WO2001090361A2 - Modulation of inflammation by protease-treated mcp-3 chemokine - Google Patents

Abstract

The invention provides methods of inhibiting the biological activity or the in vivo biological activity of CC-chemokines (including native MCP-3), such as methods of inhibiting inflammation, comprising administering to a host an effective amount of a CC-chemokine receptor antagonist of the present invention. In some embodiments, the invention may provide methods of modulating an immune response in a host, or treating inflammation or autoimmune disease in a host suffering from such diseases, comprising administering to the host an effective amount of a CC-chemokine receptor antagonist of the present invention. Another aspect of the present invention is directed to pharmaceutical compositions comprising an antagonistically effective amount of a CC-chemokine receptor antagonist of the present invention and a pharmaceutically acceptable carrier. In alternative aspects, the invention provides compounds and methods for cancer treatment that facilitate an effective immune response.

Description

MODULATION OF PROTEASE EFFECTS ON CHEMOKINE SUBSTRATES

FIELD OF THE INVENTION

The invention is in the field of therapeutic compounds and uses thereof.

BACKGROUND OF THE INVENTION

Monocyte chemoattractant protein (MCP-3) is a potent, disulfide bridged CC chemokine active in the recruitment of monocytes and other leukocytes to sites of host challenge (11). International patent publication WO9806751 discloses analogs of mammalian MCP-3 lacking amino terminal amino acids corresponding to amino acid residues 1-6, 1-7, 1-8, 1-9 or 1-10, and discusses therapeutic uses of such compounds.

A variety of metalloproteinase activators and inhibitors are known, as for example are disclosed in U.S. Patent Nos. 5,977,408 or 6,037,361 and international patent publication W09921583, all of which are incorporated herein by reference. Because metalloproteinases are thought to be involved in pathological degradation of the extracellular matrix in various diseases, it has been suggested that inhibitors of metalloproteinases may be used as anti-inflammatories in a variety of diseases. It would be contrary to this teaching to discover that metalloproteinase inhibitors may have a physiological activity that sustains an inflammatory condition.

Library screening by the yeast two-hybrid system (2) has been useful in identifying intracellular protein-protein interactions using cDNA sources ranging from bacteria to man. However, its application to extracellular interactions has been largely overlooked for disulphide cross-linked proteins. Two-hybrid library screening using a proteinase catalytic domain as bait is potentially problematic because subsequent cleavage of library encoded substrate prey proteins may prevent detection in the assay.

SUMMARY OF THE INVENTION One aspect of the present invention includes CC-chemokine receptor antagonists. Such antagonists may include truncated derivatives of native MCP-3, in which the 4 amino acids at the N-terminal have been removed (leaving amino acid 5- 76), designated MCP-3(5-76). One aspect of the present invention is directed to pharmaceutical compositions comprising an antagonistically effective amount of a CC-chemokine receptor antagonist of the present invention and a pharmaceutically acceptable carrier. In alternative aspects, the invention provides compounds and methods for cancer treatment that facilitate an effective immune response.

In alternative aspects, the present invention provides methods of inhibiting the biological activity or the in vivo biological activity of CC-chemokines, including native MCP-3, comprising administering to a host, e.g., mammal (for example, human) a therapeutically effective amount of a CC-chemokine receptor antagonist of the present invention, for a time and under conditions sufficient to inhibit the biological activity of the native molecules. In some embodiments, the invention may provide methods of modulating an immune response in a host, or treating inflammatory or autoimmune diseases in a host suffering from such diseases, comprising administering to the host, such as a mammal, a therapeutically effective amount of a CC-chemokine receptor antagonist of the present invention. Another aspect of the present invention is directed to pharmaceutical compositions comprising an antagonistically effective amount of a CC-chemokine receptor antagonist of the present invention and a pharmaceutically acceptable carrier.

In one aspect of the invention, a yeast two-assay is provided using a gelatinase A hemopexin-like C-terminal domain as bait. A cDNA library was constructed from human fibroblasts treated with the lectin Concanavalin A. To validate the efficacy of this approach with extracellular molecules, a strong interaction was first demonstrated between the gelatinase A C-domain and the tissue inhibitor of metalloproteinase-2 (TIMP-2) C-domain. Screening of the library resulted in the identification of monocyte chemoattractant protein 3 (MCP-3) as a gelatinase A C-domain binding protein. This interaction was confirmed by ELISA binding assays and chemical cross-linking. By mass spectrometry and peptide sequencing it was shown that the first 4 residues of MCP-3 are removed by gelatinase A, cleaving MCP-3 at Gly⁴-lle⁵. Removal of these residues renders MCP-3 ineffective as a chemoattractant, and the cleaved MCP-3 was shown to act as a competitor to the wild-type molecule. By calcium mobilization, chemotaxis responses, in vivo models of inflammation, and in human pathology, it is demonstrated that cleavage of MCP-3 ablates receptor activation and creates a general chemokine antagonist MCP-3(5- 76). The invention also provides methods of cloning a substrate for a proteinase using the protein-protein interaction assays, such as the two-hybrid system, wherein a non-catalytic domain of the protease is assayed for protein-protein binding activity. The invention provides methods of modulating the role MMPs play in regulating the activity of an inflammatory chemokine. In various aspects, the invention involves the manipulation of the activity of MMPs in dampening the course of inflammation by destroying chemotactic gradients and functionally inactivating chemokines. The invention also involves manipulating the activity of MMPs as effectors of an inflammatory response. In alternative aspects, the invention provides therapeutic compounds comprising a protease-resistant chemokine linked to a tumour specific ligand by a protease-cleavable peptide sequence. Alternatively, a therapeutic compound may be provided comprising a chemokine that is resistant to a protease, wherein the chemokine is linked to a tumour-specific ligand by a peptide sequence that is cleavable by the protease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Characterization of MCP-3 interactions with the gelatinase A hemopexin C domain (Hex CD). (A) In the yeast two-hybrid assay only the yeast transformants Hex CD/TIMP-2 C domain, Hex CD/MCP-3, and p53/SV40 (positive control) showed growth on medium lacking histidine. Control transformants of the individual domains showed no significant growth. (B) -Galactosidase levels (presented as Miller units) in yeast expressing the indicated fusion proteins showed significant activity in only the Hex CD/TIMP-2 C domain, Hex CD/MCP-3, and p53/SV40 transformants. Yeast strain HF7c (Clontech) has three copies of the Gal4 17-mer consensus sequence and the TATA portion of the CYC promoter fused to the lacZ reporter. (C) Glutaraldehyde cross-linking of MCP-3 and recombinant hemopexin C domain. MCP-3 either alone, or in the presence of 0.5 molar equivalent (+), 1.0 molar equivalent (++), or 2.0 molar equivalents (+++) of hemopexin C domain, was cross-linked with 0.5% glutaraldehyde for 20 min at 22 °C. (D) ELISA binding assay of 0.5 μg MCP-3 immobilized onto a 96-well plate and then incubated with recombinant gelatinase A hemopexin C domain (Hex CD) or recombinant collagen binding domain (CBD) at the concentrations indicated. Binding of the recombinant domains was monitored by -His₆ affinity purified anti- peptide antibody and quantitated at 405 nm on a plate reader. Recombinant protein domains were expressed in E. coli as before (4).

