Note: Descriptions are shown in the official language in which they were submitted.
CA 02307705 2000-08-03
MODULATION OF INFLAMMATION BY PROTEASE PRODUCTS
FIELD OF THE INVENTION
The invention is in the field of therapeutic compounds and uses thereof.
BACKGROUND OF THE INVENTION
Monocyte chemoattractant protein (MCP-3) is a potent, disulfide bridged
CC chemokine for the recruitment of monocytes and other leukocytes to sites of
host challenge (11). 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. 5977408 or 6037361 and international
patent publication W09921583, all of which are incorporated herein by
reference.
Because metalloproteinases are thought to be involved in pathological
degradation of the extracellular matrix in various diseases, it has been
suggested
that inhibitors of metalloproteinases may be used as anti-inflammatories in a
variety of diseases. It would be contrary to this teaching to discover that
metalloproteinase inhibitors may have a physiological activity that sustains
an
inflammatory condition.
Library screening by the yeast two-hybrid system (2) has been useful in
identifying intracellular protein-protein interactions using cDNA sources
ranging
from bacteria to man. However, its application to extracellular interactions
has
been largely overlooked for disulphide cross-linked proteins and to our
knowledge has never been used to identify substrates for an extracellular
proteinase. Indeed, the rationale for library screening using a proteinase
catalytic domain for bait is tenuous because subsequent cleavage of library
encoded substrate would likely prevent detection in the assay.
SUMMARY OF THE INVENTION
One aspect of the present invention includes CC-chemokine receptor
antagonists. Such antagonists may include truncated derivatives of native MCP-
3, in which the 4 amino acids at the N-terminal have been removed (leaving
amino acid 5-76), designated MCP-3(5-76).
In alternative aspects, the present invention provides therapeutic methods
of modulating an immune response in a host, comprising administering .
In alternative aspects, the present invention provides methods of inhibiting
the biological activity or the in vivo biological activity of CC-chemokines,
including
native MCP-3, comprising administering to a host, e.g., mammal (for example,
human) a therapeutically effective amount of a CC-chemokine receptor
antagonist of the present invention, for a time and under conditions
sufficient to
inhibit the biological activity of the native molecules. In some embodiments,
the
invention may provide methods of modulating an immune response in a host, or
treating inflammatory or autoimmune diseases in a host suffering from such
diseases, comprising administering to the host, such as a mammal, a
-1-
CA 02307705 2000-08-03
therapeutically effective amount of a CC-chemokine receptor antagonist of the
present invention. Another aspect of the present invention is directed to
pharmaceutical compositions comprising an antagonistically effective amount of
a CC-chemokine receptor antagonist of the present invention and a
pharmaceutically acceptable carrier.
In one aspect of the invention, a yeast two-hybrid analysis was initiated
using the gelatinase A hemopexin-like C-terminal domain as bait. A cDNA
library
was constructed from human fibroblasts treated with the lectin Concanavalin A.
To validate the efficacy of this approach with extracellular molecules, a
strong
interaction was first demonstrated between the gelatinase A C-domain and the
tissue inhibitor of metalloproteinase-2 (TIMP-2) C-domain. Screening of the
library resulted in the identification of monocyte chemoattractant protein 3
(MCP-
3) as a gelatinase A C-domain binding protein. This interaction was confirmed
by
ELISA binding assays and chemical cross-linking. By mass spectrometry and
peptide sequencing it was shown that the first 4 residues of MCP-3 are removed
by gelatinase A, cleaving MCP-3 at GIy4-IleS. Removal of these residues
renders
MCP-3 ineffective as a chemoattractant, and the cleaved MCP-3 was shown to
act as a competitor to the wild-type molecule. By calcium mobilization,
chemotaxis responses, in vivo models of inflammation, and in human pathology,
it is demonstrated that cleavage of MCP-3 ablates receptor activation and
creates a general chemokine antagonist MCP-3(5-76). The invention also
provides methods of cloning a substrate for a proteinase using the protein-
protein
interaction assays, such as the two-hybrid system, wherein a non-catalytic
domain of the protease is assayed for protein-protein binding activity. The
invention provides methods of modulating the role MMPs play in regulating the
activity of an inflammatory chemokine. In various aspects, the invention
involves
the manipulation of the activity of MMPs in dampening the course of
inflammation
by destroying chemotactic gradients and functionally inactivating chemokines.
The invention also involves manipulating the activity of MMPs as effectors of
an
inflammatory response.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Characterization of MCP-3 interactions with the gelatinase A
hemopexin C domain (Hex CD). (A) In the yeast two-hybrid assay only the yeast
transformants Hex CD/TIMP-2 C domain, Hex CD/MCP-3, and p53/SV40
(positive control) showed growth on medium lacking histidine. Control
transformants of the individual domains showed no significant growth. (B) -
Galactosidase levels (presented as Miller units) in yeast expressing the
indicated
fusion proteins showed significant activity in only the Hex CD/TIMP-2 C
domain,
Hex CD/MCP-3, and p53/SV40 transformants. Yeast strain HF7c (Clontech) has
three copies of the Gal4 17-mer consensus sequence and the TATA portion of
the CYC promoter fused to the IacZ reporter. (C) Glutaraldehyde cross-linking
of
MCP-3 and recombinant hemopexin C domain. MCP-3 either alone, or in the
presence of 0.5 molar equivalent (+), 1.0 molar equivalent (++), or 2.0 molar
equivalents (+++) of hemopexin C domain, was cross-linked with 0.5%
glutaraldehyde for 20 min at 22 °C. (D) ELISA binding assay of 0.5 Ng
MCP-3
-2-
CA 02307705 2000-08-03
immobilized onto a 96-well plate and then incubated with recombinant
gelatinase
A hemopexin C domain (Hex CD) or recombinant collagen binding domain (CBD)
at the concentrations indicated. Binding of the recombinant domains was
monitored by -Hiss affinity purified anti-peptide antibody and quantitated at
405
nm on a plate reader. Recombinant protein domains were expressed in E. coli
as before (4).
Figure 2 Gelatinase A binding and cleavage of MCP-3. (A) Gelatin
zymography of enzyme capture film assay of pro and active gelatinase A. Five
Ng each of bovine serum albumin (BSA), gelatin, TIMP-2, MCP-1, and MCP-3
were immobilized onto a 96-well plate. Recombinant gelatinase A was then
overlaid for 2 h to allow binding and the bound protein analysed by
zymography.
Overlay represents a dilution of the recombinant enzyme used. (B) Gelatin
zymography as in A, but with hemopexin-truncated gelatinase A (N-gelatinase A)
used as overlay. (C) Tricine gel analysis of MCP-3 (20) cleavage by gelatinase
A
in the presence of equimolar amounts (relative to MCP-3) of recombinant
hemopexin C domain, collagen binding domain (CBD), TIMP-2, or 10 pM
hydroxamate inhibitor BB-2275 (British Biotech Pharmaceuticals, Oxford, UK).
Only a single concentration from the full dilution range of hemopexin C domain
and CBD that was added as competitor is presented. (D) Tricine gel analysis of
human fibroblast-mediated MCP-3 cleavage. Sub-confluent fibroblast cultures
were treated with Con A (20 Ng/ml) for 24 h at 37 °C. The resultant
gelatinase A
activation was confirmed by zymography. After 16-h incubation with MCPs in the
presence of the MMP inhibitors indicated (concentrations as in C) the
conditioned
culture media were analyzed by tricine SDS-PAGE. The band at 22 kDa is the
exogenous TIMP-2. The masses of the MCP-3 forms in the culture media were
measured by electrospray mass spectrometry as shown. (E) Electrospray mass
spectrometry, N-terminal Edman sequencing, and tricine gel analysis of MCP-3
cleavage products produced by recombinant gelatinase A activity. MCP-3 (5 Ng)
was incubated with 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 100 fg
recombinant gelatinase A for 4 h at 37 °C. (F) Electrospray mass
spectrometry
and tricine gel analysis of MCP-1, -2, -3, and -4 after incubation with
recombinant
gelatinase A for 18 h at 37 °C. The N-terminal sequence of the MCPs is
shown
with the Gly-Ile scissile bond in MCP-3 in bold.
