Note: Descriptions are shown in the official language in which they were submitted.
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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-
-1-
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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
-2-
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(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
MCP-3
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.
-3-
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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.
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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-
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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,
-6-
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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
-7-
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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_
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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.
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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$rl~y
(i.e.,
phenylmethyl), Z-naphthylethyl, 2-(2-pyridyl)propyl, 6-dibenzpsuberyl, and the
like.
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The term "alkylcarbonyl", as used herein, refers to -C(t7)-alkyl. Similarly,
the term "arylcarbonyl" refers to -C(O)-aryl. The term "alkyloxycarbonyl", as
used
herein, refers to the group -C(O)O-alkyl, and the term "aryloxyrarbonyl"
refers to
-C(4~O-aryl. The tem7 "acyloxy" refers to -O-C(O)-R,, in which R7 is alkyl,
alkenyl, alkynyl, aryl, aralkyl or heterocyclyl.
The term "amino", as used herein, refers to -N(Ra)(Ra), in which RQ ant) R~
are each independently hydrpgon, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or
in
which Ra and Rs together with the nitrogen atom to which they are attached
form
a ring having 4-8 atoms. Thus, the tens "amine", as used herein, includes
unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino), and
disubstituted (e.g., dialkylamino or alkylarylarnino) amino groups. The term
"amido" refers to -C(C7)-N(Ra)(R~), in which R$ 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-
)-
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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.
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Freeman and Company, New York (1992); or Clark-~ewis, L, Dewald, ~.,
Laetscher, M., Moser, B., and Baggiolirti, M., (1994} J. giol. Chem., 289,
16075-
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~,
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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
T7
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~
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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.
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(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-
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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.
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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-
3
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
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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
MCP-3
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$o
desensitized macrophage inflammatory protein (MIP)1-alpha induced Caz'''
mobilization in THP-1 cells {not shown). Since MIP-lalpha binds CCR-1 and
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CCR-5, this confirmed the CCR-1 antagonist activity of MCP-3(5-76). As a
Control MCP-3(6-76) did not block the cak~um response to MIJC, which binds
CCR-4, a receptor not bound by MCP (Fig. 4). The physiological relevance of
MCP-3 antagonism was confirmed by cell binding assays (2~j. SCatchard
analysis showed that synthetic MCP-3(5..76) bound cells with high affinity
(fC~, of
'1$.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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2. S. Fields, O. Song, Nature 34.0, 245 (1988).
3. F.X. Gamis-Ruth et af., J. MoL Biol. 264. 558 (1986).
4. ~J.M. Wallon, G.M. werail, J. Blol. Chem. 272, 747 (1997).
5. R.V. Ward, S.J. Atkinson, J.J. Reynolds, G. Murphy, Blochem. J. 304,
263 (1994).
8. F. Wilienbrock et al., Biochemistry 32, 4330 (1993).
7. H. Sato et al., Nature 370, 61 (1994).
8. A.Y. Strongin et al_, J. Blol. Chem. 270, 5331 (1995).
9. C.M. Overall, J. Sadek, J. 8iol. Chem. 2$5, 21141 (1990).
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.
13. B. Steffensen, U.M. Wallon, C.M. Overall, J. E3iol. Chem. 270. 11555
(1995).
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.
15. Y. Itch, M.S. Binner, H. Nagase, Biochem. J. 308, 845 (1885).
1B. T.N. Young, S.V. Pizzo, S. Stack, J. Biol. Chern. 270, 999 (1995).
17. C.M. Overall et al., J. Bfof. Chem. 274, 4421 (1999).
18. S. Netzel-Amott of al., Biochemistry 32, 8427 (1993).
19. C.E. Hughes et al., J. Bivl. Chem. 267, 16011 (1982).
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CA 02316405 2000-08-18
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20. J.H. Gong, 1. Clark-Lewis, J. Exp. Med. 1$1, 631 (1995).
21. J.-H. fang et al., ~l. 6101. Chem. 271, 10521 (1996).
22. THP-1 cells (myeloid cell line, ATCC) or B Cells transfected with CCR-~
cDNA were loaded wifh Fluo-3AM fior 30 rnin at 37 °C. After addition
ofi
various full length chemakines or MCP-3(5-76) the fluorescence was
monitored with a Perkin-Elmar 660-10B spectrofluorimeter using an
excitation wavelength ofi 506 nm and an emission wavelength of 526 nm.
Desensitization assays were perfom~ed by sequential addition of MCP-
3(5-76) or buffer control, followed by the full length chemokine. THP-1 cell
migration was assessed in transwell trays (Costar) with 8.5 mm diameter
chambers of 3 pm membrane pore size. MCP-3 and MCP-3(5-76) were
added to the lower well, and THP-1 Cells (1 x 10' cellslml) were added to
the upper well. After 1.5 h, cells that had migrated to the lower well were
counted. The percent migration was calculated by dividing the mean
number of migrating cells in response to chemokine by the mean number
of cells migrating in response to medium alone.
23. 4 nM ['251j-MCP-3(1-?6) in the presence of serially diluted unlabeled MCP-
3(1-76) or MCP-3(5-76) and 0.05°~ NaN was incubated at 4 °C for
30 min
with THP-1 cells. Cell bound! and free [~z5lj-MCP-3(1-76) were separated
by centrifugation of the cells through a column of dioctyl phthalate:n-butyl
phthalate (2:3, vlv). Amounts of bound t'ZSIj-MCP-3(1-?fi) were
determined in the cell pellet by gamma counting. N4n$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
2
and 4 h by saline lavage. Cells were counted on a Coufter Counter gated
-zz-
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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.).
26. S. Struyf et al., Fur. J. Immunol. 28, 1282 (1998).
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176, 59 (1992).
_23_
