CA2316405A1 - Modulation of inflammation by protease products - 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 same 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 ecceptable carrier. In alternative aspects, the invention provides compounds and methods for cancer treatment that facilitate an affective immune response.

Description

RUG 17 2000 8:51 PM FR ~J.GEORGIR VRNCOUVER 68~ 0274 TO 18199532476 P.05 MODULATION OF INFLAMMATION BY PROTEASE I~RODUCTS
FIELD QF THE INVENTION
The invention is in the field of therapeutic compounds and uses thereof.
BACKGROUND OF THE INVENTION
Monocyte chernoattractant protein (MCP-3) is a potent, dlsulflde bridged CC chemokine far the recruitment of monocytes and other leukocytes to sites of host challenge (7 f). International patent publication W09806751 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 6,437,361 and international patent publication W49921583, all of which are incorporated herein by reference. Because metalloproteinases are thought to be involved in pathological degradation of the extracellular matrix in various disea58S, it has been suggested that inhibitory 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 ~ physiological activity that sustains an inflammatory condition.
Library screening by the yeast two-hybrid system (2) has b~en 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 and to our knowledge has never been used to Identify substrates for an extr2~cellular proteinase. Indeed, the rationale for library screening using a proteinase catalytic domain for bait is tenuous because subsequent cleavage of library encoded SUb$trate would likely prevent detection in the assay.
SUMMARY OF THE INVENTION
The invention provides methods of inhibiting the biological activity or the in vivo biologicat activity 4f CC-chemokines (including native MCP-3), such as methods of inhibiting inflammation, comprising administering to a halt an effective amount of a CC-chemokine receptor antagonist of the ptesent 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 affective 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.
One aspect of the present invention includes CC-chemokine receptor antagonists. Such antagonists may include truncated derivatives of native MCP-RUG 17 2080 8:51 PM FR ~J.GEORGIR URNCOUUER 682 0274 TO IA199532476 P.06 3, in which the 4 amino acids at the N-terminal have been removed (leaving amino acid 5-76), designated MCP-3(5-76).
in alternative aspects, the present invention provides methods of inhibiting the biological activity or the ill vlvo biological activity of CC-chemokine~, 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 sulYicient 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 effeCkive amount of a CC-chemokine receptor antagonist of the present invention. Another aspect of the present invention is directed to pharmaCeutlcal compositions comprising an antagonistically effective amount of a CC-chemokine receptor antagonist of the present invention and a phamZaceutically acceptable carrier.
In one aspect of the invention, a yeast two-hybrid analysis was initiated using the gelatinase A hemopexin-like C-terminal dom2~in 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) G-domain. Screening of the library resulted in the identification of monocyte chemoattractant protein 3 {MGP-3} as a getatinase A C-domain binding protein. This interaction was confirmed by ELISA binding assays and chemical crass-Iirlking_ By mass spectrometry and peptide sequencing it was shown that the first 4 residues of MCP-3 ar~ removed by gelatinise A, cleaving MCP-3 at Gly'-lle$. 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 vlvo models of inflammation, and in human pathology, it is demonstrated that cleavage of MCP-3 ablates receptor activation and Creates a general ch~mokine 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 involve6 the manipulation of the activity of MMP$ in dampening the cours~ of inflammation by destroying chemotactic gradients and functionally inactivating chemokines.
~'he invention also involves manipulating the activity of MMF's as effectars of an inflammatory response.
BRIEF OESCRiPTI~N OF ThIE DRAWINGS
Figure 1 Characterization of MCP-3 interactions witty the getatlnase A
hemopexin C domain (Nex CD). {A) In the yeast two-hybrid assay only the yeast transformants HeX CDrTIMP-2 C domain, Hex CDIMCP-3, and p53ISV40 RUG 17 2088 8:52 PM FR W.GEORGIR URNCOUUER 682 0274 TO 18199532476 P.07 (positive control) showed growth on medium laoking histldine. Control transformants of the individual domains showed na significant growth. (B) -Galactosidase levels (presented a5 Miller units) In yeast expressing the indicated fusion proteins showed signiftcant activity in only the Hex CDITIMP-2 C
domain, Hex CDlMCP-3, and p631SV44 transformants. Yeast strain HF7c (Clontech) has three copies of the Gal4 i7-mer consensus sequence and the TATA portion of the CYC promoter fused to the fact 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 hemapexin C domain, was oross-linked with 0.5%
glutaraldehyde for 2U min at 22 °C. (D) ELISA binding assay of 4.a Ilg immobilized onto a 9B-well plate and then incubated with recombinant gelatinise A hemopexin C domain (Hex CD) or recombinant Collagen binding domain (CBD) at the concentrations indicat8d. Binding of the recombinant domains was monit4red by -Hiss affinityr purrfied anti-peptide antibody and quantttated at 4d5 nm on a plate re2~der. Recombinant protein domains were expressed in E. coil as before (4).
Figure 2 Gelatinise A binding and cleavage of MCP-3. (A) Gelatin zymography of enzyme capture film assay of pro and active gelatinise A. Five ~Ig each of bavine serum albumin (BSA), gelatin, TIM.i-'-~, MCP-1, and M~l~-:~
were immobilized onto a 96-well plate. Recombinant gelatinise A was then overiakl for 2 h to allow binding and tile bound protein analysed by Zymography.