Figure 3 Cellular responses to MMP-cleaved MCP-3. (a) Cell receptor
binding of full length MCP-3, designated MCP-3(1-76), and MCP-3(5-76). (b)
Intracellular calcium induction by MCP-3, MCP-1, and MDC. Fluo-3AM loaded
THP-1 monocytes or a B-cell line transfected with CCR-4 (for MDC) were first
exposed to either 0 nM (left arrow, top scans) or 500 nM MCP-3(5-76) (left
arrow,
bottom scans), followed by MCP-3 (30 nM), MCP-1 (5 nM), and MDC (5 nM) as
indicated (right arrow, top and bottom scans). The data are presented as
relative
fluorescence emitted at 526 nm. (c) Chemotactic activity of MCP-3(1-76) and
MCP-3(5-76). Transwell assay of monocytes treated with MCP-3(1-76) and
MCP-3(5-76) at the indicated concentrations demonstrating dose response
antagonist action of MCP-3 (5-76). Not shown, are data that indicated loss of
-3-
CA 02307705 2000-08-03
intracellular calcium induction by MCP-3 following gelatinase A-cleavage. Fluo-
3AM loaded THP-1 monocytes were treated with 5 nM MCP-3 or MCP-1 or
respective chemokine incubated first with gelatinase A for 18 h, demonstrating
specific and complete loss of MCP-3 agonist activity.
Figure 4 Animal responses to MMP-cleaved MCP-3. Light micrographs of
haematoxylin and eosin stained subcutaneous tissue sections of mice injected
with: MCP-3(1-76) (a); gelatinase A-cleaved MCP-3 (b); 2:1 molar ratio of
gelatinase A-cleaved MCP-3:full-length MCP-3 (c); and, saline/buffer control
(d).
In paneld (d), the bar represents 20 pm; M, muscle; A, adipocyte; C, loose
connective tissue. Panel (a) clearly shows that only MCP-3(1-76) induced a
marked mononuclear cell infiltrate with associated connective tissue
disruption
surrounding the muscle layer. (e) After sub-cutaneous injections with MCP-3(1-
76) and MCP-3(5-76) mixtures the infiltrating mononuclear cells were
enumerated and expressed as cells/75,000 Nm2. (f) and (g) Haematoxylin and
eosin stained cytospins of intraperitoneal washouts of mice treated first with
zymosan A to induce peritonitis, then 24 h later injected with (f) MCP-3(5-76)
or
(g) saline for 4 h. Panel (h) shows identification of MCP-3(5-76) in human
synovial fluid by immunoprecipitation of human MCP-3/progelatinase A
complexes from inflammatory lesions. MCP-3 was pulled down using an -
MCP-3 monoclonal antibody from 200 NI synovial fluid of a patient with
seronegative spondyloarthropathy. Gelatin zymography (top panel) and western
blotting with rabbit -MCP-3(1-76) antibody (bottom panel) of the complexes.
Lane 1, active and progelatinase A standards.
DETAILED DESCRIPTION OF THE INVENTION
It has been suggested that inhibitors of metalloproteinases may be used
as anti-inflammatories in a variety of diseases. If this is done, the present
invention discloses that such inhibitors may have the counter-indicated side-
effect of sustaining an inflammatory condition, by inhibiting the proteolysis
of
MCP-3, so that MCP-3 would continue to mediate inflammation as a potent
chemoattractant cytokine. In one aspect, the present invention accordingly
provides for the co-administration of MCP-3(5-76) and a metalloproteinase
inhibitor, wherein the administration of the MCP-3(5-76) makes up for the
inhibition of the natural proteolytic effect on native MCP-3.
Metalloproteinase
inhibitors for use in such aspects of the invention may for example be
selected
from the fluorinated butyric acid compounds disclosed in U.S. Patent No.
6,037,361 or the ortho-sulfonamido aryl hydroxamic acids disclosed in U.S.
Patent No. 5,977,408 or the MMP-2 inhibitors disclosed in W09921583,
including: [f4-N-hydroxyamino}-2R-isobutyl-3S-{thienyl-thiomethyl}succinyl]-L-
phenylalanine-N-methylamide; (S)-4-dibenzofuran-2-yl-4-oxo-2-(toluene-4-
sulfonylamino)-butyric acid; (S)-2-(dibenzofuran-3-sulfonylamino)-3-methyl-
butyric acid; and 4-hydroxyimino-4-(4'-methyl-biphenyl-4-yl)-butyric acid.
Alternative MMP-2 inhibitors are disclosed in Tamura Y. et al., J. Med. Chem.,
1998, 41:640-649 and Porter J. et al., Bioorganic & Medicinal Chemistry
Letters,
1994, 4(23):2741-2746 (all of which are incorporated herein by reference).
Native
-4-
CA 02307705 2000-08-03
MMP-2 inhibitors may also be used in alternative embodiments, such as the
tissue inhibitor of metalloproteinase-2 (TIMP-2).
In alternative embodiments of the invention, proteolytic compounds, such
as proteases, may be administered therapeutically to facilitate cleavage of
native
MCP-3 to produce MCP-3(5-76), so that MCP-3(5-76) may act as a CC-
chemkine antagonist.
In some embodiments, the CC-chemokine receptor antagonists of the
invention may be substantially purified peptide fragments, modified peptide
fragments, analogues or pharmacologically acceptable salts of MCP-3 having
amino acids 1-4 truncated from the amino terminal of the native MCP-3, such
compounds are collectively referred to herein as MCP-3(5-76). MCP-3(5-76)
peptides may include homologs of the native MCP-3 sequence from amino acids
through 76, such as naturally occurring isoforms or genetic variants, or
polypeptides having substantial sequence similarity to native MCP-3 amino
acids
5-76, such as 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity
to at least a portion of the native MCP-3(5-76) sequence, the portion of
native
MCP-3 being any contiguous sequence of 10, 20, 30, 40, 50 or more amino
acids, provided the peptides have CC-chemokine receptor antagonist activity.
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). In some embodiments, peptides having an N-terminal LSY
sequence motif within 10, or 7, amino acids of the N-terminus, and/or an N-
terminal RFFESH (SEQ ID N0:5) sequence motif within 20 amino acids of the N-
terminus may be used provided they have CC-chemokine receptor antagonistic
activity. One family of such peptide antagonist candidates has an LSY motif at
amino acids 5-7. Alternative peptides further include the RFFESH (SEQ ID NO:
5) motif at amino acids 12-17. In alternative embodiments, the LSY motif is
located at positions 3-5 of a peptide. The invention also provides peptide
dimers
having two amino acid sequences, which may each have the foregoing sequence
elements, attached by a disulfide bridge within 20, or preferably within 10,
amino
acids of the N terminus, linking cysteine residues or a-aminobutric acid
residues.
It is well known in the art that some modifications and changes can be
made in the structure of a polypeptide without substantially altering the
biological
function of that peptide, to obtain a biologically equivalent polypeptide. 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. As used herein, the term
"conserved amino acid substitutions" refers to the substitution of one amino
acid
for another at a given location in the peptide, where the substitution can be
made
without loss of function. In making such changes, substitutions of like amino
acid
residues can be made on the basis of relative similarity of side-chain
substituents, for example, their size, charge, hydrophobicity, hydrophilicity,
and
the like, and such substitutions may be assayed for their effect on the
function of
the peptide by routine testing.
In some embodiments, conserved amino acid substitutions may be made
-5-
CA 02307705 2000-08-03
where an amino acid residue is substituted for another having a similar
hydrophilicity value (e.g., within a value of plus or minus 2.0), where the
following
hydrophilicity values are assigned to amino acid residues (as detailed in
United
States Patent No. 4,554,101, incorporated herein by reference): Arg (+3.0);
Lys
(+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0);
Pro (-
0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5);
Leu (-1.8);
Ile (-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: Ile
(+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 (-0.8); 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
classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met;
acidic: Asp,
Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.
