CA2152141A1 - Monocyte chemoattractant protein (mcp)-1 antagonists - Google Patents

Abstract

NH2-truncated MCP-1 analogues that function as MCP-1 antagonists are disclosed. The MCP-1 antagonist analogues may be used to inhibit MCP-1 activity and binding of MCP-1 to MCP-1 receptors. The analogues may be used in pharmaceutical preparations as anti-inflammatory agents.

Description

21~2141 .J..~YLI~ CIIEMOATTRACTANT PROTEIN (MCP~-l ANTAGONISTS
This invention relates to novel polypeptides capable of hl ~'k; n~ the affinity of monocyte chemoattractant protein -1 (referred to herein as MCP-l) 5 for MCP-1 receptors but which lack MCP-1 activity and function as MCP-1 antagonists.
Backqround of the Invention MCP-1 is an infli tory mediator and i5 characterized as a chemotactic cytokine, or ~ inP.
10 MCP-1 causes migration of monocytes and other cells such as bASIophilR and lymphocytes into sites of infl: tion.
MCP-1 has been implicated in a number of allergic and chronic infli tory ~ AR~3 such as arthritis, arteriosclerosis and various lung diseases. In such 15 conditions, monooytes inf iltration may be a key early event in the progression of the disease.
The complete amino acid sequence of MCP-1 was described in Rnh;n~)n~ E.A., et al. 198g. "Complete Amino Acid Sequence of a Eluman Monocyte Chemoattractant, 20 A Putative Nediator of ColllllAr Immune Reactions".
Proc. Natl. Acad. Sci. USA. 86: 1850-1854. MCP-l comprises a 76 amino acid polypeptide having a N~2-t~rmi nAl glutamine which spontaneous converts to pyroglutmate af ter removal of the signal peptide . MCP- 1 25 may exiAt as a monomer and as a diamer of the 76 amino acid polypeptide. ~Jnited States Patent Nos. 5,212,073 and 5,278,287 of Rollins et al ~ A~rihe a monocyte chemoattractant identif ied as JE having an amino acid sequence the same as amino acid residues 7-76 of MCP-1.
It has been suggested that substances that are capable of blocking the effect of MCP-l would be useful to moderate or inhibit i n f 1: t i on in an animal . The 21~21~1 patent application of MA 1 1 i n~krodt Medical, Inc . f iled under the Patent Cooperation Treaty and p-lhl i ch~ April 28, 1994 under W0 94/09128, describes the use of antisense MCP-l protein to block MCP-l caused restenosis 5 during use of balloon-type catheters when treating an animal. Also, antihorli~c~ to MCP-l that neutralize MCP-l were reported in Jones, M.L. et al. 1992. "Potential Role of Monocyte Chemoattractant Protein l/JE in Monocyte/Macrophage--l~p-~n~F-nt IgA Immune Complex Alveolitis in the Rat". J. Immunol. 149:2147-2154.
Another means for hl.~kin~ MCP-l activity would be the dev~l L L of antagonists that compete for binding at a MCP-l receptor. A MCP-l receptor has been identified and is reported in Charo, I. F. et al . 1994 .
"Molecular Cloning and Functional Expression of Two Monocyte ~ ~ - rA~tant Protein-l Receptors Reveals Alternative Spl i~inq of the Carboxyl-t~rm;nAl Tails" .
Proc. Natl. Acad. Sci. USA. 91: 2752-2756.
Zhang, Y.J., et al. 1994. "Structure/Activity Analysis of Human Monocyte Chemoattractant Protein-l (MCP-l~ by MutA~nec~". J.Biol. Chem. 269: 195918-15924, describes the expression of a number of mutant versions of MCP-l, including the mutant 7ND which result from the ~lef i on of the amino acid residues 2-8 of MCP-1. The 7ND protein congigted of the NE~-t~rminAl glutamine of MCP-l, followed by residues 8-76 of MCP-l.
Zhang, et al characterized mutant 7ND as capable of ,_ _ i ng with MCP-l for approximately ten percent of the NCP-l receptors on monocytes and capable of inhibiting MCP-l activity by 50t at a 75:1 molar ratio of 7ND to MCP--1 .

