Note: Descriptions are shown in the official language in which they were submitted.
21~2141
-- 1 --
.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 .
: =~
2152141
.
-- 3 --
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
-- 4 --
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).
21~2141
--5--
,
I
O
o ! ' I
~ - .
O
o
~1 --
~ ^ ~
~ # # ~ #
21~2141
-- 6 --
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.
2152141
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
-- 8 --
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
2152141
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~
-- 10 --
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
2152141
-- 12 --
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
.
-- 13 --
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
-- 14 --
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
-- 15 --
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
2152141
-- 17 --
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
21S2141
-- 18 --
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
-- 19 --
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 .