Figure 2 Gelatinase A binding and cleavage of MCP-3. (A) Gelatin zymography of enzyme capture film assay of pro and active gelatinase A. Five μg each of bovine serum albumin (BSA), gelatin, TIMP-2, MCP-1 , and MCP-3 were immobilized onto a 96-well plate. Recombinant gelatinase A was then overlaid for 2 h to allow binding and the bound protein analysed by zymography. Overlay represents a dilution of the recombinant enzyme used. (B) Gelatin zymography as in A, but with hemopexin-truncated gelatinase A (N-gelatinase A) used as overlay. (C) Tricine gel analysis of MCP-3 (20) cleavage by gelatinase A in the presence of equimolar amounts (relative to MCP-3) of recombinant hemopexin C domain, collagen binding domain (CBD), TIMP-2, or 10 μM hydroxamate inhibitor BB-2275 (British Biotech Pharmaceuticals, Oxford, UK). Only a single concentration from the full dilution range of hemopexin C domain and CBD that was added as competitor is presented. (D) Tricine gel analysis of human fibroblast-mediated MCP-3 cleavage. Sub-confluent fibroblast cultures were treated with Con A (20 μg/ml) for 24 h at 37 °C. The resultant gelatinase A activation was confirmed by zymography. After 16-h incubation with MCPs in the presence of the MMP inhibitors indicated (concentrations as in C) the conditioned culture media were analyzed by tricine SDS- PAGE. The band at 22 kDa is the exogenous TIMP-2. The masses of the MCP-3 forms in the culture media were measured by electrospray mass spectrometry as shown. (E) Electrospray mass spectrometry, N-terminal Edman sequencing, and tricine gel analysis of MCP-3 cleavage products produced by recombinant gelatinase A activity. MCP-3 (5 μg) was incubated with 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 100 fg recombinant gelatinase A for 4 h at 37 °C. (F) Electrospray mass spectrometry and tricine gel analysis of MCP-1 , -2, -3, and -4 after incubation with recombinant gelatinase A for 18 h at 37 °C. The N-terminal sequence of the MCPs is shown with the Gly-lle scissile bond in MCP-3 in bold.

Figure 3 Cellular responses to MMP-cleaved MCP-3. (a) Cell receptor binding of full length MCP-3, designated MCP-3(1-76), and MCP-3(5-76). (b) Intracellular calcium induction by MCP-3, MCP-1 , and MDC. Fluo-3AM loaded THP- 1 monocytes or a B-cell line transfected with CCR-4 (for MDC) were first exposed to either 0 nM (left arrow, top scans) or 500 nM MCP-3(5-76) (left arrow, bottom scans), followed by MCP-3 (30 nM), MCP-1 (5 nM), and MDC (5 nM) as indicated (right arrow, top and bottom scans). The data are presented as relative fluorescence emitted at 526 nm. (c) Chemotactic activity of MCP-3(1-76) and MCP-3(5-76). Transwell assay of monocytes treated with MCP-3(1-76) and MCP-3(5-76) at the indicated concentrations demonstrating dose response antagonist action of MCP-3 (5-76). Not shown, are data that indicated loss of intracellular calcium induction by MCP-3 following gelatinase A-cleavage. Fluo-3AM loaded THP-1 monocytes were treated with 5 nM MCP-3 or MCP-1 or respective chemokine incubated first with gelatinase A for 18 h, demonstrating specific and complete loss of MCP-3 agonist activity.

Figure 4 Animal responses to MMP-cleaved MCP-3. Light micrographs of haematoxylin and eosin stained subcutaneous tissue sections of mice injected with: MCP-3(1-76) (a); gelatinase A-cleaved MCP-3 (b); 2:1 molar ratio of gelatinase A- cleaved MCP-3:full-length MCP-3 (c); and, saline/buffer control (d). In paneld (d), the bar represents 20 μm; M, muscle; A, adipocyte; C, loose connective tissue. Panel (a) clearly shows that only MCP-3(1-76) induced a marked mononuclear cell infiltrate with associated connective tissue disruption surrounding the muscle layer.

(e) After sub-cutaneous injections with MCP-3(1-76) and MCP-3(5-76) mixtures the infiltrating mononuclear cells were enumerated and expressed as cells/75,000 μm².

(f) and (g) Haematoxylin and eosin stained cytospins of intraperitoneal washouts of mice treated first with zymosan A to induce peritonitis, then 24 h later injected with (f)

MCP-3(5-76) or (g) saline for 4 h. Panel (h) shows identification of MCP-3(5-76) in human synovial fluid by immunoprecipitation of human MCP-3/progelatinase A complexes from inflammatory lesions. MCP-3 was pulled down using an -MCP-3 monoclonal antibody from 200 μl synovial fluid of a patient with seronegative spondyloarthropathy. Gelatin zymography (top panel) and western blotting with rabbit -MCP-3(1-76) antibody (bottom panel) of the complexes. Lane 1 , active and progelatinase A standards. DETAILED DESCRIPTION OF THE INVENTION

It has been suggested that inhibitors of metalloproteinases may be used therapeutically as anti-inflammatories . If this is done, the present invention discloses that such inhibitors may have the counter-indicated side-effect of sustaining an inflammatory condition, by inhibiting the proteolysis of a chemokine such as MCP-3, so that the chemokine (such as MCP-3) would continue to mediate inflammation as a potent chemoattractant cytokine. In one aspect, the present invention accordingly provides for the co-administration of a chemokine-metalloproteinase cleavage produce, such as MCP-3(5-76), and a metalloproteinase inhibitor, wherein the administration of the cleavage produce, such as MCP-3(5-76), is adjusted to counteract the inhibition of the protease, such as MCP-3, so as to restore the physiological effect of the metalloproteinase on its chemokine substrate, for example to inhibit inflammation.