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Summarv of the Invention This invention provides MCP-l ~n;~l O~ R which lack MCP-l activity and are much more potent competitors of MCP-l for binding to MCP-l receptors than the mutant 5 proteins described by Zhang, et al ~supra]. The MCP-l analogues of this invention are useful as MCP-l antagonists and as anti-i nf l ~ tory agents .
Accordingly, this invention provides an analogue of MCP-l lacking ~lH~-t~rm; n~l amino acids 10 corresponding to amino acid residues 1-7, or 1-8, of MCP-1 wherein the ~n~lo~u~ lacks MCP-l biological activity and inhibits MCP-l binding to a MCP-l receptor.
Preferably, the analogue will comprise an amino acid sequence ~ubstantially equivalent to MCP-l t8-76), or MCP-l ( 9-76 ) wherein the analogue inhibits 50% of MCP-l binding to a MCP-l receptor at a 25 :1 molar ratio of analogue to MCP-l, or less. Most preferably the analogue i~ MCP-l (8-76), or MCP-l (9-76).
This invention also provides anti-; nf l l tory 20 pharmaceutical compositions c~ Ri n~ one or more of the aforementioned analogues and a rh~ utically acceptable carrier. This invention also provides a method of inhibiting MCP-l biological activity ~ Ring applying an effective amount of a MCP-l ~nAl o~ of this 25 invention to the environment of cells affected by MCP-l.
This invention also provides uses of the aforementioned analogues as MCP-l antagonists and in the preparation of anti-; nf l l tory agents . This invention also provides the use of the af orementioned analogues and 30 rh~ ~eutical compositions as anti-infl. tory agents.

21~2141 Description of the Drawinqs For a better understanding of the invention, reference may be made to the preferred ~ i Ls and examples described below, and the Arcl -nying drawings in which:
Figure 1 in which graph~ lAhel 1 o~l A and B
display receptor binding of the ;nrl;ratorl MCP-l analogues;
Figure 2 in which histograms 1 AhOl 1 od A and B
summarize Ca'+ induction and desensitization, respectively, by the indicated MCP-l analogues at the indicated conce~LL~Lions;
Figure 3 in which graphs l Ahol l ed A and B
display competitive binding to THP-l cells of llnl Ahol l ed NCP-l and MCP-l analogues titrated at the; n~; ~'Atoc~ concentrations in the presence of 4nM labelled MCP-l;
Figure 4 is a graph displaying MCP-l antagonist activity o~ the indicated MCP-l analogues titrated at the indicated con~ nf rations against MCP-l (5 x 10-~M) in a chemotaxis a3say u3ing THP-l cell3.
De3cription of the Pref erred r '; ~ ~ts Throughout this spPc;f;cAtion any reference by number to an amino acid in a MCP-l analogue will be a reference to the COLL~ 1; n~ amino acid residue number from the 76 amino acid 3equence of MCP-l 3hown in Table 1. For example, where the fir3t 7,8,9, or 10 amino acids NHl-t~-rm; nAl amino acids of MCP-l are deleted (as is the case for the analogues shown in Table 1 ) the AnAl 0~ '8 will be referred to respectively as MCP-1(8-76), MCP-l(9-76), MCP-l (10-76), and MCP-1(11-76).

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21~2141 This invention provides MCP-l antagonists, that are analogues of MCP-l truncated at the NH~-tF~rm; nllC . The AnAl og~les of this invention compete with MCP-l for binding to a MCP-l receptor but lack a MCP-l activity 5 such as the effects of MCP-l on cells of monocytic origin. The MCP-l analogues of this invention will preferably inhibit MCP-l activity by 50%, or will inhibit 50% of MCP-l binding to MCP-l receptors, at a molar ratio of antagonist/MCP-l of 25:1 or less, to allow for 10 formulation of preferred anti-; nfl t~)ry agents. Most preferably the molar ratio will be 15:1, or less.
MCP-1 analogues of the present invention may comprise an amino acid sequence that is identical to an antagonist analogue described herein, or may comprise a 15 polypeptide having a region ;rl~nti~ Al to a region of an antagonist described herein, or may comprise an amino acid sequence that is substantially equivalent to all of or a region of an analogue ~ s~ri hed herein . Analogues of this invention comprising sequences that are 20 substantially equivalent to all of (or regions of) analogues described herein are characterized as having from 1-10, (preferably 1-5) amino acid ~l~let;t~nc~
additions or substitutions that do not result in an AnAl o~ losing ability to compete with MCP-l for binding 25 to MCP-l receptors.
Three .1; -ir)nAl ---'rll;n~ techniques may be used to design and construct different MCP-l analogues of this invention . Furf h~ t, conservative ~ i f i ~ations that do not prevent an analogue from functioning as an 30 MCP-l antagonist are i n~ d within this invention.
Elowever, it is expected that it will be necessary to retain the characteristic fli~ll1rh;rl~ bridges of MCP-l and theref ore cystine residues generally corresponding in position to those of MCP-l are to be retained.

