix metalloproteinase-2 (MMP-2).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of the chlorotoxin polypeptide isolated from Leiurus quinquestriatus scorpion venom. FIG. 2 shows an exemplary polypeptide conjugate of the present disclosure, in this figure a paclitaxel-chlorotoxin conjugate.

FIG. 3 shows analysis of a paclitaxel-chlorotoxin conjugate synthesized as described in Example 1 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS).

FIG. 4 shows the cytotoxic effect of a paclitaxel-chlorotoxin conjugate synthesized as described in Example 1 against human MDA-MB-468 breast cancer cells in vitro.

FIG. 5 shows the cytotoxic effect of a paclitaxel-chlorotoxin conjugate synthesized as described in Example 1 against human PC-3 prostate cancer cells in vitro.

FIG. 6 shows systemic toxicity studies of two different paclitaxel-chlorotoxin conjugates (PTXCTX-601 and PTXCTX-701) synthesized as described in Example 1 in normal nude mice as compared to PTX at equimolar doses.

FIG. 7 shows antitumor activity of a paclitaxel-chlorotoxin conjugate synthesized as described in Example 1 in nude mice implanted with MDA-MB-468 human breast cancer tumors.

FIG. 8 shows antitumor activity of a paclitaxel-chlorotoxin conjugate synthesized as described in Example 1 in nude mice implanted with PC-3 human prostate cancer tumors.

FIG. 9 shows an exemplary polypeptide conjugate of the present disclosure, in this figure a curcumin-chlorotoxin conjugate.

FIG. 10 shows the time dependent decomposition of a paclitaxel-chlorotoxin conjugate synthesized as described in Example 1 of the present disclosure to paclitaxel and linker- chlorotoxin polypeptide at 37 degrees C in pH 7.4 phosphate buffered saline.

FIG. 1 1 shows the sequence alignment between CTX and several other closely related scorpion toxins.

DETAILED DESCRIPTION

In the following discussion certain articles and methods may be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

Definitions

As used herein, the terms "prevention", "prevent", "preventing", "suppression",

"suppress" and "suppressing" as used herein refer to a course of action (such as administering a compound or pharmaceutical composition) initiated prior to the onset of a symptom, aspect, or characteristics of a disease or condition so as to prevent or reduce such symptom, aspect, or characteristics. Such preventing and suppressing need not be absolute to be useful.

As used herein, the terms "treatment", "treat" and "treating" as used herein refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a symptom, aspect, or characteristics of a disease or condition so as to eliminate or reduce such symptom, aspect, or characteristics. Such treating need not be absolute to be useful.

As used herein, the term "in need of treatment" as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.

As used herein, the term "in need of prevention" as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a disease or condition that is preventable by a method or compound of the disclosure.

As used herein, the term "cell-mediated disease or condition" refers to a disease or condition that is caused or exacerbated by the presence or activity of at least one cell type. Any type of cell may be involved, for example a cancer cell, a bacterial cell, a protist cell, or a fungal cell.

As used herein, the terms "individual", "subject" or "patient" as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

As used herein, the term "hydrolytically stable" refers to a linkage that is stable in aqueous solutions under physiological conditions; in one embodiment, such linkages are stable for at least 24 hours, 48 hours, 96 hours, 192 hours or greater; in an alternate embodiment such linkages are stable indefinitely.

As used herein, the term "hydrolytically labile" refers to a linkage that is stable in aqueous solutions under physiological conditions for about 24 hours or less.

As used herein, the term "physiological conditions" refers to an aqueous solution having a pH from 6-8 and a temperature from 30-42 degrees Celsius. As used herein, the term "link", "linked" "linkage" or "linker" when used with respect to a polypeptide conjugate described herein, or components thereof, refers to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent linkages.

As used herein, the term "therapeutically effective amount" as used herein refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial.

As used herein, the term "protected" with respect to hydroxyl groups, amine groups, sulfhydryl groups and other reactive groups refers to forms of these functionalities which are protected from undesirable reaction with a protecting group known to those skilled in the art such as those set forth in Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G. M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) (which is incorporated herein by reference for this teaching) which can be added or removed using the procedures set forth therein. Examples of protected hydroxyl groups include, but are not limited to, silyl ethers such as those obtained by reaction of a hydroxyl group with a reagent such as, but not limited to, t- butyldimethyl-chlorosilane, trimethylchlorosilane, triisopropylchlorosilane, triethylchlorosilane; substituted methyl and ethyl ethers such as, but not limited to methoxymethyl ether, methythiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, 1-ethoxyethyl ether, allyl ether, benzyl ether; esters such as, but not limited to, benzoylformate, formate, acetate, trichloroacetate, and trifluoracetate. Examples of protected amine groups include, but are not limited to, amides such as, formamide, acetamide, trifluoroacetamide, and benzamide; imides, such as phthalimide, and dithiosuccinimide; and others. Examples of protected sulfhydryl groups include, but are not limited to, thioethers such as S-benzyl thioether, and S-4-picolyl thioether; substituted S-methyl derivatives such as hemithio, dithio and amino thio acetals; and others.

As used herein, the term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, salt of an ester, solvate or other derivative of a polypeptide conjugate of the present disclosure that, upon administration to a recipient, is capable of providing (directly or indirectly) a polypeptide conjugate of the disclosure or a metabolite or residue thereof. Particularly favored derivatives are those that increase the bioavailability of the polypeptide conjugate of the disclosure when such polypeptide conjugate is administered to a patient (e.g., by allowing an orally administered compound to be more readily absorbed into the blood), enhance delivery of the polypeptide conjugate to a given biological compartment, increase solubility to allow administration by injection, alter metabolism or alter rate of excretion. In one embodiment, the derivative is a prodrug. Exemplary prodrug forms of macrolide antibiotics are described in U.S. Patent No. 6,809,080.

As used herein, the term "pharmaceutically acceptable salt(s)", unless otherwise indicated, includes salts of acidic or basic groups that may be present in the polypeptide conjugate of the present disclosure.

As used herein, the terms "polypeptide", "peptide" and "protein", used interchangeably herein, refer to a polymer of amino acids without regard to the length of the polymer. These terms also do not specify or exclude chemical or post-expression modifications, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention. The natural or other chemical modifications, such as those listed in examples above can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini, and may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. 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 cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. Also included within the definition are polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.

As used herein, the term "alkyl", whether used alone or as part of a substituent group, includes straight hydrocarbon groups comprising from one to twenty carbon atoms. Thus the phrase includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: -CH(CH₃)₂, -CH(CH₃)(CH₂CH₃), -CH(CH₂CH₃)₂, -C(CH₃)₃, - C(CH₂CH₃)₃, -CH₂ CH(CH₃)_2> - CH₂CH(CH₃)(CH₂CH₃), -CH₂CH(CH₂CH₃)₂, -CH₂C(CH₃)₃, - CH₂C(CH₂CH₃)₃, -CH(CH₃)CH(CH₃)(CH₂CH₃), - CH₂CH₂CH(CH₃)₂, CH₂CH₂CH(CH₃)(CH₂CH₃), -CH₂CH₂CH(CH₂CH₃)₂, -CH₂CH₂C(CH₃)₃,

CH₂CH₂C(CH₂CH₃)₃, -CH(CH₃)CH₂CH(CH₃)₂, -CH(CH₃)CH(CH₃)CH(CH₃)CH(CH₃)₂, - CH(CH₂ CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The phrase also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted with straight and branched chain alkyl groups as defined above. The phrase also includes polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings substituted with straight and branched chain alkyl groups as defined above.

As used herein, the term "alkenyl", whether used alone or as part of a substituent group, includes an alkyl group having at least one double bond between any two adjacent carbon atoms.

As used herein, the term "alkynyl", whether used alone or as part of a substituent group, includes an alkyl group having at least one triple bond between any two adjacent carbon atoms.

As used herein, the term "unsubstituted alkyl", "unsubstituted alkenyl" and "unsubstituted alkynyl" refers to alkyl, alkenyl and alkynyl groups that do not contain heteroatoms.

The phrase "substituted alkyl", "substituted alkenyl" and "unsubstituted alkynyl" refers to alkyl alkenyl and alkynyl groups as defined above in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen or non-carbon atoms such as, but not limited to, an oxygen atom in groups such as alkoxy groups and aryloxy groups; a sulfur atom in groups such as, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a silicon atom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups.

As used herein, the term "unsubstituted aryl" refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as, but not limited to, phenyl, naphthyl, anthracenyl, biphenyl and diphenyl groups, that do not contain heteroatoms. Although the phrase "unsubstituted aryl" includes groups containing condensed rings such as naphthalene, it does not include aryl groups that have other groups such as alkyl or halo groups bonded to one of the ring members, as aryl groups such as tolyl are considered herein to be substituted aryl groups as described below. Unsubstituted aryl groups may be bonded to one or more carbon atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parent compound, however.

As used herein, the term "substituted aryl group" has the same meaning with respect to unsubstituted aryl groups that substituted alkyl groups had with respect to unsubstituted alkyl groups. However, a substituted aryl group also includes aryl groups in which one of the aromatic carbons is bonded to one of the non-carbon or non-hydrogen atoms, such as, but not limited to, those atoms described above with respect to a substituted alkyl, and also includes aryl groups in which one or more aromatic carbons of the aryl group is bonded to a substituted and/or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This includes bonding arrangements in which two carbon atoms of an aryl group are bonded to two atoms of an alkyl or alkenyl, group to define a fused ring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus, the phrase "substituted aryl" includes, but is not limited to tolyl, and hydroxyphenyl among others.

As used herein, the term "unsubstituted aralkyl" refers to unsubstituted alkyl or alkenyl groups as defined above in which a hydrogen or carbon bond of the unsubstituted or substituted alkyl or alkenyl group is replaced with a bond to a substituted or unsubstituted aryl group as defined above. For example, methyl (CH₃) is an unsubstituted alkyl group. If a hydrogen atom of the methyl group is replaced by a bond to a phenyl group, such as if the carbon of the methyl were bonded to a carbon of benzene, then the compound is an unsubstituted aralkyl group (i.e., a benzyl group).

As used herein, the term "substituted aralkyl" has the same meaning with respect to unsubstituted aralkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups. However, a substituted aralkyl group also includes groups in which a carbon or hydrogen bond of the alkyl part of the group is replaced by a bond to a non-carbon or a non- hydrogen atom.