Overlay represents a dilution of the recombinant enzyme used. (B) Gelatin zymography as in A, but with hemopexin-truncated gelatinise A (N-gelatinise A) used as overlay. (C) Tricine gel analysis of MCP-3 (20) cleavage by gelatinise A
in the presence of equimolar amounts (relative to MCP-3) of recombinant hemopexin C domain, collagen binding domain (CBD), TIMP-2, or 10 NM
hydroxamate inhibitor BB-2275 (British Biotech Pharmaceuticals, Oxford, UK).
only a single concentration from the full dilution range of hemopexin G 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 Irglml) for 24 h at 37 °C. The resultant gelatinise A
activation was confirmed by zymography. After 1fi-h incubation with MCPs in the presence of the MMI~ inhibitors indicated (concentrations as in G) the canditipned culture media were analyzed by triCine ADS-PAGE. The band at 22 kDa is the exogenous TIMP-2. The masses of the MCP-3 forms in the culture media w~r8 measured by electraspray mass spectrometry as shown. (E) Electrospray mass spectrometry, N-terminal Edman sequencing, and tricine gel analysis of MCP-3 cleavage products produced by recombinant gelatinise A activity. MCP-3 (5 Ng) was incubated with 100 ng, 90 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 100 fg recombinant gslatir~ase A for 4 h at 37 °C. (F) Electrospray mass spectrometry and tricine gel analysis of MCP-1, -~2, ~, and ~ after incubation with recombinant gelatinise A for 18 h at 37 °C. The N-terminal sequence of the MCPs is shown with the Gly-Ile scissile bond in MCP-3 in bold.

RUG 17 2000 8:52 PM FR IJ.GEORGIR URNCOUUER 682 0274 TD 18199532476 P.08 Figure 3 Cellular responses to MMP-cleaved MCP-3. (a) Celi receptor binding of full length MCP-3, designated MCP-3(1-76), and MCP-3(5-78). (b) Intracellular calcium induction by MCP-3, MCP-1, and M(~C. Fluo-3AM loaded THP-1 monocytes or a B-cell line transfected with OCR-4 (for MDC) were first exposed to either 0 nM (left arrow, top scetls) or 5(1Q nM MCP-3(5-76) (left arrow, bottom scans), followed by MCP-3 (34 nM), MCP-1 (6 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 speoifiC and complete loss of MCP-3 agonist activity.
Figuro 4 Animal responses t0 MMP-cleaved MCP-3. Light micrographs of haematoxylin and eosin stained subcutaneous tissue sections of mice injecked with: MCP-3(1-76) (a); gelstlnase A-cleaved MCP-3 (b); 2:1 mol2~r ratio of gelatinase A-cleaved MGP-3:full-length MCP-3 (c); and, salinelbuffer control (d).
In paneld (d), the bar represents ~0 arm; M, muscle; A, adipacyte; C, loose connective tissue. Panel (a) clearly shows that only MCP-3(1-'~fi) induced a marked mononuclear cell infiltrate with associated connective tissue disruption surrounding the muscle layer. (e) After sub-cutanQOUS il~jections with MCP-3(1-76) and MCP-3(5-76) mixtures the infiltrating mononuclear cells were enumerated and expressed as cellsl76,1)00 Nma. (f) and (g) Naematoxylin and eosin stained cytaspins of intraperitoneal Washouts of mice treated first with zymosan A to induce peritonitis, then 2d h later injected with (f) MCP-3(5-76) or (g) saline for ~t h. Panel (h) shows identification of MCP-3(5-76) in human synovial fluid by immunopreCipitation of human MCP-3lprogelatinase A
complexes from inflammatory lesions. MCP-3 was pulled d4wn using an -MCP-3 monoclonal antibody from X00 NI synovial fluid of a patient with seronegative spondyloarthropathy. Gelatin zymography (top panel) and western blQtkir~g with rabbit -MCP-3(1-76) antibody (bottom panel) of the complexes.
Lane 1, active and progelatinase A standards.
DETAILED DESCRiPTICN OF THE INVENTION
It has been suggested that inhibitors of metalloproteinases may be used therapeutically as anti-inflammatories . if this is done, the pre$ent invention discloses that such inhibitors may have the counter-indicated side-effect of sustaining an inflammatory condition, by inhibiting the proteolysis of MCP-3, so that MCP-3 would continue to mediate inflammation as a potent ohemoattractant cytokine. In one aspect, the present invention accordingly provides for the co-administration of MCP-3(5-76j and a rnetalldproteinase Inhibitor, wherein the administration of the MCP-3(5-76) is adjusted to counteract the inhibition of MCP-3 proteolysis, so as to inhibit inflammation.

RUG I7 2000 8:53 PM FR ~J.GEORGIR VRNCOUVER 682 0274 TO 18199532476 P.09 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 t~dhn-sulfnnarraido aryl hydroxafnic , __" , , _ .
acids disclosed in U.S. Patent No. 5,977,408 or the MMP-2 inhibitors disclosed in W099215$3, including: [{4-N-hydroxyamino)-2R-isobutyl-35-~thienyl-thiamethyl}suceinylj-L-pher~ylalanine-N-methylamide; (Sr4-dibenZOfuran-2-yl-4-oxo-2-(toluene-4-sulfonylamino)-butyric acid; (S)-2-(dibenzofuran-3-sulfonylamino)-3-methyl-butyric acid; and 4-hydroxyimlno-4-(4'-methyl-biphenyl-4-yl)-butyric acid. Alternative MMP-2 inhibitors ace disclosed in Tamara Y. et al., J. Mad. Chern., 1998, 41:fi40-t~49 and Porter J. et al., Bioorganic &
Medicinal Chemistry Letters, '1994, 4(23):2741-2746 (2~I1 of which are incorporated herein by reference). Native MMP-2 inhibitors may also be used in alternative embodiments, such as the ti$sue inhibitor of metalloproteinase-2 (TlMP-2).