Specific amino acid substitutions that may be tolerated include: Glu or Asn for ABP; Asp or Gln, for Glu; Arg for Lys; Lys for Arg; Asn for llis; Pro for Gly;
Gly for Pro; Asn, or Met, Leu for Gln; Gln, Ser or Ala 5 for Asn; Ser, Val or Ile for Thr; Thr or Ala for Ser; Phe for Tyr; Tyr for Phe; Ile, Val, Met for Leu; Ser for Ala;
and any combinations thereof. M-~1ifi-~tion of amino acids corr~sp~-n~lin~ to residues 13-31 of MCP-l is to be avoided, except for conservative ~ifit~tions such as 10 substituting Tyr for Phe. Preferably, modificat;~nf~ of the amino acid sequence of analogues of this invention will be made in a region beyond the regidue corr/~8pon~l i n~
to amino acid residue 35 of UCP-1.
Substantial deletion or truncation of C-15 tF~rmi n:-l residues of MCP-1 is possible in providing analogues of this invention but such deletions/truncation are preferably made in the region beyond amino acid residue 52 of MCP-1, and more preferably in the region beyond amino acid residue 68 of MCP-1.
In analogues of this invention in which the N-t~rmi n;~l amino acids COl I ~onding to the amino acid residues 1-7 of MCP-1 are not present (eg. MCP-1 8-76), the first amino acid in the analogue will preferable be Pro (which corresponds to the native amino acid residue 8 ) . Where it is desired to substitute the latter amino acid residue, it is pref erable that the substitution be Ser, Ala, Asn, Gly, Val, or Thr, but other substitutions may be accepted, inrl~ ing Leu, Ile, Met, and Cys, but not: Gln, Asp, Glu, Elis, Tyr, Trp, Arg, Lys, and Phe.
The MCP-1 analogues of this invention may be syn~h ~ ed according to the following protocol using a fully automated peptide synthesizer (Applied Biosystems 430A ). The synthesis is started with a protected C-* Tr~ ~1 rk 21~2141 t ormi nAl amino acid linked to a cross-linked polystyrene resin via a 4- ( carb~rAmi ~ hyl ) benzyl ester linkage (the so-called pam resin) (0.4 mmol of 0.8 mmol/g of aminoacyl resin). Noe-t-Boc acids with appropriate side 5 chain protecting groups are added in a stepwise fashion until the entire protected polypep~ p~hAni n is formed.
Side chain protection is as follows: benzyl (Asp, Gly, Ser, and Thr); 4-methylbenzyl (Cys); to~llon~sulfenyl (Arg); 2-chlorobenzyloxycarbonyl (Lys); 2-~ ~ yloxy-1 0 carbonyl ( Tyr ); f ormyl ( Trp ); dini LL u~ ~hl :l-y 1 ( l~is ); and none (Ala, Asn, Gly, Glen, Ile, Leu, Met, Phe, Pro, Val).
Samples may be taken after each step to retrospectively monitor the amino acid cQIl~l i n~ yields using a ninhydrin-based reaction following the ~Luce.luLes of Sarin, et al (1981) Anal. Biochem. 117: 147-157. The protected polypeptide resin i8 treated twice for 30 min with 2-mercaptoethanol (20%) in dimethylforrori-3~ containing diisopropylethylamine (5%) to remove the DNP groups from the histidine side chains. The resin i8 dried and cleaved using the "low-high" I-ydluJ~ fluoride method as described by Tam, et al ( 1984 ) J. Am. Chem. Soc . 105:
6442-6485 except for the following r--ifi~-ations: after the 25% hydrogen fluoride step, the partially protected peptide resin is filtered from the reaction mixture by using an all-Teflon' filtration apparatus fitted with a Zitex~ f ilter and washed with dichloromethane and dried before the high 9096 IIYdLUgt:II fluoride step. The ethyl acetate precipitate of the material released from the resin is dissolved in 50 ml of 6 M gllAnirlin~
hydrochloride, 0.1 M Tris-acetate, pH 8.5, and 20% 2-mercaptoethanol and stirred at 37C for 2 h and then acidif ied with 2 ml of acetic acid . Thi3 mixture is the crude peptide product.
Alternately, histidine may be protected with nbenzyloxymethyl instead of dinitrophenyl. The * Trademarks Irbenzyloxymethyl group is acid labile thus Pl;m;nAt;n~
the need for thiolysis of the dinitrophenyl group before and after llydLog~ll fluoride deprotection. Acetylation i3 carried out on the NK deprotected, but otherwise fully 5 protected peptide resin, using acetic anhydride ( 10% ) in dimethylf nrr-m; ~1P .
The crude peptide product may be purified and folded according to the following protocol. Three different C-18 silica HPLC column3 may be u3ed in the 10 pur;f;-at;on and analysis of the peptide, ;n~ 7;n~ a preparative column (22.4 x 250 mm column with at 22.4 x 100 mm guard column) packing with 12 ~m, 300-A pore size packing (Dynamax, Rainin Instrument Co., Woburn, MA. ); a semipreparative column (10 x 250 mm) Vydac~ C018 column, with 5-ym particle, 300-A pore-size packing (Separation3 Group, Hesperia, CA); and an analytical column (4.6 x 250 mm) (Vydac~) containing the same packing. The crude peptide product i8 loaded onto the preparative column and the retained material eluted with a 0-60% water-20 acetonitrile gradient in 0.1% tr;fl~loracetic acid over 4 h at a flow rate of 15 ml/min. A sample (25 zll) of fractions containing 225-nm W-absorbing material i8 rerun on the analytical column and by comparison with the profile of the crude material, fractiona ~ ntA;n;n~ the 25 major peak are pooled and lyoph; 1; 70d.
When synthP~i7;n~ MCP-1, the NH~--t~rm;nAl -glutamine may be converted to pyro~l UtAr--te by treatment for three days with 196 of acetic acid in water.
Conversion i3 monitored due to the longer HPLC retention 30 time of the pyroglutamate form and may be conf i -I by an approximate 17-Dalton difference in lPCIllAr ma33.
Alternately, pyroglutamate may be used in the 3ynthesi3 procedure .
* Trademarks 2152~4~