As used herein, the term "unsubstituted heterocyclyl" refers to both aromatic and nonaromatic ring compounds including monocyclic, bicyclic, and polycyclic ring compounds such as, but not limited to, quinuclidyl, containing 3 or more ring members of which one or more is a heteroatom such as, but not limited to, N, O, and S. Although the phrase "unsubstituted heterocyclyl" includes condensed heterocyclic rings such as benzimidazolyl, it does not include heterocyclyl groups that have other groups such as alkyl or halo groups bonded to one of the ring members, as compounds such as 2-methylbenzimidazolyl are "substituted heterocyclyl" groups as defined below. Examples of heterocyclyl groups include, but are not limited to: unsaturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl; saturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensed unsaturated heterocyclic groups containing 1 to 4 nitrogen atoms such as, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl; unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl; saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl, benzoxadiazolyl, benzoxazinyl (e.g. 2H-l ,4-benzoxazinyl etc.); unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, thiazolyi, isothiazolyl, thiadiazolyl (e.g. 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, thiazolodinyl; saturated and unsaturated 3 to 8 membered rings containing 1 to 2 sulfur atoms such as, but not limited to, thienyl, dihydrodithiinyl, dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran; unsaturated condensed heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g. 2H-l,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g. 2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8 membered rings containing oxygen atoms such as, but not limited to furyl; unsaturated condensed heterocyclic rings containing 1 to 2 oxygen atoms such as benzodioxolyl (e.g. 1 ,3-benzodioxoyl, etc.); unsaturated 3 to 8 membered rings containing an oxygen atom and 1 to 2 sulfur atoms such as, but not limited to, dihydrooxathiinyl; saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as 1 ,4-oxathiane; unsaturated condensed rings containing 1 to 2 sulfur atoms such as benzothienyl, benzodithiinyl; and unsaturated condensed heterocyclic rings containing an oxygen atom and 1 to 2 oxygen atoms such as benzoxathiinyl. Heterocyclyl group also include those described above in which one or more S atoms in the ring is double-bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include tetrahydrothiophene, tetrahydrothiophene oxide, and tetrahydrothiophene 1,1 -dioxide. Preferred heterocyclyl groups contain 5 or 6 ring members. More preferred heterocyclyl groups include morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine, thiomorpholine in which the S atom of the thiomorpholine is bonded to one or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, and tetrahydrofuran.

As used herein, the term "substituted heterocyclyl" has the same meaning with respect to unsubstituted heterocyclyl groups that substituted alkyl groups had with respect to unsubstituted alkyl groups. However, a substituted heterocyclyl group also includes heterocyclyl groups in which one of the carbons is bonded to one of the non-carbon or non-hydrogen atom, such as, but not limited to, those atoms described above with respect to a substituted alky and substituted aryl groups and also includes heterocyclyl groups in which one or more carbons of the heterocyclyl group is bonded to a substituted and/or unsubstituted alkyl, alkenyl or aryl group as defined herein. This includes bonding arrangements in which two carbon atoms of an heterocyclyl group are bonded to two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system. Examples, include, but are not limited to, 2-methylbenzimidazoIyl, 5- methylbenzimidazolyl, 5-chlorobenzthiazolyl, 1 -methyl piperazinyl, and 2-chloropyridyl among others.

As used herein, the term "unsubstituted heterocyclylalkyl" refers to unsubstituted alkyl or alkenyl groups as defined above in which a hydrogen or carbon bond of the unsubstituted alkyl or alkenyl group is replaced with a bond to a substituted or unsubstituted heterocyclyl group as defined above. For example, methyl (CH₃) is an unsubstituted alkyl group. If a hydrogen atom of the methyl group is replaced by a bond to a heterocyclyl group, such as if the carbon of the methyl were bonded to carbon 2 of pyridine (one of the carbons bonded to the N of the pyridine) or carbons 3 or 4 of the pyridine, then the compound is an unsubstituted heterocyclylalkyl group.

As used herein, the term "substituted heterocyclylalkyl" has the same meaning with respect to unsubstituted heterocyclylalkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups. However, a substituted heterocyclylalkyl group also includes groups in which a non-hydrogen atom is bonded to a heteroatom in the heterocyclyl group of the heterocyclylalkyl group such as, but not limited to, a nitrogen atom in the piperidine ring of a piperidinylalkyl group.

As used herein "carbocyclyl" refers to cyclic compounds in which all of the ring members are carbon atoms. These may be aromatic or not. An "unsubstituted carbocyclyl" refers to a cyclic compound in which all of the ring members are carbon atoms, and in which all carbon atoms are saturated. A "substituted carbocyclyl" has the same meaning with respect to unsubstituted carbocyclyl that substituted aryl groups had with respect to unsubstituted aryl groups.

As used herein, the terms "about" and "approximately" shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within 20%, within 10%, and within 5% of a given value or range of values. For biological systems, the terms refer to an acceptable standard deviation of error, preferably not more than 2-fold of a give value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

Unless explicitly stated otherwise, all references to the singular should be construed to encompass the plural, all references to the plural should be construed to encompass the singular, all references to the masculine should be construed to encompass the feminine, and all references to the feminine should be construed to encompass the masculine.

Polypeptide Conjugate

The present disclosure provides polypeptide conjugates comprising a polypeptide moiety and an agent, wherein the polypeptide moiety is linked to the agent through the a-amino group of the polypeptide moiety. The present disclosure also provides methods for the manufacture of polypeptide conjugates comprising a polypeptide moiety and an agent, wherein the polypeptide moiety is linked to the agent through the a-amino group of the polypeptide moiety. The conjugates of the instant disclosure include the conjugates that are the products of the methods of manufacture, as described below. Following the methods disclosed, the intramolecular amine functions, such as ε-ΝΗ₂ of lysine, the τ- and π-amino groups of histidine and the guanidine amines of arginine do not undergo linkage with the agent (such non-a amino groups being referred to here as "non-terminal"). Therefore, the methods disclosed provide for the selective attachment of the agent to the polypeptide moiety.

The method of attachment utilizes mild conditions to link the polypeptide moiety and the agent, thereby maintaining the overall molecular structure of the polypeptide moiety so that is biological activity and/or binding activity is maintained. Furthermore, the methods disclosed provide a polypeptide conjugate where the polypeptide moiety and the agent are present in a 1 : 1 molar ratio. As a result of N-terminal attachment of the agent to the polypeptide moiety and the presence of a single molecule of the agent linked to the polypeptide moiety, the polypeptide conjugates provided exhibit no or minimal steric hindrance from the linked agent. Therefore, the biological activity and/or binding activity of the polypeptide moiety are not significantly impacted. Finally, the methods provided are simple to implement and provide polypeptide conjugates of high purity with high yields of product recovery.

In one embodiment, the present disclosure provides a polypeptide conjugate for use in therapeutic and/or diagnostic applications. In the most general form, the polypeptide conjugate comprises a polypeptide moiety, an agent linked to the polypeptide moiety and an optional linker, wherein the agent is linked to the polypeptide moiety through the a-amino group of polypeptide moiety. Therefore, the agent is linked to the polypeptide conjugate in a specific (site directed) manner.

In certain embodiment, a linker is used to link the polypeptide moiety to the agent. Exemplary linkers are described herein. In other embodiments, a linker is not used and a functional group on the agent is linked directly to the polypeptide moiety as described.

The polypeptide conjugate may be illustrated schematically as shown in formula I below:

A-L-poly I

where A represent the agent, L represent the optional linker and poly represent the polypeptide moiety.

The nature of the linker, polypeptide moiety and the agent may be selected based on the disease to be treated or the application (diagnostic or therapeutic). Various exemplary polypeptide moieties, linkers and agents are described herein, alone and in combination with one another; however, such descriptions are provided for exemplary purposes only are not meant to limit the selection of polypeptide moiety or agent. As will be discussed herein, a variety of polypeptide moieties, agents and linkers may be used based on the teachings of the present disclosure.

The linker, when used, provides a linkage between the polypeptide moiety and the agent. In certain embodiments, a linker is useful in providing separation between the polypeptide moiety and the agent. In one embodiment, the linker may provide a hydrolytically stable linkage between the polypeptide moiety and the agent. In an alternate embodiment, the linker may provide a linkage that is hydrolytically labile. The linker has a first end and a second end. The first end contains a first functional group capable of reacting and forming a linkage with a binding partner on the agent. The second end contains a second functional group capable of reacting and forming a linkage with a-amino group of polypeptide moiety.

The linker may be illustrated schematically as shown in formula II below:

FGi-X-FG₂ II

where FGi represent the first functional group, X represent a variable sequence connecting the first and second functional groups and FG₂ represents the second functional group, wherein the second function group is an aldehyde group or a ketone group. X can be any compound that contains the first and second functional groups. In one embodiment, X is a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, aralkyl, heterocycle or heterocyclylalkyl. In one embodiment, X is a substituted or unsubstituted alkyl, alkenyl or alkynyl; in a further embodiment, the substituted or unsubstituted alkyl, alkenyl or alkynyl are C1-C6 or CI -CIO. In an alternate embodiment, X is a polymer, such as, but not limited to, a polyethylene glycol, polyoxazoline, dextran, polyvinyl, pyrrolidones, polyacrylamides, or the like. In one embodiment, X is non-immunogenic and does not produce toxic compounds when degraded in the body. Either or both of the first and second functional groups may be initially a protected group, as may be the binding partner on the agent.

In one embodiment, the first functional group is -COOH or -COZ and the binding partner on the agent is a -OH group. In an alternate embodiment, the first functional group is - COOH, -COZ, -CHO or R'COR" and the binding partner on the agent is a -NH₂ group. In still a further alternate embodiment, the first functional group is -SH, COOH, -COZ or Z-CH₂CO-Y and the binding partner on the agent is a -SH group. In yet another alternate embodiment, the first functional group is -CHO, R'COR", -COOH or COZ and the binding partner on the agent is a -NHNH₂ group. In the foregoing, Z is a halide (such as Br and I), Y is OH or OR, and R, R' and R" are independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl carbocyclyl, aromatic, or heterocyclyl groups. In one embodiment, the substituted or unsubstituted alkyl, alkenyl or alkynyl groups are C1-C6 or CI -CI O.

In a particular embodiment, the first functional group is -COOH or -COZ, the agent is paclitaxel and the binding partner is a -OH group on the paclitaxel molecule; in one embodiment the -OH group is the 2' -OH group on the paclitaxel molecule. In another particular embodiment, the first functional group is -COOH or -COZ, the agent is curcumin and the binding partner is a -OH group on the curcumin molecule; in one embodiment the -OH group is a phenolic -OH group of curcumin.

In one embodiment, the second functional group is an aldehyde group. In still a further embodiment, the second functional group is a ketone group. The aldehyde or ketone group may be initially a protected aldehyde group or a protected ketone group.

When the linker is not present, a functional group on the agent may be used to form a linkage with the a-amino group of the N-terminal amino acid residues of the polypeptide moiety. In specific embodiments, the functional group on the agent is an aldehyde group or a ketone group. As discussed above, the aldehyde group or ketone group may be initially a protected aldehyde group or a protected ketone group.

Agents

The agent can be any diagnostic agent currently known in the art or any therapeutic agent currently known in the art provided that the diagnostic or therapeutic agent has a binding partner capable of reacting with the first functional group on the linker or has a functional group capable of forming a linkage with the a-amino group of the N-terminal amino acid residues of the polypeptide moiety. Exemplary functional groups and binding partners have been described above. In certain cases a diagnostic agent may also be a therapeutic agent, and vice versa. The selection of a particular agent will depend on the particular disease to be treated or condition to be diagnosed and is within the ordinary skill in the art.