The invention provides pharmaceutical compositions containing CC-
chemokine receptor antagonists. In one embodiment, such compositions include
a CC-chemokine receptor antagonist compound in a therapeutically or
prophylactically effective amount sufficient to alter bone marrow progenitor
or
stem cell growth, and a pharmaceutically acceptable carrier. In another
embodiment, the composition includes a CC-chemokine receptor antagonist
compound in a therapeutically or prophylactically effective amount sufficient
to
inhibit a cytotoxic effect of a cytotoxic agent, such as cytotoxic agents used
in
chemotherapy or radiation treatment of cancer, and a pharmaceutically
acceptable carrier.
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 bone marrow progenitor or stem cell
multiplication, or
reduction or inhibition of a cytoxic effect of a cytoxic agent. A
therapeutically
effective amount of CC-chemokine receptor antagonist may vary according to
factors such as the disease state, age, sex, and weight of the individual, and
the
ability of the CC-chemokine receptor antagonist 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 one in which any toxic or
detrimental effects of the CC-chemokine receptor antagonist are outweighed by
the therapeutically beneficial effects.
-6-
CA 02307705 2000-08-03
A "prophylactically effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired prophylactic
result, such as preventing or inhibiting a cytoxic effect of a cytoxic agent.
Typically, a prophylactic dose is used in subjects prior to or at an earlier
stage of
disease, so that a prophylactically effective amount may be less than a
therapeutically effective amount.
In particular embodiments, a preferred range for therapeutically or
prophylactically effective amounts of CC-chemokine receptor antagonists may be
0.1 nM-0.1 M, 0.1 nM-0.05M, 0.05 nM-15NM or 0.01 nM-10 M. It is to be noted
that dosage values may vary with the severity of the condition to be
alleviated.
For any particular subject, specific dosage regimens may be adjusted over time
according to the individual need and the professional judgement of the person
administering or supervising the administration of the compositions. Dosage
ranges set forth herein are exemplary only and do not limit the dosage ranges
that may be selected by medical practicioners.
The amount of active compound in the composition may vary according to
factors such as the disease state, age, sex, and weight of the individual.
Dosage
regimens may be adjusted to provide the optimum therapeutic response. For
example, a single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or increased
as indicated by the exigencies of the therapeutic situation. It may be
advantageous to formulate parenteral compositions in dosage unit form for ease
of administration and uniformity of dosage. "Dosage unit form" as used herein
refers to physically discrete units suited as unitary dosages for subjects to
be
treated; each unit containing a predetermined quantity of active compound
calculated to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage unit forms
of
the invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular therapeutic effect
to be
achieved, and (b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in individuals.
As used herein "pharmaceutically acceptable carrier" or "exipient" includes
any and all solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that are
physiologically compatible. In one embodiment, the carrier is suitable for
parenteral administration. Alternatively, the carrier can be suitable for
intravenous, intraperitoneal, intramuscular, sublingual or oral
administration.
Pharmaceutically acceptable carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any conventional media or agent is incompatible with the active compound, use
thereof in the pharmaceutical compositions of the invention is contemplated.
7-
CA 02307705 2000-08-03
Supplementary active compounds can also be incorporated into the
compositions.
Therapeutic compositions typically must be sterile and stable under the
conditions of manufacture and storage. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure suitable to high
drug concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example, glycerol,
propylene
glycol, and liquid polyethylene glycol, and the like), and suitable mixtures
thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such
as lecithin, by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be preferable
to
include isotonic agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Prolonged absorption of the
injectable compositions can be brought about by including in the composition
an
agent which delays absorption, for example, monostearate salts and gelatin.
Moreover, the CC-chemokine receptor antagonists may be administered in a
time release formulation, for example in a composition which includes a slow
release polymer. The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic
acid and polylactic, polyglycolic copolymers (PLG). Many methods for the
preparation of such formulations are patented or generally known to those
skilled
in the art.
Sterile injectable solutions can be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion medium and
the required other ingredients from those enumerated above. In the case of
sterile powders for the preparation of sterile injectable solutions, the
preferred
methods of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. In accordance with an
alternative
aspect of the invention, a CC-chemokine receptor antagonist may be formulated
with one or more additional compounds that enhance the solubility of the CC-
chemokine receptor antagonist.
CC-chemokine receptor antagonist compounds of the invention may
include MCP-3 derivatives, such as C-terminal hydroxymethyl derivatives, O-
modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-
terminally
modified derivatives including substituted amides such as alkylamides and
hydrazides and compounds in which a C-terminal phenylalanine residue is
_g_
CA 02307705 2000-08-03
replaced with a phenethylamide analogue (e.g., Ser-Ile-phenethylamide as an
analogue of the tripeptide Ser-Ile-Phe).
Within a CC-chemokine receptor antagonist compound of the invention, a
peptidic structure (such as an MCP-3 derived peptide) maybe coupled directly
or
indirectly to at least one modifying group. The term "modifying group" is
intended
to include structures that are directly attached to the peptidic structure
(e.g., by
covalent coupling), as well as those that are indirectly attached to the
peptidic
structure (e.g., by a stable non-covalent association or by covalent coupling
to
additional amino acid residues, or mimetics, analogues or derivatives thereof,
which may flank the MCP-3 core peptidic structure). For example, the modifying
group can be coupled to the amino-terminus or carboxy-terminus of an MCP-3
peptidic structure, or to a peptidic or peptidomimetic region flanking the
core
domain. Alternatively, the modifying group can be coupled to a side chain of
at
least one amino acid residue of a MCP-3 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
residues) or a glutamic acid residue(s), through a hydroxy group of a tyrosyl
residue(s), a serine residues) or a threonine residues) or other suitable
reactive
group on an amino acid side chain). Modifying groups covalently coupled to the
peptidic structure can be attached by means and using methods well known in
the art for linking chemical structures, including, for example, amide,
alkylamino,
carbamate or urea bonds.
In some embodiments, the modifying group may comprise a cyclic,
heterocyclic or polycyclic group. The term "cyclic group", as used herein,
includes cyclic saturated or unsaturated (i.e., aromatic) group having from 3
to
10, 4 to 8, or 5 to 7 carbon atoms. Exemplary cyclic groups include
cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be
unsubstituted or substituted at one or more ring positions. A cyclic group may
for
example be substituted with halogens, alkyls, cycloalkyls, alkenyls, alkynyls,
aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides,
phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers,
sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, -CF3, -CN.
The term "heterocyclic group" includes cyclic saturated, unsaturated and
aromatic groups having from 3 to 10, 4 to 8, or 5 to 7 carbon atoms, wherein
the
ring structure includes about one or more heteroatoms. Heterocyclic groups
include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine,
piperazine,
morpholine. The heterocyclic ring may be substituted at one or more positions
with such substituents as, for example, halogens, alkyls, cycloalkyls,
alkenyls,
alkynyls, aryls, other heterocycles, hydroxyl, amino, vitro, thiol, amines,
imines,
amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,
thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, -CF3, -CN.
Heterocycles may also be bridged or fused to other cyclic groups as described
below.
_g_
CA 02307705 2000-08-03
The term "polycyclic group" as used herein is intended to refer to two or
more saturated, unsaturated or aromatic cyclic rings in which two or more
carbons are common to two adjoining rings, so that the rings are "fused
rings".
Rings that are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycyclic group may be substituted with such
substituents as described above, as for example, halogens, alkyls,
cycloalkyls,
alkenyls, alkynyls, hydroxyl, amino, vitro, thiol, amines, imines, amides,
phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers,
sulfonyls, selenoethers, ketones, aldehydes, esters, -CF3, or -CN.