Metalloproteinase inhibitors for use in various aspects of the invention may for example be selected from the fluorinated butyric acid compounds disclosed in U.S. Patent No. 6,037,361 or the ortho-sulfonamido aryl hydroxamic acids disclosed in U.S. Patent No. 5,977,408 or the MMP-2 inhibitors disclosed in W09921583, including: [{4-N-hydroxyamino}-2R-isobutyl-3S-{thienyl-thiomethyl}succinyl]-L- phenylalanine-N-methylamide; (S)-4-dibenzofuran-2-yl-4-oxo-2-(toluene-4- sulfonylamino)-butyric acid; (S)-2-(dibenzofuran-3-sulfonylamino)-3-methyl-butyric acid; and 4-hydroxyimino-4-(4'-methyl-biphenyl-4-yl)-butyric acid. Alternative MMP-2 inhibitors are disclosed in Tamura Y. et al., J. Med. Chem., 1998, 41 :640-649 and Porter J. et al., Bioorganic & Medicinal Chemistry Letters, 1994, 4(23):2741-2746 (all of which are incorporated herein by reference). Native MMP-2 inhibitors may also be used in alternative embodiments, such as the tissue inhibitor of metalloproteinase-2 (TIMP-2).

In one aspect of the invention, the dosage of a metalloproteinase inhibitor may be adjusted so that it is effective to attenuate the cleavage of a chemokine, such as MCP-3. In cancer treatments, for example, MMP-2 inhibitors may be administered at a dosage and for a time that is effective to attenuate the cleavage of MCP-3 to MCP-3(5-76), so that protease produced by the tumour, or in the vicinity of the tumour, does not inhibit an effective host immune response to the tumour. Previous suggestions for the use of metalloproteinase inhibitors in chemotherapy have not recognized that such compounds may be used to inhibit proteolysis of chemkines.

In alternative embodiments of the invention, proteolytic compounds, such as proteases, may be administered therapeutically to facilitate cleavage of native chemokines, such as MCP-3 to produce MCP-3(5-76), so that chemokine cleavage products such as MCP-3(5-76) may act as a chemokine receptor antagonists.

In a further aspect, the present invention provides protease-resistant forms of chemokines. For example, it has been discovered that murine MCP-3 is resistant to degradation by human MMP-2. Murine MCP-3 may therefore be used therapeutically as a protease-resistant chemokine. For example, in cancer, protease production by tumor cells may attenuate a beneficial host immune response. MMP-2 may for example play a role in tumour survival and metastatic spread (Collier et al., 1988, J. Biol. Chem. 263:6579-6587). In addition to basement membrane dissolution during metastasis, the present invention indicates that MMP-2-mediated cleavage of MCP-3 may contribute to cancer cell evasion of host immune system defences.

Administration of protease-resistant MCP-3 may therefore be used to counteract this effect to facilitate an effective host immune response to cancerous cells. Murine MCP-3 may for example be locally administered to a tumour to facilitate an anti- tumour immune response. In alternative embodiments, protease-resistant chemokines may be conjugated to tumour-specific ligands, such as tumour-specific antibodies, for delivery to a tumour or cancerous cell. In some embodiments, chemotherapeutic compounds, such as protease-resistant chemokines, may be attached or linked to a tumour-specific ligand by an MMP-cleavable sequence, such as an N-terminal sequence of human MCP-3. For example, murine MCP-3 may be attached to a tumour-specific monoclonal antibody by a linker comprising an N- terminal portion of human MCP-3, wherein the N-terminal portion of human MCP-3 is cleavable by MMP-2.

In some embodiments, the peptides of the invention may be substantially purified peptide fragments, modified peptide fragments, analogues or pharmacologically acceptable salts of MCP-3 having amino acids 1-4 truncated from the amino terminal of the native MCP-3, such compounds are collectively referred to herein as MCP-3(5-76) peptides. MCP-3(5-76) peptides may include homologs of the native MCP-3 sequence from amino acids 5 through 76, such as naturally occurring isoforms or genetic variants, or polypeptides having substantial sequence similarity to native MCP-3 amino acids 5-76, such as 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity to at least a portion of the native MCP-3(5-76) sequence, the portion of native MCP-3 being any contiguous sequence of 10, 20, 30, 40, 50 or more amino acids. In some embodiments, chemically similar amino acids may be substituted for amino acids in the native MCP-3 sequence (to provide conservative amino acid substitutions).

It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. For example, in one aspect of the invention, MCP-3 derived peptide antagonists of CC-chemokine receptors may include peptides that differ from a portion of the native MCP-3 sequence by conservative amino acid substitutions. Conservative amino acid substitutions of like amino acid residues can be made, for example, on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity or hydrophilicity. Such substitutions may be assayed for their effect on the function of the peptide by routine testing.

In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following hydrophilicity values are assigned to amino acid residues (as detailed in United States Patent No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (- 2.5); and Trp (-3.4).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: lie (+4.5); Va) (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (- 1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, lie, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr.

The invention provides pharmaceutical compositions, such as compositions containing CC-chemokine receptor antagonists of the invention. In one embodiment, such compositions include a CC-chemokine receptor antagonist compound in a therapeutically or prophylactically effective amount sufficient to inhibit inflammation, and a pharmaceutically acceptable carrier.

An effective amount of a compound of the invention may include a therapeutically effective amount or a prophylactically effective amount of the compound. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction of inflammation, or reduction or inhibition of monocyte chemotaxis or an alternative immune response. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also generally one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting inflammation. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

In particular embodiments, a preferred range for therapeutically or prophylactically effective amounts of compounds of the invention, such as CC- chemokine receptor antagonists, may be 0.1 nM-0.1M, 0.1 nM-0.05M, 0.05 nM- 15μM or 0.01 nM-10μM. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practicioners.