The diagnostic agent may be a fluorescent label, a radiolabel, an enzymatic label, a metallic contrast agent, or a quantum dot^® label. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, auramine, Texas Red, AMCA blue, pyrene, perylene, borodiazaindacene (BODIPY™), cyanine dyes, Alexa dyes and Lucifer Yellow. Suitable radiolabels include, alpha-, beta-, or gamma-emitting radionuclides, such as, but are not limited to, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁸⁸Y, ⁹⁰Y, ^99mTc, ¹²³I, ¹²⁵I, ^I31I, ¹⁷⁷Lu, ^{, 86}Re, and ^{, 88}Re. Suitable enzymatic labels include, but are not limited to, -glucuronidase, -D-glucosidase, - D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. Suitable metallic contrast agents include gadolinium, manganese, iron and derivatives of the foregoing and similar molecules that induce both positive and negative contrast. Diagnosis using the polypeptide conjugates may be accomplished by administering to the subject a diagnostically effective amount of the polypeptide conjugate, allowing the polypeptide conjugate to bind the target cells and detecting and measuring the level of binding to the target cells. An increased binding level as compared to a normal subject free of the disease state or condition to be diagnosed is indicative of the presence of the disease state or condition. When the label is a fluorescent label or a quantum dot^®, the label may be detected by fluorescence microscopy, fluorescence-activated cell sorting or a fluorescence plate reader. When the label is a radiolabel, the radiolabel may be detected by positron emission tomography scanning or similar methods. When the label is an enzymatic label, the enzymatic label may be detected immunohistochemically or by use of a calorimetric assay. When the label is a metallic contrast agent, the label may be detected by MRI or similar technologies.

Therapeutic agents include, but are not limited to, drugs, anti-tumor agents, cytotoxic agents, radionuclides, and metallic nuclei. An exemplary therapeutic agent described in this specification is a taxane molecule. By "taxane" it is meant to include any taxane derivatives such as paclitaxel (PTX) and docetaxel and their analogues and pharmaceutically acceptable salts. In one embodiment, the therapeutic agent PTX is described.

Another exemplary therapeutic agent is an anthracycline antibiotic. Anthracycline antibiotics are commonly used to kill or inhibit the growth of cancer cells; they are also effective against bacteria, although their high toxicity to humans has so far prevented widespread in vivo use for this purpose. Anthracycline antibiotics that are used to kill or inhibit the growth of cancer cells include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone (which is actually an anthracycline analog). In a certain embodiment of the conjugate doxorubicin is the agent; the use of doxorubicin is advantageous due to its potency as an antineoplastic agent and its elucidated pharmacokinetics.

Some embodiments of the therapeutic agent are therapeutic agents that act on the brain. Such agents may be conjugated to polypeptides capable of penetrating the blood-brain-barrier, enabling the agent to cross the barrier. This could be particularly useful for drugs capable of treating disorders such as Alzheimer's disease that are incapable of crossing the barrier, such as taxol.

Additional exemplary therapeutic agents include but are not limited to curcumin, gemcitabine, camptothecin and analogues of the foregoing. Suitable radiolabels include, alpha-, beta-, or gamma-emitting radionuclides, such as, but are not limited to, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁸⁸Y, ⁹⁰Y, ^99mTc,¹²³I, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁸⁶Re, and ¹⁸⁸Re. When injected via a systemic route, such as, an intravenous, intramuscular, intraperitoneal or subcutaneous route, the polypeptide conjugate circulates through the body until the polypeptide moiety detects and binds to a binding target on the target cell. Once bound to the target cell, the therapeutic agent acts on the target cell through internalization or proximity to the cell, causing cell damage, decreased proliferation and/or cell death. Alternatively, for treatment of disease states and conditions of the spinal cord or brain, including cancer, the polypeptide conjugate may be injected directly into the spinal cord of cranium through intrathecal or intracranial (intraventricular) routes allowing a greater dose to be delivered to the these areas for therapeutic or diagnostic purposes while minimizing systemic toxicity.

Polypeptide Moieties

The polypeptide moiety may be any polypeptide that is known in the art, provided the polypeptide contains an a-amino group capable of reacting and forming a linkage with the second functional group on the linker or the functional group on the agent.

In one embodiment, the polypeptide moiety has a targeting function. In an alternate embodiment, the polypeptide moiety has a therapeutic function. Both activities may be shared by the polypeptide moiety. The targeting function allows the polypeptide moiety to direct the polypeptide conjugate to a defined population of cells expressing a target with which the polypeptide moiety interacts, thereby allowing the agent of the polypeptide conjugate to come into contact with the target cell and exert its effects. The agent and the polypeptide moiety (when the agent has a therapeutic effect) may act through similar mechanisms or through distinct mechanisms. In one embodiment, the agent and the polypeptide moiety (when the agent has a therapeutic effect) act through distinct mechanisms.

Embodiments of the polypeptide may comprise a variant of a targeting polypeptide that retains its targeting function. Examples of such variants include variants with high levels of sequence identity, such as at least 70%, 75%, 80%, 85%, 90%, 95%, and 99%. Further examples of such variants include those than contain conservative modifications or substitutions of the native polypeptide, as explained in greater detail below.

In some embodiments of the conjugate, the polypeptide moiety is a scorpion toxin. These are generally known to target specific cell types and inhibit certain types of cellular activity. The primary amino acid sequences of exemplary scorpion toxins are found in FIG. 1 1. In one embodiment, the polypeptide moiety is a chlorotoxin (CTX) polypeptide or a fragment thereof. The amino acid sequence of the native form of CTX polypeptide is provided in FIG. 1 and SEQ ID NO: 1 (this polypeptide is sometimes referred to as CTX-601). The CTX polypeptide is a 36 amino acid peptide (MW 3950) originally isolated from Leiurus quinquestriatus scorpion venom (FIG. 1). This polypeptide contains four disulfide-bridged loops along the length of the polypeptide. The CTX polypeptide has been shown to bind specifically to matrix metalloproteinase-2 (MMP-2) (J. Biol Chem., 2003, 278(6), pp. 4135- 4144) and other molecular target, such as but not limited to, annexin A2 (J. Biol. Chem., PMID 20018898). The CTX polypeptide has also been shown to inhibit chloride transport by reducing the number of chloride receptors at the cell surface. Up-regulation of MMP-2 has been observed in tumors of neuroectodermal origin including peripheral neuroectodermal tumors (PNET) and gliomas. These include medulloblastomas, neuroblastomas, ganglioneuromas, melanomas, adrenal pheochromocytomas, primitive PNET, small cell lung carcinoma, and Ewing's sarcoma (Glia. 2002, 39(2), pp 162-173). In addition, MMP-2 is expressed cancers of the breast, colon, skin, lung, prostate and ovaries. In previous work, the CTX polypeptide was fund to bind to 74 of 79 screened human cancer types while failing to bind to 17 normal human tissues. Radiolabeled CTX selectively binds to gliomas in human subjects long after it has been eliminated from other parts of the body (Hockaday et al., J. Nuclear Med. 46(4):580 (2005); Mamelak et al., J Clin Oncol. 24:3644-3650 (2006)). Therefore, the CTX polypeptide is useful in for the treatment of a wide variety of tumor cell types as well as other diseases or conditions. These findings suggest that the CTX polypeptide is a tumor-specific polypeptide with significant therapeutic potential for cancers and other diseases. Likewise these findings suggest that the CTX polypeptide may be used as a diagnostic agent for cancers and other diseases, such as those that involve the activity of the MMP-2 and other binding targets of the CTX polypeptide.

In one embodiment, the CTX polypeptide has a targeting function to direct the polypeptide conjugate to a population of cancer cells, such as those expressing MMP-2 or another target of the CTX polypeptide. In an alternate embodiment, the CTX polypeptide has a therapeutic function and acts by inhibiting the activity and/or cell surface expression of MMP-2 and/or chloride channels. As a targeting agent, the CTX polypeptide allows the agent linked to the polypeptide conjugate to come into contact with the target cell allowing the agent to exert its effects on the target cell. The agent and the CTX polypeptide may act through similar mechanisms or through distinct mechanisms. In one embodiment, the agent and the CTX polypeptide act through distinct mechanisms.

The advantages of using scorpion toxins such as the CTX polypeptide include ease of use. They are short peptides that can be readily synthesized through means known in the art with high purity. Furthermore, the CTX polypeptide is well characterized and is amenable to molecular and structural alterations. In addition, the CTX polypeptide has shown safety and tumor specific targeting in phase I and II clinical trials. In this regard, the CTX polypeptide has shown short systemic clearance times and rapid tumor localization in animal and human studies, reducing the effects of toxicity for the polypeptide conjugate. Still further, the CTX polypeptide has been shown to bind specifically to cancerous tissues but not to normal tissues, reducing toxicity to the subject.

The present disclosure contemplates the use of CTX polypeptide having the sequence of SEQ ID NO: 1 as well as CTX polypeptide derivatives in the methods and compositions described. As defined herein, CTX polypeptide derivative includes any naturally occurring variant of the CTX due to existing polymorphism or mutations in the gene or protein as it occurs in L. quinquestriatus. A derivative of CTX also includes any variant capable of binding to MMP-2. A derivative of CTX also includes any variant comprising cysteine residues at positions 2, 5, 16, 19, 20, 28, 33, and 35 of SEQ ID NO: 1. A derivative of CTX also includes any variant comprising at least one conservative substitution of an amino acid residue at any of positions 1 , 3, 4, 6-15, 17, 18, 21-27, 29-32 and 34 of SEQ ID NO: 1. In one embodiment of the polypeptide conjugate, the CTX derivative comprises the cysteine residues at positions 2, 5, 16, 19, 20, 28, 33, and 35 of SEQ ID NO: 1 and at least one conservative substitution of an amino acid residue at any of positions 1 , 3, 4, 6-15, 17, 18, 21-27, 29-32, and 34 of SEQ ID NO: 1. In a particular embodiment, a CTX derivative includes a substitution of phenylalanine for tyrosine at position 29 of SEQ ID NO: 1. This CTX variant has been shown to retain the functions of the CTX polypeptide encoded by SEQ ID NO: 1 (this polypeptide is sometimes referred to as CTX- 701). Useful CTX polypeptide derivatives retain the ability to bind to MMP-2 or other cellular targets of CTX.

Additional embodiments of the polypeptide moiety include variants of CTX having at least 90% sequence identity with SEQ ID NO: 1 ; some such variants will be at least 33 residues in length. Further embodiments of the polypeptide moiety include a variant of CTX of at least 33 residues in length, having at least 90% sequence identity with SEQ ID NO: 1, and in which all non-identical amino acids are conservative substitutions of the native amino acids.

Other variants of CTX are fragments of at least 10 residues in length having at least 90% homology to a ten contiguous residues in SEQ ID NO: 1. Conservative modifications to the amino acid sequence of SEQ ID NO: 1 (and the corresponding modifications to the encoding nucleotides) will produce CTX polypeptide derivatives having functional and chemical characteristics similar to those of naturally occurring CTX polypeptide. For example, a "conservative amino acid substitution" for a given polypeptide may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, except for the cysteine residues at position 2, 5, 16, 19, 20, 28, 33, and 35.

Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. It will be appreciated by those of skill in the art that nucleic acid and polypeptide molecules described herein may be chemically synthesized as well as produced by recombinant means.

Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics; these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. Mol. Biol, 157: 105-131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity.

In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +1- 2 may be used; in an alternate embodiment, the hydropathic indices are with +/- 1 ; in yet another alternate embodiment, the hydropathic indices are within +/- 0.5. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a polypeptide as governed by the hydrophilicity of its adjacent amino acids correlates with a biological property of the protein.