The term "alkyl" refers to the radical of saturated aliphatic groups,
including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl
(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl
substituted
alkyl groups. In some embodiments, a straight chain or branched chain alkyl
has
20 or fewer carbon atoms in its backbone (C~-C2o for straight chain, C3-C2o
for
branched chain), or 10 or fewer carbon atoms . In some embodiments,
cycloalkyls may have from 4-10 carbon atoms in their ring structure, such as
5, 6
or 7 carbon rings. Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, having from one
to
ten carbon atoms in its backbone structure. Likewise, "lower alkenyl" and
"lower
alkynyl" have chain lengths of ten or less carbons.
The term "alkyl" (or "lower alkyl") as used throughout the specification and
claims is intended to include both "unsubstituted alkyls" and "substituted
alkyls",
the latter of which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, halogen, hydroxyl, carbonyl (such as
carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups), and
esters
(including alkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl,
acyloxy,
alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido,
amidine, imino, cyano, vitro, azido, sulfhydryl, alkylthio, sulfate,
sulfonate,
sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic or
heteroaromatic
moiety. The moieties substituted on the hydrocarbon chain can themselves be
substituted, if appropriate. For instance, the substituents of a substituted
alkyl
may include substituted and unsubstituted forms of aminos, azidos, iminos,
amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls
(including sulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl
groups, as
well as ethers, alkylthios, carbonyls (including ketones, aldehydes,
carboxylates,
and esters), -CF3, -CN and the like. Exemplary substituted alkyls are
described
below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys,
alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls described above,
but
that contain at least one double or triple bond respectively.
-10-
CA 02307705 2000-08-03
The term "aralkyl", as used herein, refers to an alkyl or alkylenyl group
substituted with at least one aryl group. Exemplary aralkyls include benzyl
(i.e.,
phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the
like.
The term "alkylcarbonyl", as used herein, refers to -C(O)-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 "aryloxycarbonyl"
refers to
-C(O)-O-aryl. The term "acyloxy" refers to -O-C(O)-R7, in which R7 is alkyl,
alkenyl, alkynyl, aryl, aralkyl or heterocyclyl.
The term "amino", as used herein, refers to -N(Ra)(Ra), in which Ra and Ra
are each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or
in
which Ra and Ra together with the nitrogen atom to which they are attached
form
a ring having 4-8 atoms. Thus, the term "amino", as used herein, includes
unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino), and
disubstituted (e.g., dialkylamino or alkylarylamino) amino groups. The term
"amido" refers to -C(O)-N(R8)(R9), in which R8 and R9 are as defined above.
The
term "acylamino" refers to -N(R'a)C(O)-R7, in which R7 is as defined above and
R'8 is alkyl.
As used herein, the term "nitro" means -NOz ; the term "halogen"
designates -F, -CI, -Br or -I; the term "sulfhydryl" means -SH; and the term
"hydroxyl" means -OH.
The term "aryl" as used herein includes 5-, 6- and 7-membered aromatic
groups that may include from zero to four heteroatoms in the ring, for
example,
phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl,
triazolyl,
pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like.
Those aryl
groups having heteroatoms in the ring structure may also be referred to as
"aryl
heterocycles" or "heteroaromatics". The aromatic ring can be substituted at
one
or more ring positions with such substituents as described above, as for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde,
ester, a
heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
Aryl
groups can also be part of a polycyclic group. For example, aryl groups
include
fused aromatic moieties such as naphthyl, anthracenyl, quinolyl, indolyl, and
the
like.
Modifying groups may include groups comprising biotinyl structures,
fluorescein-containing groups, a diethylene-triaminepentaacetyl group, a (-)-
menthoxyacetyl group, a N-acetylneuraminyl group, a cholyl structure or an
iminiobiotinyl group. A CC-chemokine receptor antagonist compound may be
modified at its carboxy terminus with a cholyl group according to methods
known
-11-
CA 02307705 2000-08-03
in the art (see e.g., Wess, G. et al. (1993) Tetrahedron Letters, 34:817-822;
Wess, G. et al. (1992) Tetrahedron Letters 33:195-198; and Kramer, W. et al.
(1992) J. Biol. Chem. 267:18598-18604). Cholyl derivatives and analogues may
also be used as modifying groups. For example, a preferred cholyl derivative
is
Aic (3-(O-aminoethyl-iso)-cholyl), which has a free amino group that can be
used
to further modify the CC-chemokine receptor antagonist compound. A modifying
group may be a "biotinyl structure", which includes biotinyl groups and
analogues
and derivatives thereof (such as a 2-iminobiotinyl group). In another
embodiment,
the modifying group may comprise a "fluorescein-containing group", such as a
group derived from reacting an MCP-3 derived peptidic structure with 5-(and 6-
)-
carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. In
various
other embodiments, the modifying groups) may comprise an N-acetylneuraminyl
group, a traps-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyl
group,
an (S)-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl group, a 2-
norbornaneacetyl group, a -oxo-5-acenaphthenebutyryl, a (-)-2-oxo-4-
thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl
group, a
diethylenetriaminepentaacetyl group, a 4-morpholinecarbonyl group, a 2-
thiopheneacetyl group or a 2-thiophenesulfonyl group.
A CC-chemokine receptor antagonist compound of the invention may be
further modified to alter the specific properties of the compound while
retaining
the desired functionality of the compound. For example, in one embodiment, the
compound may be modified to alter a pharmacokinetic property of the compound,
such as in vivo stability or half-life. The compound may be modified to label
the
compound with a detectable substance. The compound may be modified to
couple the compound to an additional therapeutic moiety. To further chemically
modify the compound, such as to alter its pharmacokinetic properties, reactive
groups can be derivatized. For example, when the modifying group is attached
to
the amino-terminal end of the MCP-3 core domain, the carboxy-terminal end of
the compound may be further modified. Potential C-terminal modifications
include those which reduce the ability of the compound to act as a substrate
for
carboxypeptidases. Examples of C-terminal modifiers include an amide group, an
ethylamide group and various non-natural amino acids, such as D-amino acids
and -alanine. Alternatively, when the modifying group is attached to the
carboxy-terminal end of the aggregation core domain, the amino-terminal end of
the compound may be further modified, for example, to reduce the ability of
the
compound to act as a substrate for aminopeptidases.
A CC-chemokine receptor antagonist compound can be further modified to
label the compound by reacting the compound with a detectable substance.
Suitable detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials and radioactive materials.
Examples
of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-
galactosidase, or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
-12-
CA 02307705 2000-08-03
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; and
examples of suitable radioactive material include'4C,'231,'241,'251,'3'l,
99mTc, 35S
or 3H. A CC-chemokine receptor antagonist compound may be radioactively
labeled with'4C, either by incorporation of'4C into the modifying group or one
or
more amino acid structures in the CC-chemokine receptor antagonist compound.
Labeled CC-chemokine receptor antagonist compounds may be used to assess
the in vivo pharmacokinetics of the compounds, as well as to detect disease
progression or propensity of a subject to develop a disease, for example for
diagnostic purposes. Tissue distribution of CXCR4 receptors can be detected
using a labeled CXCR4 antagonist compound either in vivo or in an in vitro
sample derived from a subject. For use as an in vivo diagnostic agent, a CXCR4
antagonist compound of the invention may be labeled with radioactive
technetium or iodine. A modifying group can be chosen that provides a site at
which a chelation group for the label can be introduced, such as the Aic
derivative of cholic acid, which has a free amino group. For example, a
phenylalanine residue within the MCP-3 sequence (such as aminoacid residue
13 ) may be substituted with radioactive iodotyrosyl. Any of the various
isotopes
of radioactive iodine may be incorporated to create a diagnostic agent. '231
(half-
life=13.2 hours) may be used for whole body scintigraph~y, '241 (half life=4
days)
may be used for positron emission tomography (PET), ' 51 (half life=60 days)
may
be used for metabolic turnover studies and'3'I (half life=8 days) may be used
for
whole body counting and delayed low resolution imaging studies.
In an alternative chemical modification, a CXCR4 antagonist compound of
the invention may be prepared in a "prodrug" form, wherein the compound itself
does not act as a CXCR4 antagonist, but rather is capable of being
transformed,
upon metabolism in vivo, into a CXCR4 antagonist compound as defined herein.