The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. As used herein "pharmaceutically acceptable carrier" or "exipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the compounds of the invention may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In accordance with an alternative aspect of the invention, therapeutic compounds may be formulated with one or more additional compounds that enhance the solubility of the therapeutic compounds. Peptide compounds of the invention may include derivatives, such as C- terminal hydroxymethyl derivatives, O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides and compounds in which a C-terminal phenylalanine residue is replaced with a phenethylamide analogue (e.g., Ser-lle- phenethylamide as an analogue of the tripeptide Ser-lle-Phe).

Within a peptide compound of the invention, a peptidic structure may be coupled directly or indirectly to a modifying group (e.g., by covalent coupling or a stable non-covalent association or by covalent coupling to additional amino acid residues, or mimetics, analogues or derivatives thereof, which may flank the core peptidic structure). For example, the modifying group can be coupled to the amino- terminus or carboxy-terminus of a peptidic structure, or to a peptidic or peptidomimetic region flanking the core domain. Alternatively, the modifying group may be coupled to a side chain of an amino acid residue of a peptidic structure, or to a peptidic or peptido-mimetic region flanking the core domain (e.g., through the epsilon amino group of a lysyl residue(s), through the carboxyl group of an aspartic acid residue(s) or a glutamic acid residue(s), through a hydroxy group of a tyrosyl residue(s), a serine residue(s) or a threonine residue(s) or other suitable reactive group on an amino acid side chain). Modifying groups covalently coupled to the peptidic structure can be attached using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, carbamate or urea bonds.

In some embodiments, a modifying group may comprise a cyclic, heterocyclic or polycyclic group. The term "cyclic group", as used herein, includes cyclic saturated or unsaturated (i.e., aromatic) group having from 3 to 10, 4 to 8, or 5 to 7 carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be unsubstituted or substituted at one or more ring positions. A cyclic group may for example be substituted with halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, -CF₃, -CN. The term "heterocyclic group" includes cyclic saturated, unsaturated and aromatic groups having from 3 to 10, 4 to 8, or 5 to 7 carbon atoms, wherein the ring structure includes about one or more heteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclic ring may be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, -CF_3l -CN. Heterocycles may also be bridged or fused to other cyclic groups as described below.

The term "polycyclic group" as used herein is intended to refer to two or more saturated, unsaturated or aromatic cyclic rings in which two or more carbons are common to two adjoining rings, so that the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycyclic group may be substituted with such substituents as described above, as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, -CF₃, or -CN. The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (C₁-C₂₀ for straight chain, C₃-C₂₀ for branched chain), or 10 or fewer carbon atoms . In some embodiments, cycloalkyls may have from 4-10 carbon atoms in their ring structure, such as 5, 6 or 7 carbon rings. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, having from one to ten carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have chain lengths of ten or less carbons. The term "alkyl" (or "lower alkyl") as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups), and esters (including alkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. The moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF₃, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF₃, - CN, and the like.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "aralkyl", as used herein, refers to an alkyl or alkylenyl group substituted with at least one aryl group. Exemplary aralkyls include benzyl (i.e., phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the like. The term "alkylcarbonyl", as used herein, refers to -C(0)-alkyl. Similarly, the term "arylcarbonyl" refers to -C(0)-aryl. The term "alkyloxycarbonyl", as used herein, refers to the group -C(0)-0-alkyl, and the term "aryloxycarbonyl" refers to -C(0)-0- aryl. The term "acyloxy" refers to -O-C(0)-R₇, in which R₇ is alkyl, alkenyl, alkynyl, aryl, aralkyl or heterocyclyl.

The term "amino", as used herein, refers to -N(R_α)(Rβ), in which R_α and R_β are each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or in which R_α and R_β together with the nitrogen atom to which they are attached form a ring having 4-8 atoms. Thus, the term "amino", as used herein, includes unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino), and disubstituted (e.g., dialkylamino or alkylarylamino) amino groups. The term "amido" refers to -C(O)- N(R₈)(R₉), in which R₈ and R₉ are as defined above. The term "acylamino" refers to - N(R'₈)C(0)-R₇, in which R₇ is as defined above and R'₈ is alkyl.

As used herein, the term "nitro" means -N0₂ ; the term "halogen" designates - F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; and the term "hydroxyl" means - OH.

The term "aryl" as used herein includes 5-, 6- and 7-membered aromatic groups that may include from zero to four heteroatoms in the ring, for example, phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF₃, -CN, or the like. Aryl groups can also be part of a polycyclic group. For example, aryl groups include fused aromatic moieties such as naphthyl, anthracenyl, quinolyl, indolyl, and the like.

Modifying groups may include groups comprising biotinyl structures, fluorescein-containing groups, a diethylene-triaminepentaacetyl group, a (-)- menthoxyacetyl group, a N-acetylneuraminyl group, a cholyl structure or an iminiobiotinyl group. A CC-chemokine receptor antagonist compound may be modified at its carboxy terminus with a cholyl group according to methods known in the art (see e.g., ess, G. et al. (1993) Tetrahedron Letters, 34:817-822; Wess, G. et al. (1992) Tetrahedron Letters 33: 195-198; and Kramer, W. et al. (1992) J. Biol. Chem. 267:18598-18604). Cholyl derivatives and analogues may also be used as modifying groups. For example, a preferred cholyl derivative is Aic (3-(0-aminoethyl- iso)-cholyl), which has a free amino group that can be used to further modify the CC- chemokine receptor antagonist compound. A modifying group may be a "biotinyl structure", which includes biotinyl groups and analogues and derivatives thereof (such as a 2-iminobiotinyl group). In another embodiment, the modifying group may comprise a "fluorescein-containing group", such as a group derived from reacting an MCP-3 derived peptidic structure with 5-(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. In various other embodiments, the modifying group(s) may comprise an N-acetylneuraminyl group, a trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyl group, an (S)-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl group, a 2-norbomaneacetyl group, a gamma-oxo-5- acenaphthenebutyryl, a (-)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a 4- morpholinecarbonyl group, a 2-thiopheneacetyl group or a 2-thiophenesulfonyl group.

A therapeutic compound of the invention may be modified to alter a pharmacokinetic property of the compound, such as in vivo stability or half-life. The compound may be modified to label the compound with a detectable substance. The compound may be modified to couple the compound to an additional therapeutic moiety. Examples of C-terminal modifiers include an amide group, an ethylamide group and various non-natural amino acids, such as D-amino acids and beta- alanine. Alternatively, the amino-terminal end of a peptide compound may be modified, for example, to reduce the ability of the compound to act as a substrate for aminopeptidases.