The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +/- 2 may be used; in an alternate embodiment, the hydrophilicity values are with +/- 1 ; in yet another alternate embodiment, the hydrophilicity values are within +/- 0.5.

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the CTX polypeptide, or to increase or decrease the affinity of the CTX polypeptide with a particular binding target in order to increase or decrease the activity of the CTX polypeptide.

Exemplary amino acid substitutions are set forth in the Table 1 below

Leu He, Val, Met, Ala, Phe, Norleucine He

Lys Arg, 1 ,4-diaminobutyric acid, Gin, Asn Arg

Met Leu, Phe, He Leu

Phe Leu, Val, He, Ala, Tyr Leu

Pro Ala, Gly Gly

Ser Thr, Ala, Cys Thr

Thr Ser Ser

Trp Tyr, Phe Tyr

Tyr Trp, Phe, Thr, Ser Phe

Val He, Met, Leu, Phe, Ala, Norleucine Leu

Based on this disclosure and the state of the art, one of ordinary skill in the art could formulate variants of CTX capable of effectively binding MMP-2 without undue experimentation.

It is known in the art that CTX contains two binding domains. One binding domain is located at residues 8-14 of SEQ ID NO: 1 (this sub-sequence being designated SEQ ID NO: 3), the other at residues 23-29 of SEQ ID NO: 1 (this sub-sequence being designated SEQ ID NO: 2). U.S. Patent Publication 2010/0210564 describes in paragraphs [0104]-[0109] a study of every overlapping 10-mer in SEQ ID NO: 1 to positively identify which regions have binding activity and which do not. Shorter fragments of SEQ ID NO: 2 and 3 were tested for binding, and alanine scans of each binding domain were conducted to determine which residues are critical to binding in paragraphs [011 1]-[0117]. At least one variant of CTX was identified with superior binding properties as compared to native CTX in paragraph [01 16], with the following sequence

MCMPCFTTAHAMARKCDDCCGGKGRCKCYGPQCL (SEQ ID NO: 4)

U.S. Patent Publication 2010/0210564 is incorporated by reference herein for these teachings.

Consequently, any variant of CTX comprising at least one of SEQ ID NO: 2, 3, or 4 would be expected to maintain binding activity. The polypeptide moiety may be any of these variants.

U.S. Patent Publication 2010/0210564 also teaches variants of the binding domain of

SEQ ID NO: 3 that maintain binding activity in paragraph [0044], described by the following sequence:

TTX₁X₂X₃MX₄X₅K (SEQ ID NO: 5) wherein:

X] is an acidic amino acid selected from Asp or Glu;

X₂ is any naturally occurring amino acid;

X₃ is an amide amino acid selected from Asn or Gin;

X4 is Ser, Thr, or Ala; and

X₅ is a basic amino acid selected from His, Lys, and Arg.

The polypeptide moiety of this disclosure may comprise SEQ ID NO: 5. U.S. Patent Publication 2010/0210564 is incorporated by reference herein for this teaching.

The three-dimensional structure of CTX has been elucidated (see Lewis et al., Nature Reviews: Drug Discovery 2:790 (2003)), providing additional guidance to those of ordinary skill in the art as to which variants of CTX are likely to retain their binding activity.

As stated above, CTX is related to a larger family of scorpion toxins that are short polypeptides with high mutual homology. The primary amino acid sequences of several such scorpion toxins related to CTX are provided in FIG. 1 1. The GenBank entry names in FIG. 1 1 correspond to the following scorpion toxins and GenBank accession numbers: s

Cytotoxin BmK CT Buthus martensii

Natural variants of CTX have been observed to effectively bind cancer cells. For example, the toxin of Buthus martensii (BmK CT), which shares significant homology with CTX, has been observed to bind gliomas (Fu et al., Neuroscience Letters 412:62-67 (2007)). BmK CT was reported to have the follwing amino acid sequence, which is shown next to CTX to illustrate the sequence identity between them:

M5M5B^¾HQB^SDD3¾@ @RGSSY3¾S@-@- (SEQ ID NO: 1)

in which the highlighted residues are conserved between BmK CT and CTX (although there are gaps in the sequence alignment). BmK CT has been observed to target cancer cells particularly. The polypeptide moiety of this disclosure may comprise SEQ ID NO: 7, and those variants of SEQ ID NO: 1 comprising the conserved residues between SEQ ID NO: 1 and SEQ ID NO: 7. Such variants are described by the following sequence:

CX₁PCFTTDX₁X₁MARKCX₁X₁CCGGX₁GX₂X₂KCXGPQCLCX₂RX₂ (SEQ ID NO: 8)

wherein:

Xi may be any naturally occurring amino acid; and

X₂ may be any naturally occurring amino acid, or no amino acid.

Where a given subscript appears more than once, the residue may be chosen independently in each instance.

One embodiment of the derivative of CTX comprises a 36-mer having the sequence:

X₁CX₁X₂CX₃X₄TDHQMARX₅CX₆X₆CCX₇X₇KGRGKCyX₇X₇X₈CX₉CX₁₀ (SEQ ID NO: 6) wherein:

Xi is Met, Lue, Phe, or He;

X₂ is Pro, Ala, or Gly;

X₃ is Phe, Lue, Val, He, Ala, or Tyr;

X4 is Thr or Ser;

X₅ is Lys, Arg, Gin, Asn, or 1 ,4-diaminobutyric acid;

X₆ is Asp or Glu;

X₇ is Gly, Pro or Ala;

X9 is Leu, He, Val, Met, Ala, Phe, or norleucine; and

Xio is Arg, Lys, Gin, or Asn.

Where a given subscript appears more than once, the residue may be chosen independently in each instance. This 36-mer would be expected to display binding activity, as both binding regions are conserved, all disulfide bonded cysteine residues are conserved, and all other residues are defined conservative substitutions of the corresponding residues in SEQ ID NO: 1.

A skilled artisan will be able to determine suitable derivatives of the polypeptide sequence set forth in FIG. 1 using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of the CTX polypeptide to such similar polypeptides (see, for example, FIG. 1 1). With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the CTX polypeptide that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of the CTX polypeptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in the CTX polypeptide that correspond to amino acid residues that are important for activity or structure in similar polypeptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of the CTX polypeptide.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of the CTX polypeptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test CTX polypeptide derivatives containing a single amino acid substitution at each desired amino acid residue. The derivatives can then be screened using activity assays known to those skilled in the art and as disclosed herein. Such derivatives could be used to gather information about suitable substitution. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, derivatives with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations. Numerous scientific publications have been devoted to the prediction of secondary structure from analyses of amino acid sequences (see Chou et al., Biochemistry, 13(2):222-245, 1974; Chou et al., Biochemistry, 1 13(2):211-222, 1974; Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol, 47:45-148, 1978; Chou et al., Ann. Rev. Biochem., 47:251-276, 1979; and Chou et al, Biophys. J, 26:367-384, 1979). Moreover, computer programs are currently available to assist with predicting secondary structure of polypeptides. Examples include those programs based upon the Jameson-Wolf analysis (Jameson et al., Comput. Appl. Biosci., 4(1): 181-186, 1998; and Wolf et al., Comput. Appl. Biosci., 4(1): 187-191 ; 1988), the program PepPlot.RTM. (Brutlag et al., CABS, 6:237-245, 1990; and Weinberger et al., Science, 228:740-742, 1985), and other new programs for protein tertiary structure prediction (Fetrow. et al., Biotechnology, 1 1 :479-483, 1993).

Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure (see Holm et al., Nucl. Acid. Res. , 27(l):244-247, 1999).

Additional methods of predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol, 7(3):377-87, 1997; Suppl et al., Structure, 4(1): 15-9, 1996), "profile analysis" (Bowie et al, Science, 253: 164-170, 1991 ; Gribskov et al, Meth. Enzym., 183: 146- 159, 1990; and Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355

In an additional embodiment, the polypeptide moiety may also contain a cytotoxic and/or tracing group. The cytotoxic/tracing group may be an additional polypeptide or may be a radioisotope.

In one embodiment, the cytotoxic/tracing group is a radioisotope. Suitable radioisotopes include, alpha-, beta-, or gamma-emitting radionuclides, such as, but are not limited to, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁸⁸Y, ⁹⁰Y, ^99mTc,¹²³I, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁸⁶Re, and ¹⁸⁸Re.

In an alternate embodiment, the cytotoxic/tracing group is a polypeptide, such as, but is not limited to, gelonin, ricin, saponin, pseudonomas exotoxin, pokeweed antiviral protein, diphtheria toxin, complement proteins, and green fluorescent protein. In one embodiment, when the cytotoxic/tracing group is a polypeptide, the polypeptide can be bound to the polypeptide moiety directly or through a linking molecule. In a specific embodiment, a bifunctional linking molecule is used to bind the cytotoxic/tracing group and the polypeptide moiety. In a further specific embodiment, the bifunctional linking group has to aldehyde groups and the reaction chemistry discussed above is used to join the cytotoxic/tracing and the polypeptide moiety to the bifunctional linking group. An exemplary bifunctional linking group is a dialdehyde, such as, but not limited to glyoxal, glutaraldehyde and succinicaldehyde. For example, if glyoxal is the linking group, the conjugation would be performed in two consecutive steps with conjugation of glyoxal to cytotoxic/tracing (polypeptide) group through the chemistry described above, purification of the intermediate conjugate, followed by conjugation of the polypeptide moiety by the same procedure. The polypeptide moiety may then be linked to the agent as described herein.

Various embodiments of the foregoing where the polypeptide moiety is chlorotoxin, as well as methods of attachment of the cytotoxic group to the polypeptide moiety, are described in US Patent Nos: 6,870,029, 6,667,156, 6439,187, 6,319, 891 , 6,028,174 and 5,905,027 where are hereby incorproated by reference for such teachings. When the cytotoxic/tracing group is a polypeptide, the reaction chemistries linking the polypeptide moiety to the agent may be used. Methods of Making Conjugate

As described in this disclosure, the polypeptide conjugate comprises a polypeptide moiety and an agent linked together via the a-amino group of the N-terminal amino acid residues of the polypeptide conjugate may also comprise an optional linker. The agent, polypeptide moiety and linker are described herein. This process is carried out by reacting an aldehyde group or ketone group of the agent or linker with the a-amino group of the N-terminal amino acid residues of the polypeptide moiety. The reaction medium is a buffered solution which may or may not contain an organic co-solvent such as an alcohol, dimethyl formamide, or dimethyl sulfoxide, or any other water-miscible solvent.

The methods described also discloses the use of mild reaction conditions which regenerates aldehyde groups from protected aldehyde groups (such as acetals) and ketone groups from protected ketone groups.

In one embodiment where a linker is present, the polypeptide moiety may be linked to the linker via a condensation reaction between an aldehyde group or a ketone group (the second functional group) on the linker and a-amino group of the N-terminal amino acid residues of the polypeptide moiety, such as through the formation and subsequent reduction of a Schiff base. In such embodiment, the conjugated product is an alkylamine (3), as shown in the following equation: L-CHO + NH₂-PPT→ L-CH=N-PPT-> L-CH₂-NH-PPT

1 2 3

This reaction may be generally applied to chemo selective conjugations of aldehyde- or ketone- containing molecules to the c-amino group of the N-terminal amino acid residues of the polypeptide moiety.