To fold the protein, the material i3 reconstituted in 1 M guanide hydrochloride and Tris-acetate, pH 8.5, at a CvïlC6111_LatiOn of 0.2 mg/ml and stirred vigorously overnight in an open beaker 80 the air was kept bl~hhl i n~ through the mixture by vortex action.
This procedure promoteg formation of the ~ lllfitlP
bridges by oxidation of the appropriate half-cysteines.
The material is ~ lifi~d with 2 ml of acetic acid, and half is loaded onto the semipreparative column and the retained material eluted with the same gradient as described above at a f low rate of 3 ml/min . Samples of each fraction are run on the analytical column.
Fractions c~ ; n; n~ only material with the retention time of the major peak in the folded material are pooled and lyoph; 1; 7~
An assay for free sulfhydryls using Ellman reagents, as ~ r; hed by Clark-Lewis et al ( 1988 ) Proc .
Natl. Acad. Sci. U.S.A. 65: 7897-7902, may be used to d~t~rm;n~ the extent of folding. In addition, folding 2 0 may be monitored on the analytical HPLC column by observing the appearance of a peak ~:uL r ~ onding to the f olded f orm that has a retention time approximately 3 min . earlier than the reduced f orm.
AnAl o~le purity may be assessed on an analytical HPLC column or by other means such as isoelectric focusing. A protocol for isolelectric focl-~; n~ is as follows. Mini polyacrylamide gels (ph~ ;A PHAST~ gels, IEF 3-9; ph;~ Uppsala, Sweden) are washed in 8 M urea and then in 8 M urea cnn~ ;n;n~ pH 9-11 Ampholytes (ph:~rr-~;;-), for 30 min each, either with or without 10 yM dithiothreitol. Gels are prerun for 15 V-h at 200-V, 2 . 0-mA, 3 . 0-mW maximum settings, and the samples are loaded and run for 410 v-h at 1000-V, 5.0-mA, 3.0-mW maximum settings on the * Trademark 21~21~1 phAr~ A P~AST systems for a total of 500-V with maximum settings of 2 . 0-mW, 5 . 0-mA, and lOOOV. The pM gradient may be det~rm;n~d by using a surface pll electrode. The gels may be stained with silver by using the PElASTi 5 developing systems as described in the phArr--; A- manual.
Sequence of An~log~ q may be det~rm;ned by protein sequencing, for example by using the following protocol. Protein sequences are rl~t~rm;n~d by Edman degradations using either solid-phase or gas-liquid-phase 10 methods. For solid-phase sequence analysis, reduced and caLLo,sy thylatedprotein or proteolytic cleavage frA~ --t~3 are coupled to arylamine-funct;c~nAl;7ed poly(vinyl;.1~n~if1~ ri~ ) membranes (Sequelon AA~;
M; 11 i ~n/Biosearch~ Burlington, MA) using the water-15 soluble cArhQ~l i i mi ti.o 1--ethyl--3--3 [ 3--(dimethylamino)propul]cArho-ii ;m;-l~ hydrochloride and sequenced in a Milligen/Biosearch Model 6600~ sequencer using standard protocols. For gas-liquid-phase sequence analysis, polypeptides may be applied to Polybrene-coated 20 glass fibre disks and s~qll~n-~d in an Applied Biosystems Model 477~ protein sequencer using standard protocols .
Sequencing of protected peptide resins may be carried out on l o;-d~LuLected samples by using the same methods . N-tc~rm; nA 1 solid-phase sequencing runs usually reveals a 25 major portion of the sequence. The ~ ;n;n~ sequence may be obtained by runs of the IiPLC-fractionated fragments, derived either by proteolytic cleavage with A~ nd~ .,Lease (Boehringer MAnnh~im Canada, Laval, Quebec) or by h~micAl cleavage, through pre~erential 30 hydrolysis of the Asp-Pro peptide bond in dilute formic acid .
Molecular weight of the synthetic proteins prepared as ~8~ r; hed above may be det~rm; n~t3 by electrospray mass spectrometry on a SCIE X~ triple * TrA~ rkq quadrupole Mass Spectrometer equipped with a liquid delivery apparatus. The le~u1Ar mass from the peaks corre~pon~li n~ to the charge to mass ratios of the dif f erent multiple ionized species of the protein may be 5 analyzed as described by Covey, T.R. et al. 1988. Rapid Commun . Mass . Spectrom. 2: 249-256 .
MCP-1 analogues of this invention may also be prepared through Ll ~ inAnt means, with expression in l i An or non - l i An sygtems. Portions of a DNA
10 sequence ~-nrorling MCP-l are appropriately ~if;~d to produce the desired analogue when the DNA sequence is expressed. Methods and protocols for preparation and expression of such re~: ` inAnt DNA are known in the art, including the protocol ~ rihed by Zhang, Y.J. et al.
1994. [supra] used for production of mutant MCP-1 proteins . Expression of such r~ - ' i nAnt DNA in 1 i An and nol~ 1 i An gystems is also de~rrih~3 in the aforementioned U.S. patents of Rollins, et al.
MCP-1 analogues may be assayed for MCP-1 activity by use of a cytosolic-free calcium assay, a chemotaxis assay using cells of monocytic origin, or by other assays for MCP-1 activity i nrl~ i n~ enzyme release, superoxide production or killing of microbes.
Analysis of cytosolic-f ree calcium may be carried out using the following protocol. Cells (4 x 105) are loaded with 12, 5 yg/ml Fluo-3AM in PBS saline with 0.38 mg/ml Pluronic F127~ (M~ clllAr Probes, Eugene, OR) at 37C for 30 min. After washing with PBS, the cells are resuspended in 25 mM ~epes, 140 mM NaCl, 10 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2, and 3 mM RCl, pEI 7.3. The flUULt:sCenCe i8 monitored at 7 second intervals over 150 seconds, af ter addition of test sample . Maximum Ca2~
levels are established using Fluo-3AM (designated 100%
* ~L -I rk 21~21~1 .