Compounds may be further modified to label the compound by reacting the compound with a detectable substance. Suitable detectable substances may include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or piiycoeryt m; an example of a luminescent material includes luminol; and examples of suitable radioactive material include ¹⁴C, ¹²³l, ¹²⁴l, ¹²⁵l, ¹³¹l, ^99mTc, ³⁵S or ³H. A peptide compound may be radioactively labeled with ¹ C, either by incorporation of ¹⁴C into a modifying group or one or more amino acid structures in the compound. Labelled compounds may be used to assess the in vivo pharmacokinetics of the compounds, as well as to detect disease progression or propensity of a subject to develop a disease, for example for diagnostic purposes. In an alternative chemical modification, a compound of the invention may be prepared in a "prodrug" form, wherein the compound itself does not act as a therapeutic, but rather is capable of being transformed, upon metabolism in vivo, into a therapeutic compound. A variety of strategies are known in the art for preparing peptide prodrugs that limit metabolism in order to optimize delivery of the active form of the peptide-based drug (see e.g., Moss, J. (1995) in Peptide-Based Drug Design: Controlling Transport and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter 18.

MCP-3(5-76) analogues of the invention may be prepared by standard techniques known in the art. MCP-3(5-76) analogues may be composed, at least in part, of a peptide synthesized using standard techniques (such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993); Grant, G. A. (ed.). Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992); or Clark-Lewis, I., Dewald, B., Loetscher, M., Moser, B., and Baggiolini, M., (1994) J. Biol. Chem., 269, 16075-16081). Automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Peptides may be purified by HPLC and analyzed by mass spectrometry. Peptides may be dimerized via a disulfide bridge formed by gentle oxidation of the cysteines using 10% DMSO in water. Following HPLC purification dimer formation may be verified, by mass spectrometry. One or more modifying groups may be attached to a peptide by standard methods, for example using methods for reaction through an amino group (e.g., the alpha-amino group at the amino-terminus of a peptide), a carboxyl group (e.g., at the carboxy terminus of a peptide), a hydroxyl group (e.g., on a tyrosine, serine or threonine residue) or other suitable reactive group on an amino acid side chain (see e.g., Greene, T. W. and Wuts, P. G. M. Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York (1991)).

In another aspect of the invention, peptides may be prepared according to standard recombinant DNA techniques using a nucleic acid molecule encoding the peptide. A nucleotide sequence encoding the peptide may be determined using the genetic code and an oligonucleotide molecule having this nucleotide sequence may be synthesized by standard DNA synthesis methods (e.g., using an automated DNA synthesizer). Alternatively, a DNA molecule encoding a peptide compound may be derived from the natural precursor protein gene or cDNA (e.g., using the polymerase chain reaction (PCR) and/or restriction enzyme digestion) according to standard molecular biology techniques.

The invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide of the invention. In some embodiments, the peptide may comprise an amino acid sequence having at least one amino acid deletion from the N-terminus, C-terminus and/or an internal site of MCP-3, compared to native MCP-3. Nucleic acid molecules may include DNA molecules and RNA molecules and may be single-stranded or double-stranded. To facilitate expression of a peptide compound in a host cell by standard recombinant DNA techniques, the isolated nucleic acid encoding the peptide may be incorporated into a recombinant expression vector. Accordingly, the invention also provides recombinant expression vectors comprising the nucleic acid molecules of the invention. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been operatively linked. Vectors may include circular double stranded DNA plasmids, viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (such as bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby may be replicated along with the host genome. Certain vectors may be capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors". In recombinant expression vectors of the invention, the nucleotide sequence encoding a peptide may be operatively linked to one or more regulatory sequences, selected on the basis of the host cells to be used for expression. The terms "operatively linked" or "operably" linked mean that the sequences encoding the peptide are linked to the regulatory sequence(s) in a manner that allows for expression of the peptide compound. The term "regulatory sequence" includes promoters, enhancers, polyadenylation signals and other expression control elements. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) (incorporated herein be reference). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell, those that direct expression of the nucleotide sequence only in certain host cells (such as tissue-specific regulatory sequences) and those that direct expression in a regulatable manner (such as only in the presence of an inducing agent). The design of the expression vector may depend on such factors as the choice of the host cell to be transformed and the level of expression of peptide compound desired.

The recombinant expression vectors of the invention may be designed for expression of peptide compounds in prokaryotic or eukaryotic cells. For example, peptide compounds may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Examples of vectors for expression in yeast S. cerivisae include pYepSed (Baldari et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of proteins or peptides in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39). Examples of mammalian expression vectors include pCDMδ (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

In addition to regulatory control sequences, recombinant expression vectors may contain additional nucleotide sequences, such as a selectable marker gene to identify host cells that have incorporated the vector. Selectable marker genes are well known in the art. To facilitate secretion of the peptide compound from a host cell, in particular mammalian host cells, the recombinant expression vector preferably encodes a signal sequence operatively linked to sequences encoding the amino-terminus of the peptide compound, such that upon expression, the peptide compound is synthesised with the signal sequence fused to its amino terminus. This signal sequence directs the peptide compound into the secretory pathway of the cell and is then cleaved, allowing for release of the mature peptide compound (i.e., the peptide compound without the signal sequence) from the host cell. Use of a signal sequence to facilitate secretion of proteins or peptides from mammalian host cells is well known in the art.

A recombinant expression vector comprising a nucleic acid encoding a peptide compound may be introduced into a host cell to produce the peptide compound in the host cell. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell may be any prokaryotic or eukaryotic cell. For example, a peptide compound may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. The peptide compound may be expressed in vivo in a subject to the subject by gene therapy (discussed further below).

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. Methods for introducing DNA into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (such as resistance to antibiotics) may be introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker may be introduced into a host cell on the same vector as that encoding the peptide compound or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (cells that have incorporated the selectable marker gene will survive, while the other cells die).

A nucleic acid of the invention may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake or viral-mediated transfection. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621 ; Wilson el al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor- mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel el al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126). Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include .p i.Crip, .p i.Cre, .p i.2 and .p i.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641- 647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

The genome of an adenovirus may be manipulated so that it encodes and expresses a peptide compound of the invention, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al.