For example, in this type of compound, the modifying group can be present in a
prodrug form that is capable of being converted upon metabolism into the form
of
an active CXCR4 antagonist. Such a prodrug form of a modifying group is
referred to herein as a "secondary modifying group." A variety of strategies
are
known in the art for preparing peptide prodrugs that limit metabolism in order
to
optimize delivery of the active form of the peptide-based drug (see e.g.,
Moss, J.
(1995) in Peptide-Based Drug Design: Controlling Transport and Metabolism,
Taylor, M. D. and Amidon, G. L. (eds), Chapter 18.
MCP-3(5-76) analogues of the invention may be prepared by standard
techniques known in the art. MCP-3(5-76) analogues may be composed, at least
in part, of a peptide synthesized using standard techniques (such as those
described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag,
Berlin (1993); Grant, G. A. (ed.). Synthetic Peptides: A User's Guide, W. H.
Freeman and Company, New York (1992); or Clark-Lewis, I., Dewald, B.,
Loetscher, M., Moser, B., and Baggiolini, M., (1994) J. Biol. Chem., 269,
16075-
16081 ). Automated peptide synthesizers are commercially available (e.g.,
Advanced ChemTech Model 396; Milligen/Biosearch 9600). Peptides may be
-13-
CA 02307705 2000-08-03
assayed for CC-chemokine receptor antagonist activity in accordance with
standard methods. Peptides may be purified by HPLC and analyzed by mass
spectrometry. Peptides may be dimerized via a disulfide bridge formed by
gentle
oxidation of the cysteines using 10% DMSO in water. Following HPLC
purification dimer formation may be verified, by mass spectrometry. One or
more
modifying groups may be attached to a MCP-3(5-76) derived peptidic component
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, MCP-3(5-76) peptides may be
prepared according to standard recombinant DNA techniques using a nucleic
acid molecule encoding the peptide. A nucleotide sequence encoding the peptide
may be determined using the genetic code and an oligonucleotide molecule
having this nucleotide sequence may be synthesized by standard DNA synthesis
methods (e.g., using an automated DNA synthesizer). Alternatively, a DNA
molecule encoding a peptide compound may be derived from the natural
precursor protein gene or cDNA (e.g., using the polymerase chain reaction
(PCR) and/or restriction enzyme digestion) according to standard molecular
biology techniques.
The invention also provides an isolated nucleic acid molecule comprising
a nucleotide sequence encoding a peptide of the invention. In some
embodiments, the peptide may comprise an amino acid sequence having at least
one amino acid deletion compared to native MCP-3. The term "nucleic acid
molecule" is intended to include DNA molecules and RNA molecules and may be
single-stranded or double-stranded. In alternative embodiments, the isolated
nucleic acid encodes a peptide wherein one or more amino acids are deleted
from the N-terminus, C-terminus and/or an internal site of MCP-3.
To facilitate expression of a peptide compound in a host cell by standard
recombinant DNA techniques, the isolated nucleic acid encoding the peptide may
be incorporated into a recombinant expression vector. Accordingly, the
invention
also provides recombinant expression vectors comprising the nucleic acid
molecules of the invention. As used herein, the term "vector" refers to a
nucleic
acid molecule capable of transporting another nucleic acid to which it has
been
operatively linked. Vectors may include circular double stranded DNA plasmids,
viral vectors. Certain vectors are capable of autonomous replication in a host
cell
into which they are introduced (such as bacterial vectors having a bacterial
origin
of replication and episomal mammalian vectors). Other vectors (such as non-
episomal mammalian vectors) may be integrated into the genome of a host cell
upon introduction into the host cell, and thereby may be replicated along with
the
-14-
CA 02307705 2000-08-03
host genome. Certain vectors may be capable of directing the expression of
genes to which they are operatively linked. Such vectors are referred to
herein as
"recombinant expression vectors" or "expression vectors".
In recombinant expression vectors of the invention, the nucleotide
sequence encoding a peptide may be operatively linked to one or more
regulatory sequences, selected on the basis of the host cells to be used for
expression. The terms "operatively linked" or "operably" linked mean that the
sequences encoding the peptide are linked to the regulatory sequences) in a
manner that allows for expression of the peptide compound. The term
"regulatory
sequence" includes promoters, enhancers, polyadenylation signals and other
expression control elements. Such regulatory sequences are described, for
example, in Goeddel; Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) (incorporated herein be
reference). Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell, those that
direct
expression of the nucleotide sequence only in certain host cells (such as
tissue-
specific regulatory sequences) and those that direct expression in a
regulatable
manner (such as only in the presence of an inducing agent). The design of the
expression vector may depend on such factors as the choice of the host cell to
be transformed and the level of expression of peptide compound desired.
The recombinant expression vectors of the invention may be designed for
expression of peptide compounds in prokaryotic or eukaryotic cells. For
example,
peptide compounds may be expressed in bacterial cells such as E. coli, insect
cells (using baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector may be transcribed
and
translated in vitro, for example using T7 promoter regulatory sequences and T7
polymerase. Examples of vectors for expression in yeast S. cerivisae include
pYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and
Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
Baculovirus
vectors available for expression of proteins or peptides in cultured insect
cells
(e.g., Sf 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell.
Biol.
3:2156-2165) and the pVl_ series (Lucklow, V. A., and Summers, M. D., (1989)
Virology 170:31-39). Examples of mammalian expression vectors include pCDM8
(Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO
J. 6:187-195). When used in mammalian cells, the expression vector's control
functions are often provided by viral regulatory elements. For example,
commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40.
In addition to regulatory control sequences, recombinant expression
vectors may contain additional nucleotide sequences, such as a selectable
-15-
CA 02307705 2000-08-03
marker gene to identify host cells that have incorporated the vector.
Selectable
marker genes are well known in the art. To facilitate secretion of the peptide
compound from a host cell, in particular mammalian host cells, the recombinant
expression vector preferably encodes a signal sequence operatively linked to
sequences encoding the amino-terminus of the peptide compound, such that
upon expression, the peptide compound is synthesised with the signal sequence
fused to its amino terminus. This signal sequence directs the peptide compound
into the secretory pathway of the cell and is then cleaved, allowing for
release of
the mature peptide compound (i.e., the peptide compound without the signal
sequence) from the host cell. Use of a signal sequence to facilitate secretion
of
proteins or peptides from mammalian host cells is well known in the art.
A recombinant expression vector comprising a nucleic acid encoding a
peptide compound may be introduced into a host cell to produce the peptide
compound in the host cell. Accordingly, the invention also provides host cells
containing the recombinant expression vectors of the invention. The terms
"host
cell" and "recombinant host cell" are used interchangeably herein. Such terms
refer not only to the particular subject cell but to the progeny or potential
progeny
of such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still included
within the
scope of the term as used herein. A host cell may be any prokaryotic or
eukaryotic cell. For example, a peptide compound may be expressed in bacterial
cells such as E. coli, insect cells, yeast or mammalian cells. The peptide
compound may be expressed in vivo in a subject to the subject by gene therapy
(discussed further below).
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. The terms
"transformation" and "transfection" refer to techniques for introducing
foreign
nucleic acid into a host cell, including calcium phosphate or calcium chloride
co-
precipitation, DEAE-dextran-mediated transfection, lipofection,
electroporation,
microinjection and viral-mediated transfection. Suitable methods for
transforming
or transfecting host cells can for example be found in Sambrook et al.
(Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press
(1989)), and other laboratory manuals. Methods for introducing DNA into
mammalian cells in vivo are also known, and may be used to deliver the vector
DNA of the invention to a subject for gene therapy.
For stable transfection of mammalian cells, it is known that, depending
upon the expression vector and transfection technique used, only a small
fraction
of cells may integrate the foreign DNA into their genome. In order to identify
and
select these integrants, a gene that encodes a selectable marker (such as
resistance to antibiotics) may be introduced into the host cells along with
the
gene of interest. Preferred selectable markers include those that confer
resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acids
-16-
CA 02307705 2000-08-03
encoding a selectable marker may be introduced into a host cell on the same
vector as that encoding the peptide compound or may be introduced on a
separate vector. Cells stably transfected with the introduced nucleic acid may
be
identified by drug selection (cells that have incorporated the selectable
marker
gene will survive, while the other cells die).