In one aspect of the invention, the dosage of a matalloprotelnase inhibitor may be adjusted sa 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 far a time that is effective to attenuate the cleavage of MCP-3 to MCP-3(5-76), so that protease prvdu~d 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 matalloproteinase 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 cle2waga of native chemokines, such as MCP-3 to produce MCP-3(5-76), so that chemokine cleavage products such as MCP-3(5-78) may act as a chamokine receptor antagonists.
In a further aspect, the present invention provides protease-resistant forms of chemokines. For example, it has been discovered that marine MCP-3 is resistant to degradation by human MMP-2. Marine MCP-3 may therefore be used therapeutically as a protease-r~es3stant-chart:oklt~F~r-e~campls,-in-cancer. _ _.. __-__..._.._. ... _____.___ protease production by tumor ells may attenuate a beneficial host immune response. MMP-2 rnay for example play a role in tumour survival acrd metastatic spread (Collier et al., 1985, J. Biol- Chem. 263;6579-6587). In addition to basement membrane dissolution during me'kastasis, the present Invention indicates that MMP-2-mediated cleavage of MCf~-3 may Contribute to cancer cell evasion of host immune system defences. Administration of protease-resistant MCP-3 may be usEd to counteract this effect to facilitate an effective host immune response to cancerous cells. Marine MCP-3 may for example be locally administered to a tumour to facilitate an anti-tumour immune response. In alternative embodiments, protease-resistant ahemokines may be conjugated to tumour speck ligands, such as tumour specific antibodies, far delivery to a tumour or cancerous cell. In some embodiments, chemotherapeutio compounds, such as protease-resistant chemokines, may be attached or linked to a tumour-specific ligend by an MMP-cleavable sequence, such as an N-terminal sequence of human MCP-3. For examQla, marine MCP-3 may be attached to a tumour--g-RUG 17 2000 8:53 PM FR IJ.GEORGIR VRNCOUVER 6B2 8274 TO 18199532476 P.10 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 soma embodiments, the peptides of the invention may be substantially purified peptide fragments, modified peptide fragments, analogues or pharmacologically acGeptabte salts of MCP-3 having amino acids 1-4 trtlncated from the amino terminal of the native MCP-3, such compounds are Colleotirreiy referred to herein as MCP-3(5-76) peptides. MCP-3(5-7B} peptides may include homologs of the native MCP-3 sequence from amino adds 5 through 76, such as naturally occurring iSOfomis or genetic variants, or polypeptides having substantial sequence similarity t4 native MCP-3 amino acids 5-76, suoll as 40%, 50%, 60%, 70°/6, 80%, 90%, 95% or 99% sequence identity to at least a portion of the nafive MCP-3(5-76) sequence, the portion of native MCP-3 being any contiguous sequence of 10, 20, 30, 40, 54 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 mod~cations and changes can be made in the structure of a pofypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent pt~typeptlde.
For exampl~, 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 tike 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. Sueh substitutions me~y be assayed for their effect on the function of the peptide by routine testing.
In some embodiments, conserved amino acid substitutions may ba 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 t4 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 (+4.3); Asn (+0.2); Gln (+0.2); Gly (0);
Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); CYs (-1.4); Met (-'I .3); Val (-1.5); Leu (-1.8);
IIB (-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);
Val (+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.$); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (_ 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 losses, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met acidic:
Asp, E~UG 17 2088 8:54 PM FR ~J.GEORGIR VRNCOUVER 682 8274 TO 1819953476 P.11 Glu; basic: Lye, Arg, Wis; neutral: Gly, Ser, Thr, Cys, Asn, Gln, 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 suffloient 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 prophylacctically 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 monocyta ahemotaxis 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 detrim~ntal effects of the compound are outweighed by the therapeutically bene~flCial effaCts. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the deslred 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 prophylacHcally 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-0.9 M, 0.1 nM-O.OSM, 0.03 nM-t 5uM or 0.01 nM-10pM. It is to be noted that dosage values may vary with the severity of the Corlditic~n tv be alleviated. For any particular subject, specie 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. Cosage 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 re~pon$a, 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 (ay the unique RUG 17 200Id 8:54 PM FR IJ.GEORGIR VRNCOUVER 682 0274 TD 18199532476 P.12 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 anti all Solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption deiayirig agents; ~arx! the-like-tl~rat ar'a- --physiologically compatible. In one embodiment, the carrier is suitable far parenteral administration. Alternatively, the carrier ran be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration.
Pharmaceutically acceptable oarriars include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injeot8ble 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 incvmp2tible 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 composh(on can be formulated as a solution, microemulsion, liposome, yr 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, 2nd the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the Use of a coating such as lecithin, by ihs maintenance of the roquired particle size in the case of dispersion and by the use of surPaGtants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyaloohols such as mannltol, 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, polylaCtio acid and polylactic, polyglycallc 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 enumer2~ted above. in the case of sterile powders for the preparation of sterile injectable solutions, the preferred _g_ RUG 17 2088 8:55 PM FR 1J.GEORGIR VRNCOUVER 692 8274 TO 19199532476 P.13 methods of preparation are vacuum drying and freez~-drying which yields a powder of the sctlVe ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In accnrdanoe with an alternative aspect of the invention, therapeutic compounds may be formulated with one or more additional compounds that enhance the solubility c~f tho therapeutic compounds.
Peptide compounds of the invention may include derivatives, such as C-terminal hydroxymethyl derivatives, O-mod'~led derivatives (e.g., C-terminal hydroxymethyl benZyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydraaides and compounds in which a C-terrriinai phenylalanina residue is replaoad with a phenethylamide analogue (e.g., Ser-Ile-phenethylamlde as an analogue of the trlpeptide Ser Ile-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 mimetias, 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-tcrminus of a peptidic structure, or to a pcptidic ar pepti;domimetic region flanking the care domain. Aitematively> 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 residu~e(s) or a glutamic acid residue(s), through a hydroxy group of a tyrosyl residue(s), a serine residues) or a threoninc residues) or other suitable reactive group on an amino acid side chaixr).