(1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard

(1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin el al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).

Adeno-associated virus (AAV) may be used for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32- 39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

General methods for gene therapy are known in the art. See for example, U.S. Pat. No. 5,399,346 by Anderson et al. (incorporated herein by reference). A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W., et al., Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); and , Cavazzana-Calvo et al., Science 288:669-72 (2000), all of which are incorporated herein by reference).

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The disclosed uses for various embodiments are not necessarily obtained in all embodiments, and the invention may be adapted by those skilled in the art to obtain alternative utilities.

Example 1 The two-hybrid system was used to demonstrate a strong interaction between the single disulphide bonded gelatinase A hemopexin C domain and the C domain of the tissue inhibitor of metalloproteinase (TIMP)-2 that contains 3 disulphide bonds (Fig. 1A). Deletion analyses (5) and domain swapping (6) studies have provided indirect evidence for these domain interactions in the cellular activation and localization of gelatinase A to cell surface membrane type (MT)-MMPs (7). The assay of the invention provided direct evidence for this association in the gelatinase A/TIMP-2/MT1-MMP complex (8), showing the efficacy of the yeast two-hybrid assay of the invention for revealing disulphide-containing protein interactions that normally occur extracellularly at 37 °C. Surprisingly, in accordance with the assay of the invention, protein expression and folding in yeast at 30 °C appears to generate a stable, functional protein fold despite the apparent absence of disulphide bonds.

Example 2

Concanavalin A (Con A) stimulates fibroblasts to degrade extracellular matrix components by activating gelatinase A (9). A cDNA library was constructed from Con A-treated human gingival fibroblasts. Using the gelatinase A hemopexin C domain as bait in yeast two-hybrid screens (10) MCP-3 was identified as an interactor with gelatinase A (from a full-length cDNA clone (Fig. 1). The hemopexin C domain had as strong an interaction with MCP-3 as it did with the TIMP-2 C domain in the β-galactosidase assay (Fig. 1). Chemical cross-linking (12) of MCP-3 to recombinant hemopexin C domain verified this interaction (Fig. 1). The cross- linked MCP-3-hemopexin C domain had the expected mass of a 1 :1 bimolecular complex, whereas MCP-3 alone was not significantly cross-linked. Furthermore, MCP-3 prevented hemopexin C domain oligomerization, indicating a specific interaction. This was confirmed by an ELISA-based binding assay (Fig. 1). The hemopexin C domain showed saturable binding to MCP-3. Specificity was confirmed using recombinant gelatinase A collagen binding domain protein (13), comprised of three fibronectin type II modules, which did not bind MCP-3. Using an enzyme-capture film assay (14) it was found that the full-length gelatinase A enzyme bound MCP-3 (Fig. 2), whereas a hemopexin-truncated form of the enzyme (N- gelatinase A) did not (Fig. 2). No significant interaction was observed between gelatinase A and MCP-1. As controls both the full-length and N-gelatinase A bound to gelatin and TIMP-2 by the collagen binding domain and active site (15) of both forms of the enzyme, respectively. Together, these data demonstrate a strong requirement for the hemopexin C domain of gelatinase A in binding MCP-3.

MCP-3 was shown to be a novel substrate of gelatinase A. Incubation with recombinant enzyme resulted in a small but distinct increase in electrophoretic mobility of MCP-3 on tricine gels (Fig. 2C) that the MMP specific inhibitors TIMP-2 and the synthetic hydroxamate inhibitor, BB-2275, blocked. Recombinant hemopexin C domain competed for and reduced gelatinase A cleavage of MCP-3 in a concentration dependent manner whereas the collagen binding domain had no effect (Fig. 2C). In addition, the k_cat/K_m value of MCP-3 cleavage decreased from 8,000 M^'V for full-length gelatinase A to 500 M^"1s^"1 for N-gelatinase A confirming the mechanistic importance of the hemopexin C domain binding interaction in MCP-3 degradation. Cleavage of MCP-3 by other MMPs was also assayed, illustrating alternative proteases that may be used to generate MCP-3(5-76). Matrilysin (MMP- 7), which lacks a hemopexin C domain, and the MMPs collagenase-2 (MMP-8) and gelatinase B (MMP-9) did not cleave MCP-3, but collagenase-3 (MMP-13) and MT1- MMP (MMP-14) efficiently processed MCP-3 (not shown).

In one aspect of the invention, MCP-3 may be efficiently cleaved in vivo. Indeed, MCP-3 but not MCP-1 was rapidly cleaved in cell cultures of human fibroblasts following Con A-induced gelatinase A activation, but not in untreated cells (Fig. 2D). Molar excess TIMP-2 or BB-2275 blocked this confirming MMP dependency in MCP-3 processing. The bridging interaction of TIMP-2 between the gelatinase A hemopexin C domain and MT1-MMP (8), which is central to the physiological binding, activation and activity of gelatinase A at the cell surface, did not interfere with MCP-3 binding (not shown) and cleavage (Fig. 2D).

To identify the cleavage site in MCP-3 electrospray mass spectroscopy was performed. The mass measured of the gelatinase A-cleaved MCP-3 was 8,574 Da both in cell culture (Fig. 2D) or in vitro (Fig. 2E) and differed from the mass of the full- length molecule (8,935 Da) by the exact mass of the first four N-terminal residues. N-terminal Edman sequencing confirmed that the scissile bond was at Gly⁴-lle⁵ (Fig. 2E), a preferred sequence for gelatinase A cleavage in gelatin (18) that is absent in other MCPs that were not cleaved by gelatinase A (Fig. 2F). Together, these data demonstrate the importance of the hemopexin C domain for non-collagenous substrate cleavage by any MMP. This indicates that compounds that bind to protease exosites may be used to selectively inhibit proteolytic activity against specific substrates, in accordance with an alternative aspect of the invention.