saturation) for each set of measurements by addition of 5 yM Ionomycin (Sigma rhPTnir~l Co., St. Louis, MO).
A chemotaxis assay for MCP-1 analogues may be performed according to the following protocol. Cell migration is assayed using 48-well micro-chemotaxis chambers (~ L~ be, Cabin Joh, MD). Polypeptides are dissolved in RPMI containing 0 . 5 mg/ml sSA, diluted in the same medium and 26-111 of 10~/ml suspension, are added to the upper chamber. After incubation for 2 h at 37C
in 5% CO2 in air, the filter is removed, fixed, and stained with Canco Quik Stain II (Baxter, McGaw Park~
IL). The migrated cells are counted and the chemotactic index detPrmi nPd as the ratio of the migrated cells in the presence of sample, to the control migration in the ab3ence of sample.
Inhibition of MCP-1 mediated chemotaxis may be detPrm; nPrl by using the aforementioned chemotaxis assay.
Constant amounts of MCP-1 (eg 5 x 10-~ M) are added to each well and the analogues are titrated in the assay.
. ..
Cell preparations for use in the aforementioned assays may consist of human monocytes, or monocytic cell lines such as the cell line THP-1. TEIP-1 may be obtained f rom American Type Culture Collection ( Rockville, MD ) and may be maintained in RPMI 1640 medium supplemented with 10% FCS. E~uman monocytes may be i ~ t~d from buffy coats of normal donor blood by the following protocol. A
cell suspension ig loaded onto Ficoll-~ypaque ~phAr~
Uppsala, Sweden) and centrifugated at 400g for 25 min.
followed by density centrifugation on a discontinuous Percoll (Pharmacia') gradient at 500g for 30 min. Cell fractions with a density of 1.051-1.053 (g/ml) are generally greater than 70% monocytes by morphology and may be used in the assay.
* Tr~1 rkFI