Without wishing to be bound to any hypothetical model, it is proposed that the difference in pK_a between the a-amino group and non-terminal amino groups is responsible for the highly specific reaction between the oamino group and the aldehyde or ketone. Given that the oamino group of a polypeptide will have a /? ₂, and given that each non-terminal amino group will have a ρΚ_&, the pK₂ will generally be significantly lower than the average _R. Primary and secondary amino groups exist in equilibrium between the protonated and deprotonated state. Given an ambient pR that is significantly less than both pK₂ and the average pK_K (for example, at least about 4.5 pH units below the average pK_&), at a given pH the equilibrium in the a-amino group will favor the unprotonated state to a higher degree than will the non-terminal amino groups. As protonated amines are not nucleophilic, they are unreactive with aldehydes and ketones. As the unprotonated a-amino groups are consumed by the reaction with the aldehydes or ketones, the concentration of unprotonated c -amino groups will decrease, further driving the equilibrium in the direction of the unprotonated state. Consequently, the reaction pH should be below the pK^, and low enough that the aldehyde or ketone is sufficiently protonated to be reactive. In the context of the amino group the term "reactive" generally means reactive with an aldehyde or ketone group, due to the difference between the ambient pH and the pK_R or pK of the group. In the context of the aldehyde or ketone, the term "reactive" generally means that the group is sufficiently protonated to be reactive. In some embodiments of the method a chemical buffer system may be used as the reaction solvent to retain the desired pH. In the examples below, the reaction pH is maintained about 2.0-3.5 pR units below the pK₂ of the polypeptide. In an exemplary embodiment of the method the reaction is conducted at about pH 4.8. In further embodiments of the method the reaction is conducted at from 4-6, from 4.5- 5.5, from 4.5-4.8, and about the foregoing ranges.

In some embodiments, a protected aldehyde or protected ketone group is utilized and mild conditions are utilized in regenerating the aldehyde or ketone moiety which then reacts with the oamino group of the N-terminal amino acid residues of the polypeptide moiety. The use of mild conditions provides the advantage of preserving the activity of the agent, polypeptide moiety, or other molecules of biological and/or medical interest. In one embodiment, a bifunctional acetal (i.e., the protected aldehyde) is first conjugated to the agent. The purified product is isolated and is then deprotected by conversion of the acetal to an aldehyde in a 50% aqueous solution of acetic acid at 50 °C. This mild condition readily and efficiently removes the acetal protection and regenerates the corresponding aldehyde. Other approaches to the synthesis of aldehydes may be utilized as well. Acetal formation and removal has the advantage of avoiding conditions that may adversely affect the activity of the polypeptide moiety, such as strongly oxidizing conditions.

Various methods may be employed to attach the agent to the linker, depending on the type of the functional groups present. In exemplary embodiments, the 2 '-hydroxy group of paclitaxel or a phenolic hydroxyl group of curcumin is activated by esterification. Additional functional groups on the linker and their respective binding partners are described above.

Table 3 shows some exemplary linking functionalities with their compatible counter parts.

Examples are provided describing the conjugation of paclitaxel, curcumin, and chlorotoxin to the linker. The most frequently used linkages used to link the agent to the polypeptide moiety are carboxylic ester or amide linkages, although the specific type of linkage depends on the chemical functionality present. For targeting vehicles with small MWs (<30,000 Da), such as small-molecule cell surface receptor-binding polypeptides with relatively rapid target localization and short circulation time, carboxylic ester and amide are suitable due to their relatively short release times. Another advantage of these linkages is their non-toxicity. Carboxylic ester and amide linkages are not known to be toxic to humans and other mammals. Cleavage of unhindered ester linkages usually occurs within a few hours after entering into the circulation. In the case of amide linkages, it may by slightly longer. Amide and ester linkages would have the ability to release the agent within a time window compatible with that of target localization of the polypeptide moiety. Too long a release time may result in the release of the agent only after the catabolism and excretion of the polypeptide moiety, which may lead to decreased benefit.

An additional hydrolytically labile linkage is the disulfide bond. Disulfides are thiol- dependent type linkages the rate of metabolism of which increases with an increase in the cellular levels of glutathione. Hydrolytically stable linkages exist which undergo either no metabolism or have a negligible metabolic rate in the body. Alkyls, ethers, sulfides, sulfoxides, and sulfone linkages are representatives of this group.

Embodiments of the method of linking an agent to a polypeptide comprise providing a polypeptide comprising an unprotected and reactive terminal a-amino group, and at least one non-terminal amino group, wherein all said non-terminal amino groups are unreactive; contacting the polypeptide with at least one of an agent or an agent coupled to a linker at a reaction pH, said agent or agent coupled to a linker comprising exactly one unprotected aldehyde group; creating a covalent bond selectively between the terminal a-amino group and the unprotected aldehyde group; and producing a conjugate molecule with a ratio of polypeptide to agent of 1 : 1. Further embodiments comprise providing a polypeptide comprising a terminal a-amino group having a jt?K₂ value, and at least one non-terminal amino group, each non-terminal amino group having a /?KR value; mixing said polypeptide with at least one of an agent or an agent conjugated to a linker, said agent or agent conjugated to a linker comprising exactly one unprotected aldehyde group, to form a reaction mixture; and maintaining the reaction mixture at a reaction pH, wherein the polypeptide selectively forms a covalent bond with the terminal a-amino group through a reaction between the aldehyde group and the terminal oamino group thereby producing a conjugate molecule with a ratio of polypeptide to agent of 1 : 1.

In embodiments of the method the reaction pH may be from 4-6, from 4.5-5.5, from 4.5- 4.8, 4.8, or about any of the foregoing ranges and values.

Any of the agents, linkers, and polypeptides described above may be employed in these embodiments of the method.

Methods of Treatment and Prevention

The present disclosure describes the use of a polypeptide conjugates of the present disclosure or a pharmaceutical derivative thereof to treat and/or prevent disease in a subject. Treatment or prevention of disease is accomplished by delivering the polypeptide conjugate to a cell of the subject. In one embodiment, the polypeptide moiety targets the polypeptide conjugate to a target cell. In such an embodiment the polypeptide moiety may bind to or interact with a polypeptide or other molecule expressed on the surface of the target cell. For example, CTX polypeptide has been shown to bind specifically to MMP-2 and other molecular cellular targets. In some embodiments, the polypeptide moiety may function solely as a targeting agent. In alternate embodiments, the polypeptide moiety may function as a therapeutic agent, such as cytotoxic or cytostatic agent. In still further embodiments, the polypeptide moiety may function as a targeting agent and a therapeutic agent. In this embodiment, the polypeptide conjugate comprises a therapeutic agent bound to the polypeptide moiety via the a- amino group of the N-terminal amino acid residues of the polypeptide moiety. The agent exerts an effect on the target cell that has a tendency to treat or prevent the disease. In still further embodiments, the polypeptide moiety may function to deliver the agent across the blood-brain- barrier.

The method of treatment and/or prevention comprises administering a polypeptide conjugate of the present disclosure to a subject in need thereof. The method of treatment and/or prevention may further comprise identifying a subject in need of such treatment and/or prevention. In some embodiments of the method the polypeptide conjugate is administered as a pharmaceutical composition. The pharmaceutical composition may be any that is disclosed as suitable in this disclosure or as known in the art.

In a specific embodiment, the polypeptide conjugate is administered in a therapeutically effective amount. Such administration of the polypeptide conjugate thereby treats or prevents the disease. As discussed above, the treatment and/or prevention need not be absolute to provide benefit in the methods disclosed. Furthermore, the polypeptide conjugate may be administered is a safe amount. A "safe" amount is an amount that has been determined to pose no potential negative effects to the subject or to pose potential negative effects to the subject that are warranted given the benefits to be gained by administration. In some embodiments of the method, the safe amount will be an amount that is determined to be unlikely to pose an observable negative effect on the subject. Such an amount may be the "no observable adverse effect level" or the "lowest observable adverse effect level." The safe amount may also be below a known lowest lethal dose (LD₀). However, the safe amount may be associated with significant side effects or even a significant probability of lethality, if the benefits of administration are determined by a medical professional to offset the negative effects. Such amounts are typically published in the results of clinical trials or in publically available regulatory rulemakings, and are often specific to the drug, the subject, and the disease. The principles underlying methods of measuring and characterizing the safety of chemical substances are described in C. D. laassen and D. L. Eaton, "Chapter 2: Principles of Toxicology" in Toxicology (1991) M. O. Addur, J. Doull, and C. D. Klaassen, eds., which is incorporated by reference herein for this teaching. In some embodiments of the method, the active compound is administered in an amount that is both safe and effective.

The polypeptide conjugate may be provided to the subject by any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal and pulmonary. Alternatively, for treatment of disease states and conditions of the spinal cord or brain, including cancer, the polypeptide conjugate may be injected directly into the spinal cord of cranium through intrathecal or intracranial (intraventricular) routes. The polypeptide conjugate of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the polypeptide conjugate is administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The polypeptide conjugate may also be administered to the subject more than one time per day. In addition, co-administration or sequential administration of other agents may be desirable. The polypeptide conjugate of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection or by application of a paste or cream.

Some embodiments of the disclosure provide for the treatment and/or prevention of a cell-mediated disease or condition. In one embodiment, the cell-mediated disease or condition is a neoplasm, a disease or condition associated with a neoplasm, in a subject in need of such treatment and/or prevention. Such neoplasms include, but are not limited to, cancers. The cancer may be any type of cancer, including but not limited to a tumor of neuroectodermal origin, colon cancer, skin cancer, lung cancer, prostate cancer or ovarian cancer. Furthermore, the cancer may be any cancer that expresses MMP-2. In a specific embodiment, the cancer is one of neuroectodermal origin. In embodiments in which the disease is a cancer, the target cell will in many instances be a cancer cell.

In embodiments of the method that are intended to treat cancer, the agent may be an antineoplastic agent. Such antineoplastic agents are known to those of ordinary skill in the art. Such antineoplastic agents include, but are not limited to, a radionuclide, dactinomycin, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, taxofene, tamoxifen, paclitaxel, temozolomide, curcumin, a hormone, a hormone antagonist, mitomycin, and derivatives thereof that retain at least some antineoplastic activity. Because the polypeptide conjugate will selectively target cancer cells in such embodiments, agents with general cytotoxic activity may be used that would not otherwise be suitable for antineoplastic therapy.

In embodiments of the method that are intended to target cancer cells or other cells, the polypeptide moiety of the polypeptide conjugate may be a CTX polypeptide. As explained in this disclosure, CTX polypeptide binds to MMP-2 and other cellular targets with high specificity, resulting in the specific delivery of the polypeptide conjugate to cells. In such embodiments the polypeptide moiety may be a derivative of the CTX polypeptide that retains at least partial binding specificity to its targets.

Some embodiments of the method are intended to treat a disease or condition of brain, wherein the polypeptide serves to deliver a therapeutic agent across the blood brain barrier. The therapeutic agent may be any described herein, for example taxol.

In those embodiments of the method that are methods are directed to preventing a disease, such as a neoplasm or a disease or condition associated with a neoplasm, such methods will typically be used if the subject is considered to be at risk of developing the disease, For example if the subject has experienced a previous neoplasm that is presently in remission.