To demonstrate the physiological relevance of gelatinase A association and cleavage of MCP-3, a monoclonal antibody to human MCP-3 pulled down pro- gelatinase A, but not the active enzyme, in association with full-length MCP-3 from the synovial fluid of a seronegative spondyloarthropathy patient (Fig. 3). Uncleaved MCP-3 was identified in these specific immunocomplexes using an affinity-purified anti-peptide antibody (alpha-1-76) that only recognizes the N-terminal 5 residues of MCP-3 (Fig. 3B). In order to identify gelatinase A-cleaved MCP-3 in vivo, specific antisera were raised that only recognizes the free amino group of the cleaved MCP-3 (5-76), but not the full-length MCP-3, nor another synthesized truncated MCP-3 (9- 76) as controls (Fig. 3). Using this neo-epitope antibody strategy (19) the gelatinase A-cleaved form of MCP-3 was found in human rheumatoid synovial fluid (Fig. 3C). These data demonstrate the physiological relevance of the MCP-3 interaction with gelatinase A in vivo and the pathophysiological generation of the MCP-3 cleavage product in human disease.

Activation of chemokine receptors by ligand mobilizes intracellular calcium stores and together with other signaling events leads to directed monocyte migration. MCP-3 binds CC receptors-1 , -2, and -3. Protein engineering studies have shown that N-terminal truncation at different sites has variable effects on the agonist activity of MCP-1 and MCP-3 (20, 21). To determine the effect of gelatinase A cleavage of MCP-3, we found that in calcium induction assays (22) the gelatinase A-mediated removal of the first four residues of MCP-3 resulted in the loss of receptor activation and chemokine activity. Neither gelatinase A-cleaved MCP-3 in the presence of 1/1000 gelatinase A (mole ratio enzyme/MCP-3) (Fig. 4A) nor synthetic MCP-3(5-76) (Fig. 4B) elicited a response in THP-1 cells, a monocyte cell line expressing CCR-1 and CCR-2. In addition to loss of CCR agonist activity, MCP-3(5-76) antagonized the subsequent response to both uncleaved MCP-3 and MCP-1 , which binds CCR-2 (Fig. 4B). MCP-3(5-76) also desensitized macrophage inflammatory protein (MIP)1- alpha induced Ca²⁺ mobilization in THP-1 cells (not shown). Since MIP-1 alpha binds CCR-1 and CCR-5, this confirmed the CCR-1 antagonist activity of MCP-3(5- 76). As a control MCP-3(5-76) did not block the calcium response to MDC, which binds CCR-4, a receptor not bound by MCP-3 (Fig. 4). The physiological relevance of MCP-3 antagonism was confirmed by cell binding assays (23). Scatchard analysis showed that synthetic MCP-3(5-76) bound cells with high affinity ( of 18.3 nM) similar to that of MCP-3 (K_ά of 5.7 nM) (Fig. 4C).

To determine the cellular response to gelatinase A cleavage of MCP-3, monocyte chemotaxis responses were measured. In transwell cell migration assays (22) MCP-3(5-76) was not chemotactic, even at a 100-fold higher dose than full- length MCP-3, which elicited the typical chemotactic response (Fig. 4). Consistent with the calcium mobilization experiments, synthetic MCP-3(5-76) (Fig. 4) and gelatinase A-cleaved MCP-3 (not shown) also functioned as antagonists in a dose dependent manner to inhibit the chemotaxis directed by full-length chemokine. Thus, inactivation of MCP-3 generates a broad-spectrum antagonist for CC- chemokine receptors that retains strong cellular binding affinity and modulates the response to a number of related chemoattractants.

To examine the biological consequences of MMP cleavage of MCP-3 in inflammation, a series of subcutaneous injections were performed in mice (24) of various mole ratios of full-length MCP-3 and gelatinase A-cleaved or synthetic MCP- 3(5-76). On analysis of tissue sections MCP-3, but not gelatinase A cleaved MCP-3 induced a marked infiltration of mononuclear inflammatory cells with associated degradation of matrix at 18 h (Fig. 4). ANOVA analysis of morphometric counts showed the statistically significant dose dependent reduction in the mononuclear cell infiltrate in response to as little as a 1:1 mixture of MCP-3(5-76) with MCP-3 (Fig. 4). In a separate mouse model of inflammation, the cellular infiltrate in 24-h zymosan A- induced peritonitis (24) was significantly attenuated after intraperitoneal injection with MCP-3(5-76). Consistent with morphometric examination of the lavage cytospins (Fig. 4), FACS analysis (25) of the peritoneal washouts showed that macrophage (F4/80+) cell counts were significantly reduced by -40% at both 2 and 4 hours following MCP-3(5-76) treatment (Fig. 4). The present example demonstrates of the extracellular inactivation of a cytokine in vivo by MMP activity.

In various aspects of the invention, the relative amounts of intact and cleaved MCP-3 that are present after pathophysiological cleavage will determine chemotactic and inflammation outcomes. Thus, gelatinase A expression, which is induced in tissues at the later stages of inflammation (34) by cytokines from macrophages and other earlier participants in the inflammatory reaction, may also serve to dampen inflammation by destroying the MCP-3 chemotactic gradient. This in turn can functionally inactivate the gradients of other CC chemokines having similar CCR usage. Of note, gelatinase A is largely stromal-cell derived and not usually expressed by leukocytes (35) which express MMP-8 and gelatinase B, both of which are not active on MCP-3. References and Notes

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10. Yeast strain HF7c (Clontech) was transformed as per suppliers' instructions with cDNA encoding the protein domains described fused to the Gal4 DNA- binding domain and the Gal4 transactivation domain. Transformants were selected on appropriate growth media, then tested on media lacking the metabolite histidine. Colony growth was monitored after 4 days incubation at 30 °C and the plate was photographed. Yeast growth indicates a positive interaction between proteins fused to the Gal4 domains. Quantitative analysis of interactions was done by liquid -galactosidase assays as per supplier instructions.

11. G. Opendaker et al. , Biochem. Biophys. Res. Commun. 191 , 535 (1993).

12. MCP-3 (0.1 mg/ml) and gelatinase A hemopexin C domain were combined at various mole ratios for 10 min at room temperature. Glutaraldehyde was then added to a final concentration of 0.5% for 20 min at room temperature. The reaction was terminated by the addition of Tris containing SDS-PAGE sample buffer. Samples were electrophoresed in 15% SDS-PAGE Tricine gels and stained with silver nitrate. MCP-3 was chemically synthesized using solid phase methods, the polypeptide was purified by reverse phase HPLC and folded using air oxidation. 13. B. Steffensen, U.M. Wallon, CM. Overall, J. Biol. Chem. 270, 11555 (1995). 14. The enzyme capture film assay is a modification of an ELISA-based binding assay. Proteins to be tested for binding were immobilized onto a 96-well plate. Following blocking by bovine serum albumin, enzyme solutions were overlaid onto wells for 2 h at room temperature to allow binding. After extensive washes to reduce non-specific interactions, bound enzyme was recovered with SDS-PAGE sample buffer and assayed for gelatinolytic activity by gelatin zymography. Recombinant human progelatinase was expressed in CHO cells and purified by gelatin-Sepharose chromatography. N-gelatinase

A was produced by autocatalytic degradation of recombinant full-length gelatinase A at 37 °C, after activation by 1 mM 4-aminophenylmercuric acetate in the presence of 1.0 % TX-100, and dialyzed for 16 h to remove the reactants. 15. Y. Itoh, M.S. Binner, H. Nagase, e/ocietr/. J. 308, 645 (1995).