Modifying groups covalently coupled to the peptidic structure can be attached using methods well known in tht alt for linking chemical structures, including, for example, amide, alkylamino, carbamate or urea bonds.
In some embodiments, a modifying group may comprise a cyclic, lleterocyclic or polycyclic group. The term "cyclic group'', as used herein, includes cyclic saturated or unsaturated (i.e., aromatic) group having from 3 to 10, ~1 to 8, or S to 7 carbon atoms.
Exemplary cyclic groups include eyelopropyl, cyclaburyl, cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be unsubstituted ox substituted at one or more ring positions. A cyclic group may for example be substituted with halogens, alkyls, cyclvalkyls, alkenyls, alkynyls, aryls, hcterocycles, 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 gmups having from 3 to 10, 4 to $, or 5 to 7 carbon atoms, wherein the ring structtwe includes about one or more heteroato~ms- Heterocyclio groups include pyrrolidirie, oxolane, thialane, imidazole, oxazole, piperidine, piperazine, morpholi~ne.
The heterocyclic ring may be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cycloalkyls, slkenyls, alkynyls, tuyls, other hcterocyclcs, hydroxyl, amino, vitro, thiol, amines, imines, amides, phosphonatcs, phosphmes, carbonyls, carboxyls, silyls, ethers, thioethexs, sulfonyls, selenoethers, ketones, aldeltydes, esters, -CF3, -CN. Hettroeycles xnay also be bridged or fused to other cyclic groups as described below.
-g-RUG 17 2888 8:55 PM FR IJ.GEORGIR VRNCOUUER 682 8274 TD 18199532476 P.14 The term "polycycllc 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 Gammon to two adjoining rings, so that the rings are "h.~sed 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, vitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carbonyls, 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 (alicydlc) groups, alkyl substituted cyoloalkyl 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 (Ci-C2o for straight chain, Ca-Czo for branched chain), cr 9p cr fewer carbon atoms . In same embodiments, cyGoalkyls may have from 4-1 D 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") a$ uthroughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having subst'rtuents replacing a hydrogen on one or more carbons of the hydrrbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and aryicarbonyl groups), and altars (including alkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, vitro, azido, sutfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sutfonamido, 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), -GF3, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyl$ can be further substituted with alkyls, alkenyls, alkoxys, aikylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3. -CN, and the like.
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length arid 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 b$rl~y (i.e., phenylmethyl), Z-naphthylethyl, 2-(2-pyridyl)propyl, 6-dibenzpsuberyl, and the like.

RUG 17 2000 8:56 PM FR ~J.GEORGIR VRNCOUVER 682 0274 TO 18199532476 P.15 The term "alkylcarbonyl", as used herein, refers to -C(t7)-alkyl. Similarly, the term "arylcarbonyl" refers to -C(O)-aryl. The term "alkyloxycarbonyl", as used herein, refers to the group -C(O)O-alkyl, and the term "aryloxyrarbonyl"
refers to -C(4~O-aryl. The tem7 "acyloxy" refers to -O-C(O)-R,, in which R7 is alkyl, alkenyl, alkynyl, aryl, aralkyl or heterocyclyl.
The term "amino", as used herein, refers to -N(Ra)(Ra), in which RQ ant) R~
are each independently hydrpgon, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or in which Ra and Rs together with the nitrogen atom to which they are attached form a ring having 4-8 atoms. Thus, the tens "amine", as used herein, includes unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino), and disubstituted (e.g., dialkylamino or alkylarylarnino) amino groups. The term "amido" refers to -C(C7)-N(Ra)(R~), in which R$ and Rg are as defined 2~bove.
The term "aoylamino" refers to -N(R's)C(O)-R,, in which R~ is as defined above and R'$ is alkyl.
As used herein, the term "vitro" means -N02 ; the term "halogen"
designates ~F, -CI, -Br or -I; the term "sulfhydryl" means -8H; and the term "hydroxyl" means -OH.
The tens "aryl" as used herein includes 5-, ~- and 7-membered aromatic groups that may include from aero to four heteroatoms in the ring, for example, phenyl, pyrrolyl, furyl, thiophenyl, imldaZOlyl, oxazole, #hiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like.
Those aryl groups having heteroatoms in the r(ng 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, alkonyl, alkynyl, cycloalkyl, hydroxyl, amino, vitro, sulfhydryl, imino, amido, phosphonate, phasphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aklehyde, ester, a heterocyclyl, an aromatic or heteroarornatic moiety, -CF3, -CN, ar tho like.
Aryl groups can also be part of a polycyclic group. For example, aryl groups include fused aromati4 moieties such as naphthyl, anthracenyl, quinolyl, indolyl, and the like.
Modifying groups may include groups comprising biofinyl structures, fluorescein-containing groups, a diethylene-triaminepentaacetyl group, a (-)-msnthoxyacetyl group, a N-acetylneuraminyi group, a Cholyl structure or an iminiobiotinyl group. A CC-ohomokine receptor antagonist compound may be modified at its carboxy terminus with a cholyl group according to methods known in the art (see e.g., bless, G. et al. (1993) Tetrahedron Letters, 3~4:$1'~-$~z;
bless, G. et al. (1992) Tetrahedron Letters 33:995-198; and Kramer, W, et al.