Methods of Diagnosis

The present disclosure describes the use of a polypeptide conjugates of the present disclosure or a pharmaceutical derivative thereof to diagnose a disease or condition in a subject. Such diagnosis is accomplished by delivering the polypeptide conjugate to a subject. In one embodiment, the polypeptide moiety targets the polypeptide conjugate to a target cell. In such an embodiment the polypeptide moiety may bind to or interact with a polypeptide or other molecule expressed on the surface of the target cell. For example, CTX polypeptide has been shown to bind specifically to MMP-2 and other cellular targets. For example, CTX polypeptide has been shown to bind specifically to a variety of tumors, including tumors of neuroectodermal origin as well as to breast, colon, pancreas, prostate, and some other human carcinomas. In this embodiment, the polypeptide conjugate comprises a diagnostic agent bound to the polypeptide moiety via the -amino group of the N-terminal amino acid residues of the polypeptide moiety. The diagnostic agent may then be detected indicating the presence of the target cell.

The method of diagnosis comprises administering a polypeptide conjugate of the present disclosure to a subject in need thereof. The method of treatment and/or prevention may further comprise identifying a subject in need of such diagnosis. In some embodiments of the method the polypeptide conjugate is administered as a pharmaceutical composition. The pharmaceutical composition may be any that is disclosed as suitable in this disclosure or as known in the art. Methods of diagnosis using the polypeptide conjugates may be accomplished by administering to the subject a diagnostically effective amount of the polypeptide conjugate, allowing the polypeptide conjugate to bind the target cells and detecting and measuring the level of binding to the target cells. An increased binding level as compared to a normal subject free of the disease state or condition to be diagnosed is indicative of the presence of the disease state or condition.

In a specific embodiment, the polypeptide conjugate is administered in a diagnostically effective amount. Such administration of the polypeptide conjugate thereby allows detection of the target cell desired to be detected. The polypeptide conjugate may be provided to the subject by any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal and pulmonary. Alternatively, for diagnosis of disease states and conditions of the spinal cord or brain, including cancer, the polypeptide conjugate may be injected directly into the spinal cord of cranium through intrathecal or intracranial (intraventricular) routes. The polypeptide conjugate of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the polypeptide conjugate is administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The polypeptide conjugate may also be administered to the subject more than one time per day. In addition, co-administration or sequential administration of other agents may be desirable. The polypeptide conjugate of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection or by application of a paste or cream.

Some embodiments of the method are methods of diagnosing a cell-mediated disease or condition. Some embodiments of the disease or condition are a neoplasm, a disease or condition associated with a neoplasm, in a subject in need of such diagnosis. Such neoplasms include, but are not limited to, cancers. The cancer may be any type of cancer, including but not limited to a tumor of neuroectodermal origin, colon cancer, skin cancer, lung cancer, prostate cancer or ovarian cancer. Furthermore, the cancer may be any cancer that expresses MMP-2 or other targets of the CTX polypeptide. In a specific embodiment, the cancer is one of neuroectodermal origin. In embodiments in which the disease is a cancer, the target cell will in many instances be a cancer cell.

In embodiments of the method that are intended to diagnose cancer cells or other cells expressing MMP-2 on the cell surface, the polypeptide moiety of the polypeptide conjugate may be a CTX polypeptide. As explained in this disclosure, CTX polypeptide binds to MMP-2 with high specificity, resulting in the specific delivery of the active compound to cells comprising MMP-2 on the cell surface. In such embodiments the polypeptide moiety may be a derivative of the CTX polypeptide that retains at least partial binding specificity to MMP-2.

Method of Delivering an Agent to a Target Cell

The disclosure provides a method of delivering an agent to a cell comprising contacting the cell with a polypeptide conjugate described in this disclosure. The polypeptide moiety of the polypeptide conjugate may specifically or preferentially bind to a component of the cell. Such components may be presented on the outer surface of the plasma membrane, or in other parts of the cell. The agent may be any that is described in this disclosure as a suitable part of the polypeptide conjugate. The agent may modulate the metabolism of the cell. For example, the agent may exert a cytotoxic effect on the cell. The active compound may be present in a concentration effective to permit the agent to exert a detectable effect on the cell. The cell may be of any type, including a bacterial cell, a fungal cell, a protist cell, an animal cell, or a plant cell. In some embodiments of the method, the cell is a pathogen cell. In some embodiments of the method, the cell is a neoplastic cell of an animal or a plant. The method may be an in vitro method or an in vivo method.

The present disclosure describes the use of a polypeptide conjugates of the present disclosure or a pharmaceutical derivative thereof to deliver an agent to a cell or tissue in a subject. Such delivery is accomplished by delivering the polypeptide conjugate to a cell of the subject via the polypeptide moiety. In one embodiment, the polypeptide moiety targets the polypeptide conjugate to a target cell. In such an embodiment the polypeptide moiety may bind to or interact with a polypeptide or other molecule expressed on the surface of the target cell. For example, CTX polypeptide has been shown to bind specifically to MMP-2 as well as other cellular targets and target a variety of tumors, as discussed above. The polypeptide conjugate comprises an agent bound to the polypeptide moiety via the a-amino group of the N-terminal amino acid residues of the polypeptide moiety. The agent exerts an effect on the target cell as described herein.

In some embodiments the polypeptide functions to permit the conjugate to cross the blood-brain-barrier, allowing agents that otherwise could not reach the brain to do so.

The method of delivery comprises administering a polypeptide conjugate of the present disclosure to a subject in need thereof. The method of delivery may further comprise identifying a subject in need of such delivery. In some embodiments of the method the polypeptide conjugate is administered as a pharmaceutical composition. The pharmaceutical composition may be any that is disclosed as suitable in this disclosure or as known in the art.

In a specific embodiment, the polypeptide conjugate is administered to deliver the agent in a therapeutically effective amount. Such delivery of the agent thereby treats or prevents a disease or condition or diagnoses a disease or condition. As discussed above, such treatment and/or prevention need not be absolute to provide benefit in the methods disclosed. Furthermore, the polypeptide conjugate may be administered to deliver the agent in a safe amount. A "safe" amount is an amount that has been determined to pose no potential negative effects to the subject or to pose potential negative effects to the subject that are warranted given the benefits to be gained by administration. In some embodiments of the method, the safe amount will be an amount that is determined to be unlikely to pose an observable negative effect on the subject. Such an amount may be the "no observable adverse effect level" or the "lowest observable adverse effect level." The safe amount may also be below a known lowest lethal dose (LDo). However, the safe amount may be associated with significant side effects or even a significant probability of lethality, if the benefits of administration are determined by a medical professional to offset the negative effects. Such amounts are typically published in the results of clinical trials or in publically available regulatory rulemakings, and are often specific to the drug, the subject, and the disease. The principles underlying methods of measuring and characterizing the safety of chemical substances are described in C. D. Klaassen and D. L. Eaton, "Chapter 2: Principles of Toxicology" in Toxicology (1991) M. O. Addur, J. Doull, and C. D. Klaassen, eds., which is incorporated by reference herein for this teaching. In some embodiments of the method, the active compound is administered in an amount that is both safe and effective.

In one embodiment, the teachings of the present disclosure provide for the delivery of an agent to treat and/or prevent a neoplasm, a disease or condition associated with a neoplasm, in a subject in need of such treatment and/or prevention. Such neoplasms include, but are not limited to, cancers. The cancer may be any type of cancer, including but not limited to a tumor of neuroectodermal origin, colon cancer, skin cancer, lung cancer, prostate cancer or ovarian cancer. Furthermore, the cancer may be any cancer that expresses MMP-2 and other molecular target within the tissue. In a specific embodiment, the cancer is one of neuroectodermal origin. In embodiments in which the disease is a cancer, the target cell will in many instances be a cancer cell.

In embodiments of the method that are intended to target cancer cells or other cells expressing MMP-2 or other cellular targets, the polypeptide moiety of the polypeptide conjugate may be a CTX polypeptide. As explained in this disclosure, CTX polypeptide binds to MMP-2 with high specificity, resulting in the specific delivery of the active compound to cells comprising MMP-2 on the cell surface. In such embodiments the polypeptide moiety may be a derivative of the CTX polypeptide that retains at least partial binding specificity to MMP-2. Compositions

Useful compositions of the present disclosure may comprise one or more compounds useful in the methods of the present disclosure, such as, but not limited to, those compounds identified in the present disclosure or identified by a screening method of the present disclosure. In one embodiment, such compounds decrease the expression, in whole or in part, of one or more genes involved in the process of producing such chemotactic peptides, such as PGP, so as to reduce the levels of such proteins in the subject. In one embodiment, such genes encode for a MMP, including but not limited to, MMP-8 and MMP-9, or a serine protease, including but not limited to, PE. In an alternate embodiment, such compounds decrease the activity, in whole or in part, of one or more enzymes involved in the process of producing such chemotactic peptides, such as PGP, so as to reduce the activity of such enzymes in the subject. In one embodiment, such enzymes are an MMP, including but not limited to, MMP-8 and MMP-9, or a serine protease, including but not limited to, PE. In yet another alternate embodiment, such compounds decrease the activity, in whole or in part, of neutrophils or cells activated by neutrophils as described above. In one embodiment, such compositions are pharmaceutical compositions. The compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington: The Science and Practice of Pharmacy (20^th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor). To form a pharmaceutically acceptable composition suitable for administration, such compositions will contain a therapeutically effective amount of compound.

The pharmaceutical compositions of the disclosure may be used in the treatment and prevention methods of the present disclosure. Such compositions are administered to a subject in amounts sufficient to deliver a therapeutically effective amount of the compound(s) so as to be effective in the treatment and prevention methods disclosed herein. The therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject's condition, weight, sex and age. Other factors include the mode and site of administration. The pharmaceutical compositions may be provided to the subject in any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, and intramuscular. The compositions of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The compositions may also be administered to the subject more than one time per day. The therapeutically effective amount of the nucleic acid molecules and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects. In addition, coadministration or sequential administration of other agents may be desirable.

The compositions of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection or by application of a paste or cream.

The compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and/or or decrease the toxicity of the compounds(s). Examples of such agents are described in a variety of texts, such a, but not limited to, Remington: The Science and Practice of Pharmacy (20^th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor).

The compositions of the present disclosure can be administered in a wide variety of dosage forms for administration. For example, the compositions can be administered in forms, such as, but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous administration or injection. Other dosage forms include administration transdermally, via patch mechanism or ointment. Any of the foregoing may be modified to provide for timed release and/or sustained release formulations.

In the present disclosure, the pharmaceutical compositions may further comprise a pharmaceutically acceptable carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrating agents and carriers, as well as accessory agents, such as, but not limited to, coloring agents and flavoring agents (collectively referred to herein as a carrier). Typically, the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use. The pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.

For instance, for oral administration in solid form, such as but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, or granules, the compound(s) may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier, such as, but not limited to, inert fillers, suitable binders, lubricants, disintegrating agents and accessory agents. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthum gum and the like. Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid as well as the other carriers described herein. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

For oral liquid forms, such as but not limited to, tinctures, solutions, suspensions, elixirs, syrups, the nucleic acid molecules of the present disclosure can be dissolved in diluents, such as water, saline, or alcohols. Furthermore, the oral liquid forms may comprise suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like. Moreover, when desired or necessary, suitable and coloring agents or other accessory agents can also be incorporated into the mixture. Other dispersing agents that may be employed include glycerin and the like.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound(s) may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkylbeta-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight.