16. T.N. Young, S.V. Pizzo, S. Stack, J. Biol. Chem. 270, 999 (1995).

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18. S. Netzel-Amett et al. , Biochemistry 32, 6427 (1993).

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21. J.-H. Gong et al., J. Biol. Chem. 271 , 10521 (1996).

22. THP-1 cells (myeloid cell line, ATCC) or B cells transfected with CCR-3 cDNA were loaded with Fluo-3AM for 30 min at 37 °C After addition of various full length chemokines or MCP-3(5-76) the fluorescence was monitored with a Perkin-Elmer 650-10B spectrofluorimeter using an excitation wavelength of

506 nm and an emission wavelength of 526 nm. Desensitization assays were performed by sequential addition of MCP-3(5-76) or buffer control, followed by the full length chemokine. THP-1 cell migration was assessed in transwell trays (Costar) with 6.5 mm diameter chambers of 3 μm membrane pore size. MCP-3 and MCP-3(5-76) were added to the lower well, and THP-1 cells (1 x

10⁷ cells/ml) were added to the upper well. After 1.5 h, cells that had migrated to the lower well were counted. The percent migration was calculated by dividing the mean number of migrating cells in response to chemokine by the mean number of cells migrating in response to medium alone. 23. 4 nM [¹²⁵l]-MCP-3(1-76) in the presence of serially diluted unlabeled MCP- 3(1-76) or MCP-3(5-76) and 0.05% NaN₃ was incubated at 4 °C for 30 min with THP-1 cells. Cell bound and free [¹²⁵l]-MCP-3(1-76) were separated by centrifugation of the cells through a column of dioctyl phthalate.n-butyl phthalate (2:3, v/v). Amounts of bound [¹²⁵l]-MCP-3(1-76) were determined in the cell pellet by gamma counting. Nonspecific binding was determined in the presence of a 100-fold concentration of unlabeled ligand and was subtracted from the total. The data were analyzed by Scatchard analysis. 24. CD-4 mice (5 per group) were injected at two subcutaneous sites (500 ng/100 μl pyrogen free saline) with either full-length MCP-3 [designated MCP-3(1- 76)], gelatinase A-cleaved MCP-3 [designated MCP-3(5-76)], 2:1 molar ratio of gelatinase A-cleaved MCP-3:MCP-3(1-76), or saline/buffer control. In other experiments, 6 replicate mice per group were injected as before, but with 100 μl MCP-3(1-76)/MCP-3(5-76) mixtures as follows: 500 ng/0, 0/500 ng, 500 ng/500 ng, 500 ng/1000 ng, 500 ng/2500 ng, or saline. Mice were sacrificed 18 h post-injection and paraffin sections transverse to the skin were analyzed. Sections were stained with haematoxylin and eosin and examined by light microscopy. Morphometric cell counts per 75,000 μm² field of mononuclear cell infiltrates in the loose connective tissue immediately above the muscle layer of skin were performed double blind and used to calculate the mean and the standard error of the mean. Peritonitis was induced in mice using zymosan A (1 mg/500 μl saline) injected in the peritoneal cavity. At 24 h an intraperitoneal 5 ml saline lavage was performed to collect infiltrating cells that increased ~40-fold compared to saline controls. In experiments, 50 μg MCP-

3(5-76) or saline was administered to the peritoneal cavity 24 h after the induction of peritonitis. Infiltrating cells were collected after 2 and 4 h by saline lavage. Cells were counted on a Coulter Counter gated at 5-10 μm and 100 μl cytospins were examined by light microscopy after haematoxylin and eosin staining.

25. Peritoneal cells were stained for 60 min. on ice with 20 μg/ml of rat anti- mouse F4/80 mAb or rat lgG2b isotype control. After extensive washing, cells were stained with FITC-conjugated anti-rat IgG for 45 min. on ice, extensively washed, and analyzed by flow cytometry using a FACScan analyzer (Becton Dickinson, U.K.).

26. S. Struyf et al., Eur. J. Immunol. 28, 1262 (1998).

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30. C.L. Wilson et al., Science 286, 113 (1999).

31. M. Baggiolini, B. Dewald, B. Moser, Annu. Rev. Immunol. 15, 675 (1997).

32. E.F. Foxman, J.J. Campbell, E.C Butcher, J. Cell Biol. 139, 1349 (1997). 33. B. Lu et al., J. Exp. Med. 187, 601 (1998).

34. R.G. Paul et al., Int. J. Biochem. Cell Biol. 29, 211 (1997).

35. G. Openakker, S. Masure, B. Grillet, J. Van Damme, Lymphokine Cytokine Res. 10, 317 (1991).

36. I.E. Collier et al, J. Biol. Chem. 263, 6579 (1988). 37. J. Van Damme, P. Proost, J.P. Lenaerts, G. Opdenakker, J. Exp. Med. 176, 59 (1992).

Claims

WHAT IS CLAIMED IS:

1. MCP-3(5-76).

2. The use of MCP-3(5-76) to treat inflammation.

3. A method of inhibiting inflammation comprising administering to a patient an effective amount of MCP-3(5-76).

4. The use of murine MCP-3 to treat a cancer in a human patient.

5. The use of a protease-resistant chemokine to treat a cancer.

6. A therapeutic composition comprising a chemokine modified to be protease resistant.

7. The use of MCP-3(5-76) to formulate a medicament.

8. The use of MCP-3(5-76) to formulate a medicament to treat inflammation.

9. The use of murine MCP-3 to formulate a medicament.

10. The use of murine MCP-3 to formulate a medicament to treat a cancer.