(1992) J. Biol. Chem. 267:1859$-1$604). Cholyl derivatives end analogues may also be used as modifying groups. For example, a preferred choly) derivative is Aic (3-(O-aminoethyl-iso)-cholyl), which has a free amino group That can be used to further modify the CC-chemokine receptor antagonist compound. I~ modifying group may be a "biatiny) structure", which includes biotinyl groups and analogues and derivatives thereof ($uph 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 structc~re with 5-(and 8-)-RUG 17 2000 8:57 PM FR ~J.GEORGIR URNCOUVER 682 0274 TO 18199532476 P.16 carboxyfluorescein, succinimidyl ester or flu4rescein isothioeyanate. In various other embodiments, the modifying groups) may comprise an N-acetylneurar'ninyl group, a traps-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyi group, an (S)-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl group, a 2-norbomaneacetyl group, 8i gamma-oxo-5-acenaphthenebutyryl, a (-)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a 4-merpholinecarbonyl 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 bs modified to couple the compound to an additional theraQeutic 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 aminop~tidases.
Compounds may be further modified to label th4 compound by reacting the c4mpound with a detectable Substance. Suitable detestable substances may include various enzymes, prosthetic groups, fluorescent materials, IuminesCertt materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinestera5e; examples of suitable prosthetic group complexes include streptavidinlbiotin and avidinlbiotin; examples of suitable fluorescent materials include umbelllferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotrlazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radiOaCdve material include 140.'231, l2di, 1251, ~3~1, 9s'"Tc. ~S or 3H. A pe~tide compound may be radioactively labeled with'°C, either by incorporation of C 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, a$ well as to detect disease progression or propensity of a subject to develop a disease, for example for diagnostic purposes.
In an alternative chemical mod~cation, 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 vrvo, into a therapeutic compound. A variety of strategies are known in the art for preparing peptide prodrug& th2~t 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 (ads), Chapter 18.
MCP-3(5-76) analogues of the inventian 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 strandard techniques (such as those described in Bodansky, M. Principles of Peptide Synthesis, 5pringer VErlag, Berlin (1993); Grant, O. A. (ed.). Synthetic Peptides: A User's Guide, W. ti.

RUG 17 2000 8:57 PM FR W.GEORGIR URNCOUUER 682 0274 TO 18199532476 P.17 Freeman and Company, New York (1992); or Clark-~ewis, L, Dewald, ~., Laetscher, M., Moser, B., and Baggiolirti, M., (1994} J. giol. Chem., 289, 160~1). Automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligcn/SiosearGh 9600). Peptides may be purified by HPLC and analyzed by mass spectrometry. Peptides may be dimerized via a disulfide bridgo formed by gentle oxidation of the cysteiner 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 u9ing 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 nueleoxide sequence may be synthesized by standard DNA synthesis methods (e.g., using an automated DNA synthesiser}. 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 tc standard molecular biology techniques.
The invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide of theinr~ntion;-in-games--- --- ---------embodiments, the peptide may comprise an amino acid sequence having at least one amino aei~f deletion from the N-terminus, C-terminus andlor an internal site of MCP-3, compared to native MCP-3. Nucleic acid molecules may include DNA
molecules and RNA molecules arid may be single-stranded or double-stranded.
Tp 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 expres$ion vector. Accordingly, the invention also provides recombinant expression vectors comprising the nucleic acid molecules of the invention. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucl~ic acid to which it has been operatively linked. Vectors may include Circular double stranded DNA plasmids, viral vectors. Certain vectors are capable of autonomous replication in a host Dell into which they are introduced (such as bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome cf a, host cell upon introduction into the host cell, and thereby may be replicated along with the host genome. certain vectors may be capable of directing the exprc$cion of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors~, RUG 17 2000 8:58 PM FR W.GEORGIR URNCOUUER 682 0274 TO 18199532476 P.18 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 bast 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 sequences) in a manner that alknrvs for expression of the peptide compound. The term "regufatary sequence" includes promoters, enhancers, poiyadenylation signals and other expression control elements. Such regulatory sequences are described, for eicample, in C3oeddel; Gene Expression Technology: Methods in Enzymology 1$6, Academic Press, San Diego, Calif. (1990) {incorporated herein be reference). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of hpst cell, those that direct expression of the nucleotide sequence only in certain host cells (such as tissue-specific regulatory sequences) and those that direct expression in a regulatable manner (such as only in the pr$sence pf 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 eukaryatic 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 ~cpression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
('f 990). Altemativeiy, the recombinant expression vector may be transcribed and tr2~r~Slr~ted in vitro, for example using T7 promoter regulatory sequences and polymerase. Examples of vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari et al., (1987) EMBC J. B:229-234), pMFa (Kurjan and I-terskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1$87) Gene 54:113-123), and pYES2 (Invitrogen Corporation, $an Diego, Calif.).
Baculovirus vectors available far expression of proteins or peptides in cultured insect cells (e.g., St 9 cells) include the pAc series (Smith et al., ("19$3) Mol. Cell.
Siol.
3:2158-2185) and the pVt_ series {l_ucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39). Examples of mammalian expression vectors include pCDMB
(Seed, B., (1987} Nature 329:840) and pMT2PC (Kaufman et al. (18$7), EMBO
J. 6:'187-19a). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Viru$ 40.
In addition to n~gulatory control sequences, recombinant expression vectors may contain additional nucleotide sequence, 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 calls, 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 seduen~

RUG 17 2888 8:58 PM FR W.GEORGIR VRNCOUVER 682 8274 TO 18199532476 P.19 fused to its amino terminus. This signal sequence directs the peptide compound into the seaetory pathway of the cx~ll and is then cleaved, allowing for release of the mature peptide compound (i.e., the peptide compound without the signet sequence) from the host cell. Use of a signal sequence to facilitate secretion of proteins or peptides from mammalian host cells is weN known in the art.