Topical dosage forms, such as, but not limited to, ointments, creams, pastes, emulsions, containing the nucleic acid molecule of the present disclosure, can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. Inclusion of a skin exfoliant or dermal abrasive preparation may also be used. Such topical preparations may be applied to a patch, bandage or dressing for transdermal delivery or may be applied to a bandage or dressing for delivery directly to the site of a wound or cutaneous injury.

The compound(s) of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Such liposomes may also contain monoclonal antibodies to direct delivery of the liposome to a particular cell type or group of cell types.

The compound(s) of the present disclosure may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include, but are not limited to, polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of natural or synthetic biodegradable polymers useful in achieving controlled release of a drug, for example, poly(amino acids), polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross- linked or amphipathic block copolymers of hydrogels.

EXAMPLES

Example 1. Conjugation of Chlorotoxin to Paclitaxel

Commercially supplied PTX (1 eq) was dissolved in dry pyridine followed by the addition of finely powdered succinic anhydride (6 eq) to produce PTX-2'-hemisuccinate. The solution was kept at room temperature (RT) overnight. The solvent was distilled in vacuum and the residual oil was purified by silica gel (Si0₂) column chromatography in a 0%-10% methanol gradient in chloroform (MTL:CHL). The purified PTX-2'-hemisuccinate was reacted with a commercially supplied 2-amino acetaldehyde diethyl acetal (linker) in a carbodiimide-mediated coupling reaction at RT. The product was purified by Si0₂ chromatography to afford the protected PTX aldehyde. The latter compound was dissolved in 50:50 (v/v) aqueous solution of acetic acid under argon atmosphere and the clear and colorless solution was gently stirred at 50 °C overnight. The solvents were distilled in vacuum and the oily residue was purified on Si0₂ with 1 % - 5% (MTL:CHL) to afford the PTX aldehyde. Chlorotoxin peptide and the PTX aldehyde were mixed in a reaction vessel under argon and absolute ethanol (1 part) and a 150 mM, pH 4.80 ammonium acetate buffer (1 part) were added to a clear solution which was stirred in dark for 15 min. A calculated volume of a 1 M solution of NaCNBH₃ in water was added to an overall concentration of 50 mM. Stirring was then continued at room temperature or below for 12 h at which time analytical reversed-phase (RP) HPLC indicated the formation of a new product. The paclitaxel-chlorotoxin conjugate is shown in FIG. 2.

This product was purified by preparatory RP-HPLC and was identified by matrix- assisted laser desorption/ionization mass spectrometry (MALDI MS) to confirm the target conjugate structure. The result was a paclitaxel-chlorotoxin conjugate at a 1 : 1 molar ratio as detected by MALDI MS (FIG. 3)

Example 2. Identification of the aldehvde-peptide conjugates

Although molecular weight (MW) analyses by MALDI MS indicated formation of drug- peptide conjugates with 1 : 1 molar ratios, the site of conjugation to the chlorotoxin peptide was not determined. The chlorotoxin peptide contains 4 amine (3 core lysines and an N-terminal methionine) and 3 imines (2 core and 1 C-terminal arginine) groups. Each of these functionalities is capable of reacting with the aldehyde group to form the observed conjugates.

To determine the site of conjugation on the chlorotoxin peptide PTX aldehyde (1 eq) was added to a mixture of methionine (1 eq), N^-Z-arginine (3 eq), and iV^-Boc-lysine (3 eq), each dissolved in absolute ethanol (2 mL) and ammonium acetate buffer (150 mM, pH 4.80, 2 mL). The solution was stirred under argon and NaCNBH₃ in water was added to an overall concentration of 50 mM. The reaction was stirred at RT overnight after which time it was purified by RP-HPLC. Chromatographic (HPLC) and mass spectrometric analyses of the purified products indicated formation of the Λ^-ΡΤΧ-linker-methionine adduct (MW 1 128) as the major product confirming conjugation occurred at the N-terminus of the chlorotoxin peptide. Example 3- Effect of Paclitaxel-Chlorotoxin Conjugate (PTX-CTX) on MDA-MB-468 cells

The cytotoxic activity of the PTX-CTX conjugate synthesized as set forth in Example 1 was tested against MDA-MB-468 cells to evaluate the cytotoxicity of the compound. MDA- MB-468 human breast cancer cells were plated in 24-well dishes using their respective cell culture medium containing 10% fetal bovine serum and 2 mM L-glutamine. When the cells attain 50% confluence, PTX (10 nM) and PTX-CTX conjugate (1, 5 and 10 nM) were added to the culture medium in quadruplicate. DMSO (solvent for the water-insoluble PTX) was maintained at 0.5% in all treatment groups, including the untreated control group. The cells were exposed to the treatment groups for 24 h. After the incubation period, the culture medium was removed, the cells were washed with PBS, and drug-free medium was added to the cells. Following a 96-h post-treatment incubation under standard conditions, the cells were washed and the viable cells were counted using a particle counter (Beckman Coulter, Inc, Fullerton, CA). The cell numbers were normalized to the percent of untreated controls. The normalized number of surviving cells represents cytotoxicity of the respective treatment.

The results indicate that the PTX-CTX conjugate was more effective than the unconjugated PTX in inhibiting the growth of MDA-MB-468 cells (FIG. 4). For example, the PTX-CTX conjugate at 5 nM showed greater cytotoxicity against MDA-MB-468 cells than 10 nM unconjugated PTX.

Example 4- Effect of Paclitaxel-Chlorotoxin Conjugate (PTX-CTX) on PC-3 cells

The cytotoxic activity of the PTX-CTX conjugate synthesized as set forth in Example 1 was tested against PC-3 cells to evaluate the cytotoxicity of the compound. PC-3 human prostate cancer cells were plated in 24-well dishes using their respective cell culture medium containing 10% fetal bovine serum and 2 mM L-glutamine. When the cells attain 50% confluence, PTX- CTX conjugate (1 , 5 and 10 nM) were added to the culture medium in quadruplicate. DMSO was maintained at 0.5% in all treatment groups, including the untreated control group. The cells were exposed to the treatment groups for 24 h. After the incubation period, the culture medium was removed, the cells were washed with PBS, and drug-free medium was added to the cells. Following a 96-h post-treatment incubation under standard conditions, the cells were washed and the viable cells were counted using a particle counter (Beckman Coulter, Inc, Fullerton, CA). The cell numbers were normalized to the percent of untreated controls. The normalized number of surviving cells represents cytotoxicity of the respective treatment.

The results indicate that the PTX-CTX conjugate was effective in inhibiting the growth of PC-3 cells (FIG. 5).

Example 5- Effect of Paclitaxel-Chlorotoxin Conjugate (PTX-CTX) on Systemic Toxicity Nude Mice

To determine the systemic toxicity of the paclitaxel-chlorotoxin conjugate in nude mice, groups of 8 normal female nude mice were injected intravenously during the treatment period with 100 μ]_, of saline (untreated controls, group 1), unconjugated paclitaxel (Taxol^®, Bristol- Myers-Squibb, Princeton, NJ, group 2), the paclitaxel-chlorotoxin conjugate of Example 1 (PCXCTX-601, group 3) and a second paclitaxel-chlorotoxin conjugate where the tyrosine residue at position 29 of the CTX polypeptide (FIG. 1) was replaced with a phenylalanine (PTXCTX-701, group 4; prepared as described in Example 1). All treatments were injected as solutions in 100 μΐ_^ of saline. The paclitaxel dose was at an equimolar dose with the conjugate (3.4 mg/kg). The mice were observed for a period of 40-50 days for weight loss and survival as a measure of treatment toxicity. As shown in FIG. 6, neither paclitaxel-chlorotoxin conjugate showed signs of systemic toxicity during the experiment.

Example 6- Effect of Paclitaxel-Chloro toxin Conjugate (PTX-CTX) on Antitumor Activity in Nude Mice

Female nude mice (3-6 weeks-old) were subcutaneously implanted in the flank with either MDA-MB-468 human breast or PC-3 prostate cancer cells (2 x 10⁶ cells/mouse). With the tumors at a 5 mm cross-diameter, the mice were randomized into 3x8 groups. Animals were injected iv with 100 μΐ_^ of saline (untreated controls, group 1), the unconjugated paclitaxel (Taxol^®, Bristol-Myers-Squibb, Princeton, NJ), (group 2), and the paclitaxel-chlorotoxin conjugate of Example 1 (group 3). All treatments were injected as solutions in 100 μϋ of saline. The paclitaxel dose was at an equimolar dose with the conjugate. A total of 5 injections were administered every 48 h. Tumor volumes were measured 3 times weekly by calipers and the growth rates in treated groups, as compared to the untreated control, were plotted against time to represent the antitumor activity of the treatment. The results were then statistically analyzed for validity.

As can be seen in FIG. 7, the paclitaxel-chlorotoxin conjugate was effective in reducing tumor size in this model, while unconjugated paclitaxel was not effective. The reduction in tumor size was statistically significant as compared to the saline group (p 0.001) and taxol group (p O.01).

As can be seen in FIG. 8, the paclitaxel-chlorotoxin conjugate was effective in reducing PC-3 tumor size in this model, while unconjugated paclitaxel was not effective. The reduction in tumor size was statistically significant as compared to the saline group (p 0.0009) and taxol group (p 0.007).

Example 7. Conjugation of Chloro toxin to Curcumin

2-Amino acetaldehyde diethyl acetal was coupled to bis-(4-nitrophenyl) carbonate in dichloromethane and in the presence of N, N-diisopropyl ethylamine and the product was purified by Si0₂ chromatography. In a second coupling, this compound was transesterified to commercially supplied curcumin (CCMN) through one of the phenolic oxygen atoms of CCMN. This protected CCMN aldehyde was then deprotected and conjugated to the chlorotoxin peptide through the procedures described above in Example 1. This product was purified by preparatory RP-HPLC and was identified by matrix- assisted laser desorption/ionization mass spectrometry (MALDI MS) to confirm the target conjugate structure. The result was a curcumin-chlorotoxin conjugate at a 1 :1 molar ratio. The curcumin-chlorotoxin conjugate is shown in FIG. 9.

Example 8. Conjugation of Chlorotoxin to Gemcitabine.

The hydroxyl groups of Gemcitabine (GEM, 2', 2'-difluorodeoxycitidine) were blocked by di-tert-butyl carbonate in a mixture of dioxane and water, according to the procedure of Guo et al. (J Org Chem, 1999, 64, 8319-8322). The amine function was then coupled to N-(2, 2- diethoxyethyl)succinamide through a carbodiimide reaction, using dicyclohexyl carbodiimide, catalyzed by dimethyl aminopyridine and in methylene chloride (DCM) as solvent. The product was treated with trifluoroacetic acid in DCM to remove the tert-bytyloxy carbonyl protecting groups. The product was then treated with the 50:50 (v/v), acetic acid:water solution as described above in Example 1. The resulting aldehyde was N-terminally conjugated to CTX as described above in Example 1.