21521ql MCP-1 receptor binding may be det~rm; n~d by the following protocol . MCP-l ( lOug) i8 labelled with io~ i n A ted Bolton-Munter reagent ( spe~; f i ~1 activity 2,200 Ci/mmol; DuPont, Wilmington, DB~ at 4C for 30 5 min., to provide 3pecific activity of 12sI-l ~hel 1 ed MCP-l of 130 Ci/mmol. To determine the binding kinetics, monocytic cells (such as THP-l) at (5 x 106 cells) in 200 ul of binding buffer (RPMI 1640, 0.5 mg/ml BSA, 50 mM
Mepes, and 0.1% NaNl) are in~ llhs~ted with varying 10 concentrations of l2sI-MCP-l at 4C for 30 min. The cells are p~ tPd through a mixture of diacetylrhth~l~te and dibutylphthalate and radioactivity that i8 cell A~so~ t~3 is counted (total binding). Nonspecific binding i8 det~rm; ne~l in the presence of a 100-fold 15 concentration of llnl;~h.oll-~d ligand and subtracted from the total binding . Kinetic parameters ( Kd and receptor number) are ~tf~rrn;n~d by Scatchard analysis.
Competitive receptor binding by MCP-l ~nF~l og may be measured by carrying out the ai~orementioned 20 receptor binding protocol wherein various concentrations of MCP-1 analogues are added to the cells in the prGE_,lce of 4nM l25I-MCP-1. Non-specific binding is subtracted from total binding and the result may be expressed as a percent of maximum specific binding.
A further assay that may be carried out to determine whether a non-chemotactic analogue binds to MCP-1 receptors is to measure the ability of an analogue to desensitize calcium ~ tion by MCP-l . Fol l ~ -;
a first treatment with a MCP-l receptor ligand, the calcium Le~lL8e will be temporally desensitized to a second treatment with a MCP-1 receptor ligand. This may be det~rm; ne~l by carrying out the aforementioned cytosolic-free calcium assay with addition of a first ligand, followed by a second treatment after 150 second 35 using either the ~ame of a different ligand. A MCP-1 21~21~1 antagonist will not of itself stimulate calcium induction but when used as the first ligand, will desensitize the cells to subsequence stimulation by MCP-1.
Methods of in vitro use of the antagonist of this invention will be readily apparent from the ~ a herein and the assays described above. Furthermore, antagonists of this invention will be useful as anti-;nfli tory agents, part;c~ srly in humans. MCP-l antagonists of this invention may be delivered as a nasal spray for upper respiratory treatments or as an aerosol inhaler f or lung conditions . The antagonists may also be used in topical applications. Alternatively, antagonists of this invention may be delivered by injection (preferably intl ~cl-lAr but may be subcutaneous, intr~ r~ , intraperitoneal, or intraarticular). Dose will depend upon manner of delivery and the nature of the disease being treated. For example, if local concentrations of MCP-l that cause ~;~n;f;~isnt pathology in the body are of the order of lOnM in the endothelium, then it would take in the order of 400nM of MCP-l ( 9-76 ) to inhibit the MCP-l if delivery is 10096 f~ff;~ipnt~
Therefore, a MCP-l (9-76) dose of 0.003-0.03mg for a nasal spray and 0 . 03-0 . 3mg for other forms of delivery may be required.
The exact doses of MCP-1 to be used in a particular application may be det~rm; nf~d by accepted ~h;lr~^qeutical methodg known to those skilled in the f ield . This may be carried out by measuring antagonist blood concentration and det~rm; ni n~ the antagonist half life. It is anticipated that antagonists of this invention may have what would be otherwise an u~ ;i edly high half life for similar polypeptides due to binding of the analogues to cel 1 ll1 isr receptors.