A recombinant expression vector comprising a nucleic acid encoding a peptide compound may be introduced into a host cell to p~'oduce the peptide compound in the host cell. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The term$
"host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular sqbject 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. Far example, a peptide compound may be expressed in bacterial cells such as E. ooli, 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 4r eukaryoiac cells via ~nventional transformation or transfection techniques. The terms "transformation" and "transfaction" refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated transfectlon, lipofaction, 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. ~refen~t selectable markers include those that confer resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker may be introduced into a host tail on the same vector as that encoding the peptide compound or may be introduced on a separate vector. Cells stabty transfected with the introduced nucleic acid may be ident~ed 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 tie delivered to cells Jn vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake or viral-madiated transfection. Direct injection has been used to introduce naked DNA
into cells in vivo (see e.g., Aosadi et al. (1991) Nature 332:815-81$; Wolff et al.
-1h-AUG l7 2000 8:59 PM FR W.GEORGIA URNCOUVER 682 8274 TO 18199532476 P.20 (1990) Scierloe 247:1465-1488), A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially avallabla (e.g., from BioRad). Naked DNA may also be introduced into cells by eomplexing the DNA to a ration, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (19$$) J. Biol. them. 2$3:1d$21; Wilson el al. (1992) J. Biol. Chem.
287:9fi3-987; and U.S. Pat. No. b,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 links 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 Guriel el al. (1991) Proc. Natl. Acad. 5ci. USA 88:8850; Cristiano et al. (1993) Proc.
Natl.
Acad. Sci. USA 90:2122-212fi).
Defective retroviruses are well characterized for use in gone transfer for g~ne therapy pufposes (for a review see Miller, A. D. (1990) Blood 76:271 ).
Protocols for producing recombinant retraviruses and far infeotlng cells In vitro ar in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. tads.) Grasps Publishing Associates, (i989), 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.
F~camples of suitable packaging virus lines include .p i.Crip, .p i.Cre, .p 1.2 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 man-ow cells, in vitro andlor in vivo (see for example Eglitis, et al. (1985) Science 280:1395-1398; papas and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8460-8484; Vllilson et al. (1988) Proc.
Natl.
Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Aead. Scl.
USA 87'6141-8145; Huber et al. (1991) Proc. Natl. Acad_ Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8$81; Chawdhuty et al. (1991 ) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl.
Acad. Sci. USA 89:7640-7644; Kay et al. (1892) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA $9:10$92-10895; Hwu et al.
(1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,8B8,11g; U.S. Pat. No.
4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT
Application WO $9105845; and PCT Application WO 92107573).
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:$1fi; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 88:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or othar strains of adenavirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled ire the art. E~ecombinarlt adenoviruses are advanxageaus in that they d4 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), andothellal cells (t_emarchand et al. (1992) Proc. Natl. Acad. Sci. USA
89:8482-RUG 17 2000 8:59 PM FR W.GEORGIR URNCOUUER 682 0274 TO 18199532476 P.21 6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA
90:2812-2$16) and muscle Cells (4uantin el al. (1992) Proc. Natl. Acad. Sci.
USA
$9:2581-2584).
Adeno-associated virus (AAV) may be used for delivery of DNA for gene therapy purposes. AAV is a naturally oCCUrring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus far efficient replication and a pr~uctive life cycle (Muzyczka et al. Curr. Topics in Micro. and Irnmunol. (1992) 15$:97-129). AAV may be used to integrate DNA
into non-dividing cells (see fot example Fl4tte et al. (1992) Am. J. Respir.
Cell.
Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and MGLaughlin et al. (1989) J. Virol. 62:'1963-1973). An AAV vector such as that described in Tratschin et al. (198x) Mol. Cell. 8iol. 5:3251-3260 may be usad to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl.
Acad. Sci. USA $1:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (198$) Moi. EndoCrinol_ 2:$2-39; Tratschin et ai.
(1984) J.
~rol. 51:611-G19; and Flotte et al. (1993) J. Biol. Chem. 2fi8:3781-3790).
General methods for gene therapy are known in the art. See for example, U.S. Pat. NQ. 5,399,346 by Anderson et al. (incorporated herein by reference).
A
biocompatible capsule for delivering genetic material is described in PCT
Publication WO 95105452 by Baetge et al. Methods of gene transfer into hernatopoietic cells have also previously been reported (see Clapp, D. W., et al., Blood 78: 1132-1739 (1991); Anderson, Science 288:627-9 (2000); and , Cavazzana-Caivo ef al., Science 2B8:8B9-72 (x000), all of which are incorporated her$tn by reference).
Although various embodiments of the inventipn 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 modlttcations 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 daims, 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 inventior~ may be adapted by those skilled in the art to obtain alternative utilities.
Exampl~ 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 All'IMP-21MT1-MMP complex (8), showing the eft3cacy of the yeast two-hybrid assay of the invention for revealing disulphide-containing protein interactions that normally occur extracellularly at 37 °C.

RUG l7 2000 9:00 PM FR ~J.GEORGIR URNCOUUER 682 0274 TO 18199532476 P.22 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 2~ppar'ant absence of disulphide bonds.
Example 2 Concanavalin A (Con A) stimulates flbroblasts to degrade extracellular matrix components by activating gelatinise A (9). A cDNA library was constructed from Con A-treated human gingival febroblasts. Using the gelatinise A, hemopexin C domain as bait in yeast two-hybrid screens (~a) MCP-3 was identified as an intaractor with gelatinise A (from a full-length cDNA clone (Fig.
i). The hemopexin C domain had aS StfOng an interaction with MCP-3 as it did with the TIMP-2 C domain in the (i~alactosidase assay (Fig. 1). Chemical cross-linking (92) of MCP-~ to recombinant hemopexin C domain verifiEd this interaction (Fig. 1). The cross-linked MCP-3-hemapexin C domain had the expected mass of a 1:1 bimolecular complex, whereas MCP-3 alone wa$ not sign~cantty cross-linked. Furthermore, MCP~3 prevented hemapexin C domain oligomerization, indicating a specific interaCticn. 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 gelatinise A collagen binding domain protein (93), comprised of three fibronectin type ll modules, which did not bind MGP-3. Using an enzyme-capture fitlm assay (i4) it was found that the full-length gelatinise A enzyme bound MCP-3 (Fig.