This product was purified by preparatory RP-HPLC and was identified by matrix- assisted laser desorption/ionization mass spectrometry (MALDI MS) to confirm the target conjugate structure. The result was a gemcitabine-chlorotoxin conjugate at a 1 :1 molar ratio. Example 9. Conjugation of Chlorotoxin to Camptothecin.

Camptothecin was esterified on the 20-hydroxyl group with N-(2, 2- diethoxyethyl)succinamide and through a carbodiimide reaction, using dicyclohexyl carbodiimide and catalyzed by dimethyl aminopyridine and in methylene chloride (DCM) as solvent. The product was deprotected in the 50:50 (v/v), acetic acid:water solution as described above in Example 1. The resulting aldehyde was N-terminally conjugated to CTX as described above in Example 1.

This product was purified by preparatory RP-HPLC and was identified by matrix- assisted laser desorption/ionization mass spectrometry (MALDI MS) to confirm the target conjugate structure. The result was a camptothecin-chlorotoxin conjugate at a 1 : 1 molar ratio. Example 10- Time-dependent decomposition of a Paclitaxel-Chlorotoxin Conjugate

In order to determine the stability of the paclitaxel-chlorotoxin conjugate of the present disclosure, a paclitaxel-chlorotoxin conjugate (B) was incubated at 37 °C in pH 7.4 PBS. The decomposition to paclitaxel (C) and linker-attached chlorotoxin polypeptide (A) was monitored. The paclitaxel-chlorotoxin conjugate was prepared as in Example 1. Samples were analyzed by reversed-phase HPLC every hour from 1 hour post-incubation (bottom trace) to 6 hour post- incubation (top trace). The results are shown in FIG. 10 and show a time-dependent pattern of decomposition of the paclitaxel-chlorotoxin conjugate to its constituting peptide and drug.

The foregoing description illustrates and describes the methods and other teachings of the present disclosure. Additionally, the disclosure shows and describes only certain embodiments of the methods and other teachings disclosed, but, as mentioned above, it is to be understood that the teachings of the present disclosure are capable of use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the teachings as expressed herein, commensurate with the skill and/or knowledge of a person having ordinary skill in the relevant art. The embodiments described hereinabove are further intended to explain best modes known of practicing the methods and other teachings of the present disclosure and to enable others skilled in the art to utilize the teachings of the present disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses. Accordingly, the methods and other teachings of the present disclosure are not intended to limit the exact embodiments and examples disclosed herein. All references cited herein are incorporated by reference as if fully set forth in this disclosure.

Claims

CLAIMS What is claimed:

1. A method of linking an agent to a polypeptide, comprising:

(a) providing a polypeptide comprising

(i) an unprotected and reactive terminal α-amino group, and

(ii) at least one non-terminal amino group, wherein all said non-terminal amino groups are unreactive;

(b) contacting the polypeptide with at least one of an agent or an agent coupled to a linker at a reaction pR, said agent or agent coupled to a linker comprising exactly one unprotected aldehyde group;

(c) creating a covalent bond selectively between the terminal α-amino group and the unprotected aldehyde group; and

(d) producing a conjugate molecule with a ratio of polypeptide to agent of 1 : 1.

2. A method of linking an agent to a polypeptide, comprising:

(a) providing a polypeptide comprising

(i) a terminal a- amino group having a />K₂ value, and

(ii) at least one non-terminal amino group, each non-terminal amino group having a pK_& value;

(b) mixing said polypeptide with at least one of an agent or an agent conjugated to a linker, said agent or agent conjugated to a linker comprising exactly one unprotected aldehyde group, to form a reaction mixture; and

(c) maintaining the reaction mixture at a reaction pH, wherein the polypeptide selectively forms a covalent bond with the terminal oamino group through a reaction between the aldehyde group and the terminal a-amino group thereby producing a conjugate molecule with a ratio of polypeptide to agent of 1 : 1.

3. The method of claim 1 or 2, wherein the reaction pH is from about 4-6.

4. The method of claim 1 or 2, wherein the reaction pH is from about 4.5 to 5.5.

5. The method of claim 1 or 2, wherein the reaction pH is from about 4.5 to 4.8

6. The method of claim 1 or 2, wherein the reaction pH is 4.8.

7. The methods of any one of claims 1 -3, wherein the agent is a therapeutic agent.

8. The methods of any one of claims 1 -3, wherein the agent is selected from the group consisting of paclitaxel, curcumin, gemcitabine, doxorubicin, and camptothecin.

9. The methods of any one of claims 1 -3, wherein the agent is a diagnostic agent.

10. The methods of any one of claims 1 -6, wherein the polypeptide comprises a fragment of at least 10 residues in length having at least 90% sequence identity to a fragment of SEQ ID NO: 1.

1 1. The methods of any one of claims 1-6 wherein the polypeptide comprises a fragment of at least 33 residues in length having at least 90% sequence identity to SEQ ID NO: 1.

12. The method of any one of claims 1-6, wherein the polypeptide comprises SEQ ID NO: 6.

13. The method of any one of claims 1 -6, wherein the polypeptide comprises:

(a) at least 34 amino acid residues;

(b) the cysteine residues corresponding to positions 2, 5, 16, 19, 20, 28, 33, and 35 of SEQ ID NO: l ; and

(c) at least one of SEQ ID NO: 2, 3, and 5.

14. The method of any one of claims 1 -6, wherein the polypeptide is at least 34 residues in length, has at least 90% sequence homology to SEQ ID NO: 1 , and comprises the cysteine residues corresponding to positions 2, 5, 16, 19, 20, 28, 33, and 35 of SEQ ID NO: 1.

15. The methods of any one of claims 1 -6, wherein the polypeptide has at least 90% sequence homology to at least of of SEQ ID NO: 1 -8.

16. The methods of any one of claims 1-6, wherein the polypeptide has at least 95% sequence identity to a scorpion toxin selected from the group consisting of SEQ ID NO: 1 and 9-28.

17. A conjugate of a polypeptide and an agent that is the product of the process of any one of claims 1-13.

18. A polypeptide conjugate, said polypeptide conjugate having the structure A-Li-poly, wherein A is an agent, L is a linker, poly is a polypeptide moiety and 1 is 0 or 1 , wherein the agent is linked to the polypeptide moiety, either directly or through L, to an alpha amino group of the polypeptide moiety and wherein the agent and polypeptide moiety are present at a ratio of polypeptide to agent of 1 : 1.

19. The conjugate of claim 15, wherein the L is hydrolytically stable or hydrolytically labile.

20. The conjugate of claim 15, wherein L is FG_!-X-FG₂, wherein FGi is a first functional group capable of forming a linkage with a binding partner on the agent, FG₂ is a second functional group capable of forming a linkage with an alpha-amino group on the polypeptide moiety and X is a sequence linking FGi and FG₂.

21. The conjugate of claim 17, wherein FG₂ is a ketone group or FG₂ is an aldehyde group.

22. The conjugate of claim 17, wherein FGj is -COOH or -COZ and the binding partner on the agent is -OH, FG, is COOH, -COY, -CHO or -R'COR" and the binding partner on the agent is NH2, FGj is -SH, -COOH, -COZ or -Z-CH₂CO-Y and the binding partner on the agent is -SH, or FGi is -CHO, -R'COR", -COOH or -COZ and the binding partner on the agent is -NHNH₂, wherein Z is a halide, Y is OH or OR, and R, R' and R" are independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocyclyl, aromatic, or heterocyclyl groups.

23. The conjugate of claim 15 having the structure A-FGi-X-FG₂-poly, wherein A is an agent, FGj is a first functional group capable of forming a linkage with the agent, FG₂ is a second functional group capable of forming a linkage with the polypeptide moiety, X is a sequence linking FG) and FG₂ and poly is a polypeptide moiety.

24. The conjugate of claim 20, wherein FG₂ is a ketone group or FG₂ is an aldehyde group.

25. The conjugate of claim 20, wherein FGi is -COOH or -COZ and the binding partner on the agent is -OH, Fd is COOH, -COY, -CHO or -R'COR" and the binding partner on the agent is NH2, FGj is -SH, -COOH, -COZ or -Z-CH₂CO-Y and the binding partner on the agent is -SH, or FGi is -CHO, -R'COR", -COOH or -COZ and the binding partner on the agent is -NHNH₂, wherein Z is a halide, Y is OH or OR, and R, R' and R' ' are independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl carbocyclyl, aromatic, or heterocyclyl groups.

26. The conjugate of any one of claims 15-22, wherein the agent is a diagnostic agent.

27. The conjugate of any one of claims 15-22, wherein the agent is a therapeutic agent.

28. The conjugate of claim 24 wherein the therapeutic agent is paclitaxel, curcumin, gemcitabine, doxorubicin, or camptothecin.

29. The conjugate of any one of claims 15-25, wherein the polypeptide moiety comprises a fragment of at least 10 residues in length having at least 90% sequence identity to a fragment of SEQ ID NO: 1.

30. The conjugate of any of claims 15-25, wherein the polypeptide moiety comprises a fragment of at least 33 residues in length having at least 90% sequence identity to SEQ ID NO: 1

31. The conjugate of any one of claims 15-25, wherein the polypeptide moiety comprises SEQ ID NO: 6.

32. The conjugate of any one of claims 15-25, wherein the polypeptide moiety comprises:

(a) at least 34 amino acid residues;

(b) the cysteine residues corresponding to positions 2, 5, 16, 19, 20, 28, 33, and 35 of SEQ ID NO: 1 ; and (c) at least one of SEQ ID NO: 2, 3, and 5.

33. The conjugate of any one of claims 15-25, wherein the polypeptide moiety has at least 34 amino acid residues, has at least 90% sequence homology to SEQ ID NO: 1, and comprises the cysteine residues corresponding to positions 2, 5, 16, 19, 20, 28, 33, and 35 of SEQ ID NO: 1.

34. The conjugate of any one of claims 15-25 wherein the polypeptide moiety has at least 90% sequence homology to at least one of SEQ ID NO: 1-8.

35. The conjugate of any one of claims 15-25, wherein the polypeptide moiety has at least 95% sequence identity to a scorpion toxin selected from the group consisting of SEQ ID NO: 1 and 9-28.

36. A pharmaceutical composition comprising the conjugate of any of claims 14-32 or a pharmaceutically acceptable derivative thereof.

37. A method of delivering an agent to a cell comprising contacting the cell with the conjugate of any of claims 14-32.

38. The method of claim 34, wherein the cell is a cancer cell that is known to expresses MMP-2

39. A method for the treatment, prevention, or diagnosis of a cell-mediated disease or condition, said method comprising:

(a) identifying a subject in need of such treatment, prevention, or diagnosis; and

(b) administering the pharmaceutical composition of claim 33 to the subject.

40. A use of the conjugate of any of claims 14-32 in the manufacture of a pharmaceutical composition for the treatment, prevention, or diagnosis of a cell-mediated disease or condition.

41. The use of claim 37, wherein the cell-mediated disease is a neoplastic disease.

42. The use of claim 38, wherein the neoplastic disease is a cancer known to express MMP-2.

43. An ex vitro method for diagnosing a neoplastic disease comprising contacting a sample from a subject in need of such diagnosis with the conjugate of any of claims 14-32.

44. The methods of any of claims 35 and 40 wherein the cell-mediated disease is a neoplastic disease.

45. The method of claim 41, wherein the neoplastic disease is a cancer known to express MMP-2.