21521~1 phArr-Aeutical compositions comprising MCP-1 antagonists of this invention and suitable rhArr~ tica carriers may be f~ 1 at~d by persons skilled in this field taking into c~n~ rat; on the nature of the 5 polypeptide ~ of this invention and the desired mode of administration. Generally, the antagonists of this invention are soluble and therefore, pharmaceutical compositions in which antagonists of this invention are 501llh; l; ~ 1 such as in physiological saline, or other 10 phy~ir)loq;~AAl buffers, may be formulated. Alternatively, and without limiting the scope of use of this invention antagonists of this invention may be f ormulated in sustained release delivery systems or topical formulations containing an aqueous - _ l-nt.
15 ExamPles MCP-1 and various NEI2-t~rm; nA 1 truncated analogues were synth~ d and fielded using the methods Ar; hF-d above. The following truncated AnAl oguP~ were produced: MCP-1 (2-76) , t3-76), (4-76), (5-76), (6-76), (7-76), (8-76), (9-76), (10-76) and (11-76). In addition, a peptide equivalent to amino acids 1-10 of MCP-1 was syn~h~ d. The full length MCP-1 protein was converted to the pyroglutamate f orm bef ore f olding .
Overall yields of pure folded protein were 20-50 mg. The 25 synthetic product3, including all of the MCP-1 analogues were found to fold spontAnf~o~l~ly as indicated by the absence of free thiols and the characteristic shift in EIPLC retention time.
The chemotactic activities of the analogues 30 were compared to MCP-1 using p~r;rh~rAl blood monocytes or TllP-cells as targets. NCP-1 consistently gave the highest level of migration and was the most potent inducer of both cell ~ources. In general, results with THP-l cells were found to parallel fintlin~E with monocytes .
When Py~m;npd for THP-l chemotaxis, the natural form MCP-l had the highest activity, the (2-76) analogue was 300-fold lower whereas the (3-76) and (4-76) An~lo~les had only marginal activity. The (5-76) analogue had readily detectable activity of approximately 2/3 the level of migration of MCP-l, and was only four fold less potent than MCP-l. The (6-76) analogue had lower but R;qn;f;r~nt chemotactic activity. The L. ; n i n~ analogues lacked detectable chemotactic activity as did the peptide COLL~ 1; n~ to residues 1-10 of MCP-1. A c~ , -r; ~n of the chemotactic activity of MCP-l and the NH~-tprm; nAl truncated analogues on THP-l cells is displayed in Figure 1 wherein the results are reprP~Pnt~;ve of 3 experiments and the indicated ~h lr; n~ c~,..cellLLationg are ghown ag the mean + SD of triplicate determinations. Similar results were obtained with monocytes as target cells.
Calcium 'i l i 7~tion and desensitization by MCP-l and the NH~-tprm;n~l truncated ~n~ P~ was detPrmi n~d according to the above dP~-r; hP(l methods .
MCP-l was the most efficient in; n~ ; n~ cytosolic calcium hi 1 i ~S~tion of all the proteins tested but the (5-76) analogue also induced a ~iqni fi~nt calcium rise in THP-l cells. The (2-76), (4-76), and (6-76) i~n:~lo~
induced a lower re~yullse. The (3-76), (7-76), (8-76), (9-76), (10-76), (11-76) analogues and the (1-10) peptide did not induce 3iqnifi~.?nt calcium rise at levels up to lOOOnM. Figure 2 summarizes the results of 3timulation and desensitization of calcium induction by the indicated proteins. Desensit;7~ti~n is displayed a3 percentage desensif;~ of a subsequent treatment by lOnM MCP-l by a f irst treatment with the i n~ tPd protein at the 35 indicated concentration. Maximal desensitization of fluorescellce was obtained with MCP-l and was designated as 100% desensiti7Atin~. The maximal fluoLescence induced by lOnM MCP-l was designated as 0%
desensitization. All the truncated AnAlg~l~P~
5 desensitized T~IP-l cells to a subsequent MCP-l rhAl 1 ~n~e but the MCP-l (1-10) peptide did not attenuate the calcium respon3e . NCP- l ( 3-7 6 ), ( 7 -7 6 ), ( 8-7 6 ), ( 9 -7 6 ) and ( 10-76 ) desensitized, but did not induce. Of the non-inducing analogues, MCP-l (9-76) was consistently the 10 most ef fective at desensitization.
Several of the truncated analogues were tested for MCP-l receptor binding by competition for l2sI-MCP-l.
The results are displayed in Figure 3 in which the ;nt3irAted ~ ol.c~llLL,-tions of analogues were added to TEIP-l 15 cells in the presence of 4nM l2sI-MCP-l. ~on-gper;f;r-binding was subtracted from total binding and the result ss~d as a percentage of maximum specific binding with the results being representative of two ~-rr~r; Ls.
The (2-76) and (3-76) analogues had a Rd of 385 and 20 487nM, respectively which was much higher than MCP-l (Rd2.8nM). The (5-76) analogue had only an 8-fold higher Rd(23nM) than MCP-l. The inactive (9-76) analogue had 3-fold higher Kd than MCP-l (approximately 8nM). The Kd of the (10-76) and (11-76) analogues were 13 and 48 fold 25 higher than MCP-l respectively. The MCP-l (1-10) peptide did not bind. Direct binding ag8ay8 u8ing lAh~ ( 9-76) and (11-76) analogues were performed with results correlating to the calcium desensif i 7Ai- j nn study.
To test analogues for competitive binding to 30 MCP-l receptors resulting in blocking of NCP-l biological response, the (8-76), (9-76), (10-76), and (11-76) AnAl O~ B were titrated in the presence of 5nM MCP-l in the chemotaxis assay as described ~bove. The analogues inhibited MCP-l stimulated chemotaxis in a dose ~F.p~nrl~nt 35 manner as shown in Figure 4. The (9-76) AnAls~u~ was the 2I~21~1 manner as shown in Figure 4 . The ( 9-76 ) analogue was the most potent (ICs = 20nM; analogue/MCP-l ratio = 4). The (8-76) analogue was 3-fold less potent (ICs = 60nM;
analogue/MCP-l ratio = 12). The (10-76) and (11-76) 5 analogues were less potent again ( ICs = O . 6 and l~lM, respectively). The (1-10) peptide did not R;~n;fi~ntly inhibit chemotaxis.
On the basis of the foregoing ~ _ 1PY, the two ~H2-t~rm;n~l truncated analogues, MCP-l (8-76) and (9-76) 10 are potent inhibitors of MCP-l. The analogues are MCP-l antagonists which bind to, but cannot activate, MCP-l receptors .
Various changes and ~;f;ca~innR may be made in practising this invention as disclosed herein without 15 parting f rom the substance and scope thereof .