2), whereas a hemopexin-truncated form of the enzyme (N-gelatinise A) did not (Fig. 2)_ No significant interaction was observed between gelatin2se A and MCP-1. As controls both the full-length and N-gelatinise A bound to gelatin and TIMP-2 by the collagen binding domain and active site (95) of both forms of the enzyme, respectively. Together, these data demonstrate a strong requirement for the hemopexin C domain of gelatinise A in binding MCP-3.
MCP-3 was shown to be a novel substrate of gelatinise A. Incubation with recombinant enayrme resulted in a Small but distinct increase in electrophoretic mobiiity of MCP-3 on tricine gets (Fig. 2C) that the MMP
specific inhibitors TIME'-2 and the synthetic hydroxamate inhibitor, BB-2275, blocked.
Recombinant hemopexin C domain competeCl for and reduced gelatinise A
cleavage of MCP-3 in a concentration dependent manner whereas the collagen binding domain had no effect (Fig 2C). In addition, the k~,,~lKm value of MCP-cleavage decreased from $,000 M-~s-~ far full-length gelatinise A to 500 M-~s' for N-gelatinise 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 altematfve proteases that may be used to generate MCP-3(5-7fi). Matrilysin (MMP-7), which lacks a hemopexin C domain, and the MMPs collagenase-2 (MMP-8) and gelatinise 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 vlvo.
indeed, MCP-3 but not MCP-1 was rapidly cleaved in cerll cultures of human fibroblasts following Con A-induced gelatinise A activation, but not in untreated _ 18-RUG 17 2000 9:00 PM FR 1J.GEORGIR VRNCOUVER 6B2 0274 TO 18199532476 P.23 cells (Fig. 2D). Molar excess TfMP-2 or BB-2276 blocKed this coniitrnlng MMP
dependency in MCP-3 processing, The bridging Interaction of TIMP-2 between the gelatin~lse A hemopexin C domain and MTi-MMP (8), which is central to the physiological binding, activation and activity of gelatinise 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 gelatinise 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-tenninal residues. N-terminal Edman sequencing confirmed that the scissile bond was at GIy4-IleS (Fig. 2E), a preferred sequence for gelatinise A
cleavage in gelatin (78) that is absent in other MCPs that were not cleaved by gelatinise A
(Fig. 2F). Together, these data demonstrate the importance of the hemOpexin C
domain far 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 gelatinise A association and cleavage of MCP-3, a monoclonal antibody to human MCP pulled down pro-gelatinise A, but not the active enzyme. in a$soaation with full-length from the synovial fluid of a seronegative spondyloarthropathy patient (Fig.
3}.
Uncleaved MCP-3 was identified in these specf0c 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 gelatinise A-cleaved MCP-3 in viva, specfic 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 (79) the gelatinise A-cieaved form of MCP-3 was found in human rheumatoid synovial fluid (Fig. 3C). These data demonstrate the physiological relevance of the MCPT3 interaction with gelatinise A in vivo and the pathophysiofogical 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. Prot~in engineering studies have shown that N-terminal truncation at different sites has variable effects on the agonfst activity of MCP-1 and MCP-$ (~Q, 21). To determine the effect of gelatinise A cleavage of MCP-3, we found that in calcium induction assays (22) the gelatinise A-mediated removal of the first four residues of MCP,3 resulted in __- .. the--fuss-flf-r-activation-a~rd-th~a~rnokirre-aotivity. --Neitherwgetatin~W-cleaved MCP-3 in the presence of 111000 gelatinise A (mole ratio enzymeIMCP-3) (Fig. 4A) nor synthetic MCP-3(5-76) (Fig. 4B) elicited a response in THP-1 cells, a monocyte cell Ilne expressing CGR-1 and GGR-2. In addition to loss of CCR agonist activity, MCP-3{5-76) antagonised the subsequent response to both uncleaved MCP-3 and MCP-1, which binds CCR-2 (Fig. 4B). MCP-3(5-76) al$o desensitized macrophage inflammatory protein (MIP)1-alpha induced Caz''' mobilization in THP-1 cells {not shown). Since MIP-lalpha binds CCR-1 and RUG l7 2000 9:01 PM FR 1J.GEORGIR VRNCGUVER 682 0274 TO 18199532476 P.24 CCR-5, this confirmed the CCR-1 antagonist activity of MCP-3(5-76). As a Control MCP-3(6-76) did not block the cak~um response to MIJC, which binds CCR-4, a receptor not bound by MCP (Fig. 4). The physiological relevance of MCP-3 antagonism was confirmed by cell binding assays (2~j. SCatchard analysis showed that synthetic MCP-3(5..76) bound cells with high affinity (fC~, of '1$.3 ~7M) similar to that of MCP-3 (Kd of 6.7 nM) (Fig. 4C).