A nucleic acid of the invention may be delivered to cells in vivo using
methods such as direct injection of DNA, receptor-mediated DNA uptake or viral-
mediated transfection. Direct injection has been used to introduce naked DNA
into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff
et al.
(1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for
injecting DNA into cells in vivo may be used. Such an apparatus may be
commercially available (e.g., from BioRad). Naked DNA may also be introduced
into cells by complexing the DNA to a cation, such as polylysine, which is
coupled to a ligand for a cell-surface receptor (see for example Wu, G. and
Wu,
C. H. (1988) J. Biol. Chem. 263:14621; Wilson el al. (1992) J. Biol. Chem.
267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex
to the receptor may facilitate uptake of the DNA by receptor-mediated
endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt
endosomes, thereby releasing material into the cytoplasm, may be used to avoid
degradation of the complex by intracellular lysosomes (see for example Curiel
el
al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc.
Natl.
Acad. Sci. USA 90:2122-2126).
Defective retroviruses are well characterized for use in gene transfer for
gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:271 ).
Protocols for producing recombinant retroviruses and for infecting cells in
vitro or
in vivo with such viruses can be found in Current Protocols in Molecular
Biology,
Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections
9.10-
9.14 and other standard laboratory manuals. Examples of suitable retroviruses
include pLJ, pZIP, pWE and pEM which are well known to those skilled in the
art.
Examples of suitable packaging virus lines include .p i.Crip, .p i.Cre, .p i.2
and
.p i.Am. Retroviruses have been used to introduce a variety of genes into many
different cell types, including epithelial cells, endothelial cells,
lymphocytes,
myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for
example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan
(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc.
Natl.
Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci.
USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-
8043; Ferry et al. (1991 ) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury
et
al. (1991 ) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl.
Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-
647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al.
(1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.
4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT
Application WO 89/05345; and PCT Application WO 92/07573).
-17-
CA 02307705 2000-08-03
The genome of an adenovirus may be manipulated so that it encodes and
expresses a peptide compound of the invention, but is inactivated in terms of
its
ability to replicate in a normal lytic viral life cycle. See for example
Berkner et al.
(1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and
Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived
from
the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,
Ad2,
Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant
adenoviruses are advantageous in that they do not require dividing cells to be
effective gene delivery vehicles and can be used to infect a wide variety of
cell
types, including airway epithelium (Rosenfeld et al. (1992) cited supra),
endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-
6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA
90:2812-2816) and muscle cells (Quantin el al. (1992) Proc. Natl. Acad. Sci.
USA
89:2581-2584).
Adeno-associated virus (AAV) may be used for delivery of DNA for gene
therapy purposes. AAV is a naturally occurring defective virus that requires
another virus, such as an adenovirus or a herpes virus, as a helper virus for
efficient replication and a productive life cycle (Muzyczka et al. Curr.
Topics in
Micro. and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA
into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir.
Cell.
Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and
McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such as that
described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 may be used
to
introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl.
Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-
2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.
(1984) J.
Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
General methods for gene therapy are known in the art. See for example,
U.S. Pat. No. 5,399,346 by Anderson et al. (incorporated herein by reference).
A
biocompatible capsule for delivering genetic material is described in PCT
Publication WO 95/05452 by Baetge et al. Methods of gene transfer into
hematopoietic cells have also previously been reported (see Clapp, D. W., et
al.,
Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); and ,
Cavazzana-Calvo et al., Science 288:669-72 (2000), all of which are
incorporated herein by reference).
Although various embodiments of the invention are disclosed herein,
many adaptations and modifications may be made within the scope of the
invention in accordance with the common general knowledge of those skilled in
this art. Such modifications include the substitution of known equivalents for
any
aspect of the invention in order to achieve the same result in substantially
the
same way. Numeric ranges are inclusive of the numbers defining the range. In
the claims, the word "comprising" is used as an open-ended term, substantially
-18-
CA 02307705 2000-08-03
equivalent to the phrase "including, but not limited to". The disclosed uses
for
various embodiments are not necessarily obtained in all embodiments, and the
invention may be adapted by those skilled in the art to obtain alternative
utilities.
Example 1
The two-hybrid system was used to demonstrate a strong interaction
between the single disulphide bonded gelatinase A hemopexin C domain and the
C domain of the tissue inhibitor of metalloproteinase (TIMP)-2 that contains 3
disulphide bonds (Fig. 1A). Deletion analyses (5) and domain swapping (6)
studies have provided indirect evidence for these domain interactions in the
cellular activation and localization of gelatinase A to cell surface membrane
type
(MT)-MMPs (7). The assay of the invention provided direct evidence for this
association in the gelatinase A/TIMP-2/MT1-MMP complex (8), showing the
efficacy of the yeast two-hybrid assay of the invention for revealing
disulphide-
containing protein interactions that normally occur extracellularly at 37
°C.
Surprisingly, in accordance with the assay of the invention, protein
expression
and folding in yeast at 30 °C appears to generate a stable, functional
protein fold
despite the apparent absence of disulphide bonds.
Example 2
Concanavalin A (Con A) stimulates fibroblasts to degrade extracellular
matrix components by activating gelatinase A (9). A cDNA library was
constructed from Con A-treated human gingival fibroblasts. Using the
gelatinase
A hemopexin C domain as bait in yeast two-hybrid screens (10) MCP-3 was
identified as an interactor with gelatinase A (from a full-length cDNA clone
(Fig.
1 ). The hemopexin C domain had as strong an interaction with MCP-3 as it did
with the TIMP-2 C domain in the f3-galactosidase assay (Fig. 1). Chemical
cross-
linking (12) of MCP-3 to recombinant hemopexin C domain verified this
interaction (Fig. 1 ). The cross-linked MCP-3-hemopexin C domain had the
expected mass of a 1:1 bimolecular complex, whereas MCP-3 alone was not
significantly cross-linked. Furthermore, MCP-3 prevented hemopexin C domain
oligomerization, indicating a specific interaction. This was confirmed by an
ELISA-based binding assay (Fig. 1 ). The hemopexin C domain showed
saturable binding to MCP-3. Specificity was confirmed using recombinant
gelatinase A collagen binding domain protein (13), comprised of three
fibronectin
type II modules, which did not bind MCP-3. Using an enzyme-capture film assay
(14) it was found that the full-length gelatinase A enzyme bound MCP-3 (Fig.
2),
whereas a hemopexin-truncated form of the enzyme (N-gelatinase A) did not
(Fig. 2). No significant interaction was observed between gelatinase A and MCP-
1. As controls both the full-length and N-gelatinase A bound to gelatin and
TIMP-
2 by the collagen binding domain and active site (15) of both forms of the
enzyme, respectively. Together, these data demonstrate a strong requirement
for the hemopexin C domain of gelatinase A in binding MCP-3.
MCP-3 was shown to be a novel substrate of gelatinase A. Incubation
with recombinant enzyme resulted in a small but distinct increase in
electrophoretic mobility of MCP-3 on tricine gels (Fig. 2C) that the MMP
specific
-19-
CA 02307705 2000-08-03
inhibitors TIMP-2 and the synthetic hydroxamate inhibitor, BB-2275, blocked.
Recombinant hemopexin C domain competed for and reduced gelatinase A
cleavage of MCP-3 in a concentration dependent manner whereas the collagen
binding domain had no effect (Fig. 2C). In addition, the k~t/Km value of MCP-3
cleavage decreased from 8,000 M-'s-' for full-length gelatinase A to 500 M-'s-
' for
N-gelatinase A confirming the mechanistic importance of the hemopexin C
domain binding interaction in MCP-3 degradation. Cleavage of MCP-3 by other
MMPs was also assayed, illustrating alternative proteases that may be used to
generate MCP-3(5-76). Matrilysin (MMP-7), which lacks a hemopexin C domain,
and the MMPs collagenase-2 (MMP-8) and gelatinase B (MMP-9) did not cleave
MCP-3, but collagenase-3 (MMP-13) and MT1-MMP (MMP-14) efficiently
processed MCP-3 (not shown).