Claims (21)

1. An analogue of MCP-1 lacking NH2-terminal amino acids corresponding to amino acid residues 1-7, or 1-8, of MCP-1 wherein the analogue lacks MCP-1 biological activity and inhibits MCP-1 binding to a MCP-1 receptor.

2. The analogue of claim 1 which inhibits 50% of MCP-1 binding to a MCP-1 receptor at a 25:1 molar ratio of analogue to MCP-1, or less.

3. The analogue of claim 1 which inhibits 50% of MCP-1 binding to a MCP-1 receptor at a 15:1 molar ratio of analogue to MCP-1, or less.

4. The analogue of claim 1,2 or 3 comprising MCP-1 (8-76), or a fragment thereof.

5. The analogue of claim 1 which is MCP-1 (8-76).

6. The analogue of claim 1,2 or 3 comprising MCP-1 (9-76), or a fragment thereof.

7. The analogue of claim 1 which is MCP-1 (9-76).

8. An anti-inflammatory pharmaceutical composition comprising an analogue of claim 1,2 or 3 and a pharmaceutically acceptable carrier.

9. The anti-inflammatory pharmaceutical composition of claim 8 wherein the analogue comprises MCP-1 (8-76), or a fragment thereof.

10. The anti-inflammatory pharmaceutical composition of claim 8 wherein the analogue comprises MCP-1 (9-76), or a fragment thereof.

11. An anti-inflammatory pharmaceutical composition comprising MCP-1 (8-76), and a pharmaceutically acceptable carrier.

12. An anti-inflammatory pharmaceutical composition comprising MCP-1 (9-76), and a pharmaceutically acceptable carrier.

13. A method of inhibiting MCP-1 biological activity, comprising applying an effective amount of an analogue according to claim 1, 2, 3, 5, or 7 to the environment of cells affected by MCP-1.

14. The use of the analogue of claim 1, 2, 3, 5, or 7 in the preparation of an anti-inflammatory agent.

15. The use of the analogue of claim 4 in the preparation of an anti-inflammatory agent.

16. The use of the analogue of claim 6 in the preparation of an anti-inflammatory agent.

17. The use of the analogue of claim 1, 2, 3, 5, or 7 as an anti-inflammatory agent.

18. The use of the analogue of claim 4 as an anti-inflammatory agent.

19. The use of the analogue in claim 6 as an anti-inflammatory agent.

20. The use of the pharmaceutical composition of claim 8 as an anti-inflammatory agent.

21. The use of the pharmaceutical composition of claims 9,10,11 or 12 as an anti-inflammatory agent.