To determine the cellular response to gelatinise A cleavage of MCP-3, monocyte chemotaxis responses were measured. In transwell cell migration assays (22) MCP-~(5-7S) 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 gelatinise A-Cleaved MCP-8 (net shpwn) also functioned as antagonists in a dose dependent manner to inhibit the chemotaxis direct~i by fuN-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 gelatinise A-cleaved or synthetic MCP-3(5-78). On analysis of tissue sections MCP-3, but not gelatinise 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 iri 24-h zymosan A-induced pedtonitts (24j 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/$0+) cell counts were significantly reduced by ~40°~ at both 2 and 4 hours following MCP-3(5-7B) 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 cleavag~ will determine ch~motactic and inflammation outcomes. Thus, gelatinise A expression, which is induced in tissues $t the l2~ter stages of inflammation (34j by cytokines from macrophages and other earlier participants in the inflammatory reaction, may alsp 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. C3f note, gelatinise A is largely stromal-cell derived and net usually expr~,ssed by leukocytes (35) which express MMP-8 and gelatinise 9, both of which are not active on MCP-3.
References and Notes 9 . K.S. Lam et al., Nature 354, 82 (1991 ).

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10. Yeast strain HF7c (Glontech) was transformed as per supplier' instructions with cDNA encoding th~ protein domains described fused to the Gal4 DNA-binding domain and the Gal4 transactivation d4main.
Trartsformants were selected on appropriate growth media, then tested on media lacking the metabolite histldine. Calany growth was monitored after 4 days incubation at 30 °C and the plate was photographed. Yeast g~'awth indi~tes a positive interaction between proteins fused to the Gal4 domains. Quantitative analysis of Interactions was done by liquid -galactosidase assays as per supplier instruCtior~s.
11. G. Opendaker et al., Blochem. Biophys. Res. Commun. 191, 635 (1993).
12. MCP-3 (0.1 mglml) and gelatinase A hemopexin C domain were combined at various mote 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 chemit~lly synthesized using solid phase methods, the polypsptide was purified by reverse phase HPLC and folded using air oxidation.
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14. The enzyme capture film assay is a modificatipn 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-speck interactions, bound enzyme was recovered with SDS-PAGE sample buffer and assayed f4r gelatirrolytic activity by gelatin zymography. Recombinant human progelatinase was expressed in CH4 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 9~o TX-100, and dialyzed for 16 h tv rernvve the reactants.
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22. THP-1 cells (myeloid cell line, ATCC) or B Cells transfected with CCR-~
cDNA were loaded wifh Fluo-3AM fior 30 rnin at 37 °C. After addition ofi various full length chemakines or MCP-3(5-76) the fluorescence was monitored with a Perkin-Elmar 660-10B spectrofluorimeter using an excitation wavelength ofi 506 nm and an emission wavelength of 526 nm.
Desensitization assays were perfom~ed 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 8.5 mm diameter chambers of 3 pm membrane pore size. MCP-3 and MCP-3(5-76) were added to the lower well, and THP-1 Cells (1 x 10' cellslml) 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 ['251j-MCP-3(1-?6) in the presence of serially diluted unlabeled MCP-3(1-76) or MCP-3(5-76) and 0.05°~ NaN was incubated at 4 °C for 30 min with THP-1 cells. Cell bound! and free [~z5lj-MCP-3(1-76) were separated by centrifugation of the cells through a column of dioctyl phthalate:n-butyl phthalate (2:3, vlv). Amounts of bound t'ZSIj-MCP-3(1-?fi) were determined in the cell pellet by gamma counting. N4n$pecNic binding was determined in the presence of a 100-fold concentration of unlabeled ligand and was subtracted from the total. The data were analyzed by Scatchard analysis.
2~t. CD-4 mice (5 per group) were injected ~It two subCUtaneaus sites (a00 ng1100 WI pyrogen free saline) with either full-length MCP-3 [designated MCP-3(1-78)j, gelatinise A-clmaved MCP-3 [designated MCP-3(5-76)], 2:1 molar ratio of gelatinise A-cleaved MCP-3:MCP-3(1-76), or salinelbuffer control. In other experiments, 6 replicate mic~ per group were injected as bolero, but with 140 NI MCP-3(1-76)IMCP-3(5-?6) mixtures as follows: 500 ngl0, 01500 ng, 500 ng1500 ng, 500 ng11000 ng, 500 ngl2500 ng, or saline. Mice were sacrificed 18 h post-injection and paraffin sections transverse to the skin were analysed. Sections were stained with haemataxylin and eosin and examined by light microscopy.
Morphometric cell counts per 75,000 Nm2 field of mananuclear cell infiltrates in the loose connective tissue immediately above the muscle layer of skin were p~rtormed double blind and used to calculate the mean and the standard error of the mean. Peritonitis was induced in mice using zymosan A (1 mg/500 pl saline) Injected in the peritoneal cavity. At 24 h an intraperitoneal 5 ml Saline lavage was performed t4 collect infiltrating cells that inoreaeed --40-fold compared to saline controls. In experiments, 50 pg MCP-3(5-7fi) or saline was administered to the peritoneal cavity 24 h after the induction of peritonitis. Infiltrating cells were collected after and 4 h by saline lavage. Cells were counted on a Coufter Counter gated -zz-RUG 17 2000 9:02 PM FR ~J.GEORGIR VRNCOUVER 682 0274 TO 18199532476 P.27 at 5-10 arm and 194 NI cytospins were examined by light micro$copy after haematoxylin and eosin staining.
25. Peritoneal Calls were st8~ined for 69 min. on ice with 20 Irglml of rat anti-mouse F4180 mAb or rat IgG2b isotype control. After exE;ensive 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, IJ.K.).
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_23_

Claims (4)

1. A method 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.

2. MCP-8(5-76).

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

4. A method of inhibiting inflammation comprising administering to a patient an effective amount of MCP-3(5-76).
6. The use of routine MCP-3 to treat a cancer in a human patient.
B. The use of a protease-resistant chemokine to treat a cancer.
7. A therapeutic compound comprising a protease-resistant chemokine linked to a tumour specific ligand by a protease-cleavable peptide sequence.
8. A therapeutic compound 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 pretense.