In one aspect of the invention, MCP-3 may be efficiently cleaved in vivo.
Indeed, MCP-3 but not MCP-1 was rapidly cleaved in cell cultures of human
fibroblasts following Con A-induced gelatinase A activation, but not in
untreated
cells (Fig. 2D). Molar excess TIMP-2 or BB-2275 blocked this confirming MMP
dependency in MCP-3 processing. The bridging interaction of TIMP-2 between
the gelatinase A hemopexin C domain and MT1-MMP (8), which is central to the
physiological binding, activation and activity of gelatinase A at the cell
surface,
did not interfere with MCP-3 binding (not shown) and cleavage (Fig. 2D).
To identify the cleavage site in MCP-3 electrospray mass spectroscopy
was performed. The mass measured of the gelatinase A-cleaved MCP-3 was
8,574 Da both in cell culture (Fig. 2D) or in vitro (Fig. 2E) and differed
from the
mass of the full-length molecule (8,935 Da) by the exact mass of the first
four N-
terminal residues. N-terminal Edman sequencing confirmed that the scissile
bond was at GIy4-Ile5 (Fig. 2E), a preferred sequence for gelatinase A
cleavage
in gelatin (18) that is absent in other MCPs that were not cleaved by
gelatinase A
(Fig. 2F). Together, these data demonstrate the importance of the hemopexin C
domain for non-collagenous substrate cleavage by any MMP. This indicates that
compounds that bind to protease exosites may be used to selectively inhibit
proteolytic activity against specific substrates, in accordance with an
alternative
aspect of the invention.
To demonstrate the physiological relevance of gelatinase A association
and cleavage of MCP-3, a monoclonal antibody to human MCP-3 pulled down
pro-gelatinase A, but not the active enzyme, in association with full-length
MCP-3
from the synovial fluid of a seronegative spondyloarthropathy patient (Fig.
3).
Uncleaved MCP-3 was identified in these specific immunocomplexes using an
affinity-purified anti-peptide antibody (alpha-1-76) that only recognizes the
N-
terminal 5 residues of MCP-3 (Fig. 3B). In order to identify gelatinase A-
cleaved
MCP-3 in vivo, specific antisera were raised that only recognizes the free
amino
group of the cleaved MCP-3 (5-76), but not the full-length MCP-3, nor another
synthesized truncated MCP-3 (9-76) as controls (Fig. 3). Using this neo-
epitope
antibody strategy (19) the gelatinase A-cleaved form of MCP-3 was found in
human rheumatoid synovial fluid (Fig. 3C). These data demonstrate the
physiological relevance of the MCP-3 interaction with gelatinase A in vivo and
the
pathophysiological generation of the MCP-3 cleavage product in human disease.
-20-
CA 02307705 2000-08-03
Activation of chemokine receptors by ligand mobilizes intracellular calcium
stores and together with other signaling events leads to directed monocyte
migration. MCP-3 binds CC receptors-1, -2, and -3. Protein engineering studies
have shown that N-terminal truncation at different sites has variable effects
on
the agonist activity of MCP-1 and MCP-3 (20, 27). To determine the effect of
gelatinase A cleavage of MCP-3, we found that in calcium induction assays (22)
the gelatinase A-mediated removal of the first four residues of MCP-3 resulted
in
the loss of receptor activation and chemokine activity. Neither gelatinase A-
cleaved MCP-3 in the presence of 1/1000 gelatinase A (mole ratio enzyme/MCP-
3) (Fig. 4A) nor synthetic MCP-3(5-76) (Fig. 4B) elicited a response in THP-1
cells, a monocyte cell line expressing CCR-1 and CCR-2. In addition to loss of
CCR agonist activity, MCP-3(5-76) antagonized the subsequent response to both
uncleaved MCP-3 and MCP-1, which binds CCR-2 (Fig. 4B). MCP-3(5-76) also
desensitized macrophage inflammatory protein (MIP)1-alpha induced Ca2+
mobilization in THP-1 cells (not shown). Since MIP-1alpha binds CCR-1 and
CCR-5, this confirmed the CCR-1 antagonist activity of MCP-3(5-76). As a
control MCP-3(5-76) did not block the calcium response to MDC, which binds
CCR-4, a receptor not bound by MCP-3 (Fig. 4). The physiological relevance of
MCP-3 antagonism was confirmed by cell binding assays (23). Scatchard
analysis showed that synthetic MCP-3(5-76) bound cells with high affinity (Kd
of
18.3 nM) similar to that of MCP-3 (Kd of 5.7 nM) (Fig. 4C).
To determine the cellular response to gelatinase A cleavage of MCP-3,
monocyte chemotaxis responses were measured. In transwell cell migration
assays (22) MCP-3(5-76) was not chemotactic, even at a 100-fold higher dose
than full-length MCP-3, which elicited the typical chemotactic response (Fig.
4).
Consistent with the calcium mobilization experiments, synthetic MCP-3(5-76)
(Fig. 4) and gelatinase A-cleaved MCP-3 (not shown) also functioned as
antagonists in a dose dependent manner to inhibit the chemotaxis directed by
full-length chemokine. Thus, inactivation of MCP-3 generates a broad-spectrum
antagonist for CC-chemokine receptors that retains strong cellular binding
affinity
and modulates the response to a number of related chemoattractants.
To examine the biological consequences of MMP cleavage of MCP-3 in
inflammation, a series of subcutaneous injections were performed in mice (24)
of
various mole ratios of full-length MCP-3 and gelatinase A-cleaved or synthetic
MCP-3(5-76). On analysis of tissue sections MCP-3, but not gelatinase A
cleaved MCP-3 induced a marked infiltration of mononuclear inflammatory cells
with associated degradation of matrix at 18 h (Fig. 4). ANOVA analysis of
morphometric counts showed the statistically significant dose dependent
reduction in the mononuclear cell infiltrate in response to as little as a 1:1
mixture
of MCP-3(5-76) with MCP-3 (Fig. 4). In a separate mouse model of
inflammation, the cellular infiltrate in 24-h zymosan A-induced peritonitis
(24) was
significantly attenuated after intraperitoneal injection with MCP-3(5-76).
Consistent with morphometric examination of the lavage cytospins (Fig. 4),
FACS
analysis (25) of the peritoneal washouts showed that macrophage (F4/80+) cell
counts were significantly reduced by ~40% at both 2 and 4 hours following MCP-
-21 -
CA 02307705 2000-08-03
3(5-76) treatment (Fig. 4). The present example demonstrates of the
extracellular
inactivation of a cytokine in vivo by MMP activity.
In various aspects of the invention, the relative amounts of intact and
cleaved
MCP-3 that are present after pathophysiological cleavage will determine
chemotactic and inflammation outcomes. Thus, gelatinase A expression,
which is induced in tissues at the later stages of inflammation (34) by
cytokines from macrophages and other earlier participants in the
inflammatory reaction, may also serve to dampen inflammation by
destroying the MCP-3 chemotactic gradient. This in turn can functionally
inactivate the gradients of other CC chemokines having similar CCR
usage. Of note, gelatinase A is largely stromal-cell derived and not usually
expressed by leukocytes (35) which express MMP-8 and gelatinase B,
both of which are not active on MCP-3.
References and Notes
1. K.S. Lam etal., Nature 354, 82 (1991).
2. S. Fields, O. Song, Nature 340, 245 (1988).
3. F.X. Gomis-Ruth et al., J. Mol. Biol. 264, 556 (1996).
4. U.M. Wallon, C.M. Overall, J. Biol. Chem. 272, 747 (1997).
5. R.V. Ward, S.J. Atkinson, J.J. Reynolds, G. Murphy, Biochem. J. 304,
263 (1994).
6. F. Willenbrock et al., Biochemistry 32, 4330 (1993).
7. H. Sato et al., Nature 370, 61 (1994).
8. A.Y. Strongin et al., J. Biol. Chem. 270, 5331 (1995).
9. C.M. Overall, J. Sodek, J. Biol. Chem. 265, 21141 (1990).
10. Yeast strain HF7c (Clontech) was transformed as per suppliers'
instructions with cDNA encoding the protein domains described fused to
the Gal4 DNA-binding domain and the Gal4 transactivation domain.
Transformants were selected on appropriate growth media, then tested on
media lacking the metabolite histidine. Colony growth was monitored after
4 days incubation at 30 °C and the plate was photographed. Yeast growth
indicates a positive interaction between proteins fused to the Gal4
domains. Quantitative analysis of interactions was done by liquid -
galactosidase assays as per supplier instructions.
11. G. Opendaker et al., Biochem. Biophys. Res. Commun. 191, 535 (1993).
12. MCP-3 (0.1 mg/ml) and gelatinase A hemopexin C domain were combined
at various mole ratios for 10 min at room temperature. Glutaraldehyde
was then added to a final concentration of 0.5% for 20 min at room
temperature. The reaction was terminated by the addition of Tris
containing SDS-PAGE sample buffer. Samples were electrophoresed in
15% SDS-PAGE Tricine gels and stained with silver nitrate. MCP-3 was
chemically synthesized using solid phase methods, the polypeptide was
purified by reverse phase HPLC and folded using air oxidation.
13. B. Steffensen, U.M. Wallon, C.M. Overall, J. Biol. Chem. 270, 11555
(1995).
-22-
CA 02307705 2000-08-03
14. The enzyme capture film assay is a modification of an ELISA-based
binding assay. Proteins to be tested for binding were immobilized onto a
96-well plate. Following blocking by bovine serum albumin, enzyme
solutions were overlaid onto wells for 2 h at room temperature to allow
binding. After extensive washes to reduce non-specific interactions,
bound enzyme was recovered with SDS-PAGE sample buffer and
assayed for gelatinolytic activity by gelatin zymography. Recombinant
human progelatinase was expressed in CHO cells and purified by gelatin-
Sepharose chromatography. N-gelatinase A was produced by
autocatalytic degradation of recombinant full-length gelatinase A at 37
°C,
after activation by 1 mM 4-aminophenylmercuric acetate in the presence
of 1.0 % TX-100, and dialyzed for 16 h to remove the reactants.
15. Y. Itoh, M.S. Binner, H. Nagase, Biochem. J. 308, 645 (1995).
16. T.N. Young, S.V. Pizzo, S. Stack, J. Biol. Chem. 270, 999 (1995).
17. C.M. Overall et al., J. Biol. Chem. 274, 4421 (1999).
18. S. Netzel-Arnett et al., Biochemistry 32, 6427 (1993).
19. C.E. Hughes et al., J. Biol. Chem. 267, 16011 (1992).
20. J.H. Gong, I. Clark-Lewis, J. Exp. Med. 181, 631 (1995).
21. J.-H. Gong et al., J. Biol. Chem. 271, 10521 (1996).
22. THP-1 cells (myeloid cell line, ATCC) or B cells transfected with CCR-3
cDNA were loaded with Fluo-3AM for 30 min at 37 °C. After addition of
various full length chemokines or MCP-3(5-76) the fluorescence was
monitored with a Perkin-Elmer 650-10B spectrofluorimeter using an
excitation wavelength of 506 nm and an emission wavelength of 526 nm.
Desensitization assays were performed by sequential addition of MCP-
3(5-76) or buffer control, followed by the full length chemokine. THP-1 cell
migration was assessed in transwell trays (Costar) with 6.5 mm diameter
chambers of 3 Nm membrane pore size. MCP-3 and MCP-3(5-76) were
added to the lower well, and THP-1 cells (1 x 10' cells/ml) were added to
the upper well. After 1.5 h, cells that had migrated to the lower well were
counted. The percent migration was calculated by dividing the mean
number of migrating cells in response to chemokine by the mean number
of cells migrating in response to medium alone.
23. 4 nM ['251]-MCP-3(1-76) in the presence of serially diluted unlabeled MCP-
3(1-76) or MCP-3(5-76) and 0.05% NaN3 was incubated at 4 °C for 30 min
with THP-1 cells. Cell bound and free ['251]-MCP-3(1-76) were separated
by centrifugation of the cells through a column of dioctyl phthalate:n-butyl
phthalate (2:3, v/v). Amounts of bound ['251]-MCP-3(1-76) were
determined in the cell pellet by gamma counting. Nonspecific binding was
determined in the presence of a 100-fold concentration of unlabeled ligand
and was subtracted from the total. The data were analyzed by Scatchard
analysis.
24. CD-4 mice (5 per group) were injected at two subcutaneous sites (500
ng/100 NI pyrogen free saline) with either full-length MCP-3 [designated
MCP-3(1-76)], gelatinase A-cleaved MCP-3 [designated MCP-3(5-76)],
2:1 molar ratio of gelatinase A-cleaved MCP-3:MCP-3(1-76), or
-23-
CA 02307705 2000-08-03
saline/buffer control. In other experiments, 6 replicate mice per group
were injected as before, but with 100 NI MCP-3(1-76)/MCP-3(5-76)
mixtures as follows: 500 ng/0, 0/500 ng, 500 ng/500 ng, 500 ng/1000 ng,
500 ng/2500 ng, or saline. Mice were sacrificed 18 h post-injection and
paraffin sections transverse to the skin were analyzed. Sections were
stained with haematoxylin and eosin and examined by light microscopy.
Morphometric cell counts per 75,000 Nm2 field of mononuclear cell
infiltrates in the loose connective tissue immediately above the muscle
layer of skin were performed double blind and used to calculate the mean
and the standard error of the mean. Peritonitis was induced in mice using
zymosan A (1 mg/500 NI saline) injected in the peritoneal cavity. At 24 h
an intraperitoneal 5 ml saline lavage was performed to collect infiltrating
cells that increased ~40-fold compared to saline controls. In experiments,
50 Ng MCP-3(5-76) or saline was administered to the peritoneal cavity 24
h after the induction of peritonitis. Infiltrating cells were collected after
2
and 4 h by saline lavage. Cells were counted on a Coulter Counter gated
at 5-10 Nm and 100 NI cytospins were examined by light microscopy after
haematoxylin and eosin staining.
25. Peritoneal cells were stained for 60 min. on ice with 20 Ng/ml of rat anti-
mouse F4/80 mAb or rat IgG2b isotype control. After extensive washing,
cells were stained with FITC-conjugated anti-rat IgG for 45 min. on ice,
extensively washed, and analyzed by flow cytometry using a FACScan
analyzer (Becton Dickinson, U.K.).
26. S. Struyf et al., Eur. J. Immunol. 28, 1262 (1998).
27. J.E. Ehlert, J. Gerdes, H.D. Flad, E. Brandt, J. Immunol. 161, 4975
(1998).
28. P. Proost et al., J. Biol. Chem. 274, 3988 (1999).
29. A.J. Gearing et al., Nature 370, 555 (1994).
30. C.L. Wilson et al., Science 286, 113 (1999).
31. M. Baggiolini, B. Dewald, B. Moser, Annu. Rev. Immunol. 15, 675 (1997).
32. E.F. Foxman, J.J. Campbell, E.C. Butcher, J. CeIIBioI. 139, 1349 (1997).
33. B. Lu et al., J. Exp. Med. 187, 601 (1998).
34. R.G. Paul et al., Int. J. Biochem. Cell Biol. 29, 211 (1997).
35. G. Openakker, S. Masure, B. Grillet, J. Van Damme, Lymphokine
Cytokine Res. 10, 317 (1991 ).
36. I.E. Collier et al., J. Biol. Chem. 263, 6579 (1988).
37. J. Van Damme, P. Proost, J.P. Lenaerts, G. Opdenakker, J. Exp. Med.
176, 59 (1992).
-24-
