U.S. patent application number 10/662061 was filed with the patent office on 2005-09-29 for method of inhibiting stenosis and restenosis.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Horvath, Christopher J., Rao, Patricia E..
Application Number | 20050214299 10/662061 |
Document ID | / |
Family ID | 24104941 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050214299 |
Kind Code |
A1 |
Horvath, Christopher J. ; et
al. |
September 29, 2005 |
Method of inhibiting stenosis and restenosis
Abstract
The invention relates to a method of inhibiting stenosis or
restenosis in a subject. In one embodiment, an agent which inhibits
recruitment and/or adhesion of neutrophils and mononuclear cells to
a site of vascular injury is administered to a subject in need
thereof. In another embodiment, a first agent which inhibits
recruitment and/or adhesion of neutrophils to a site of vascular
injury, and a second agent which inhibits recruitment and/or
adhesion of mononuclear cells to a site of vascular injury are
administered to a subject in need thereof. In particular
embodiments, the agents are antibodies or antigen-binding fragments
thereof which bind to CD18 or CCR2.
Inventors: |
Horvath, Christopher J.;
(Taunton, MA) ; Rao, Patricia E.; (Acton,
MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
24104941 |
Appl. No.: |
10/662061 |
Filed: |
September 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10662061 |
Sep 12, 2003 |
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09809739 |
Mar 15, 2001 |
|
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6663863 |
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09809739 |
Mar 15, 2001 |
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09528267 |
Mar 17, 2000 |
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Current U.S.
Class: |
424/152.1 ;
424/144.1; 604/500 |
Current CPC
Class: |
A61P 9/14 20180101; A61M
25/104 20130101; A61P 9/10 20180101; A61K 2039/505 20130101; C07K
2317/76 20130101; C07K 16/2866 20130101; C07K 2319/00 20130101;
C07K 2317/24 20130101; C07K 16/2845 20130101 |
Class at
Publication: |
424/152.1 ;
424/144.1; 604/500 |
International
Class: |
A61K 039/395; A61M
031/00 |
Claims
1-48. (canceled)
49. A method of inhibiting stenosis in a human blood vessel, the
method comprising administering to the human an anti-CD18 antibody
which binds specifically with at least the CD18 portion of a
mammalian protein which comprises CD18, whereby stenosis is
inhibited in the vessel.
50. The method of claim 49, wherein the anti-CD18 antibody has an
epitopic specificity which is the same as or similar to that of
monoclonal antibody 1B4.
51. The method of claim 49, wherein the protein is a leukocyte
cell-surface antigen.
52. The method of claim 51, wherein the antigen is selected from
the group consisting of Mac-1, LFA-1, p150,95, and CD11d/CD18.
53. The method of claim 51, wherein binding of the anti-CD18
antibody with the antigen inhibits binding of a natural ligand of
the antigen therewith.
54. The method of claim 53, wherein the ligand is selected from the
group consisting of ICAM-1, ICAM-2, ICAM-3, C3bi, factor X, fibrin,
and fibrinogen.
55. The method of claim 49, wherein binding of the anti-CD18
antibody with the protein modulates at least one function normally
associated with binding of a natural ligand of the protein
therewith.
56. The method of claim 55, wherein the function is selected from
the group consisting of binding of leukocytes with vascular
endothelium, translocation of leukocytes through vascular
endothelium, infiltration of leukocytes into intimal vascular
tissue, release of a chemotactic factor from leukocytes in a
vascular tissue, release of a growth factor from leukocytes in a
vascular tissue, leukocyte-binding-associated release of a
chemotactic factor from a vascular tissue, and
leukocyte-binding-associ- ated release of a growth factor from a
vascular tissue.
57. The method of claim 49, wherein the blood vessel is a vessel in
which the vascular endothelium has been traumatically
perturbed.
58. The method of claim 49, wherein the blood vessel is a vessel in
which the vascular endothelium has non-traumatically
deteriorated.
59. The method of claim 49, wherein the anti-CD18 antibody is an
antibody fragment.
60. The method of claim 49, wherein the anti-CD18 antibody is a
chimeric antibody.
61. The method of claim 49, wherein the anti-CD18 antibody is a
humanized antibody.
62. The method of claim 49, wherein the anti-CD18 antibody is a
human antibody.
63. The method of claim 49, wherein the stenosis is restenosis
following an angioplastic intervention performed upon the
human.
64. A kit for assessing stenosis in a human blood vessel, the kit
comprising an anti-CD18 antibody having a detectable label and an
instructional material which describes detecting the anti-CD18
antibody in a blood vessel of the human.
65. The kit of claim 64, wherein the detectable label is a gamma
radiation source.
66. A method of inhibiting interaction of a leukocyte having a
CD18-containing cell-surface protein with vascular endothelium in a
human, the method comprising contacting the leukocyte with an
anti-CD18 antibody, whereby interaction of the leukocyte with
vascular endothelium is inhibited.
67. The method of claim 66, wherein the leukocyte is selected from
the group consisting of lymphocytes, monocytes, granulocytes,
neutrophils, T cells, and basophils.
68. A method of assessing the presence of leukocytes associated
with vascular stenosis in blood obtained from a human, the method
comprising contacting the blood with an anti-CD18 antibody and
detecting binding of the anti-CD18 antibody with leukocytes in the
blood, wherein binding of the anti-CD18 antibody with leukocytes in
the blood is an indication of the presence of leukocytes associated
with vascular stenosis in the blood.
69. The method of claim 68, wherein binding of the anti-CD18
antibody with leukocytes in the blood is quantified.
70. A method of alleviating a disorder associated with stenosis in
a blood vessel of a human, the method comprising administering to
the human an anti-CD18 antibody which binds specifically with at
least the CD18 portion of a mammalian protein which comprises CD18,
whereby stenosis is alleviated in the vessel and the disorder is
thereby alleviated.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/809,739, filed Mar. 15, 2001, which is a continuation-in-part of
application Ser. No. 09/528,267, filed on Mar. 17, 2000
(abandoned). The entire teachings of each of the foregoing
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] A stenosis is a stricture of a canal or duct. In the context
or the vascular system a stenosis is a narrowing of the lumen of a
blood vessel. A stenosis can severely restrict blood flow and
promote thrombosis which can lead to myocardial infarction or
stroke, for example. A common type of primary stenosis is
artherosclerotic plaque. Several therapeutic methods have been
developed to improve circulation and hemostasis in stenotic vessels
including by-pass surgery and revascularization procedures.
Revascularization procedures (e.g., balloon angioplasty,
atherectomy, rotorary ablation (rotoblation)) serve to improve
blood flow by reducing or removing the stenosis. However, these
procedures frequently injure the blood vessel. The biological
response to the injury is a multifactorial fibro-proliferative
process that is similar to wound healing, and includes the
elaboration of growth factors from a variety of cell types,
infiltration of leukocytes, migration and proliferation of smooth
muscle cells, the production of extracellular matrix and tissue
remodeling (Anderson, Vessels, 2:4-14 (1996)). The process can
result in the formation of a thick neointima within the vessel wall
which reduces the luminal area of the vessel (i.e., restenosis).
Restenosis occurs following about 20-50% of coronary angioplasty
procedures (Anderson, Vessels, 2:4-14 (1996)).
[0003] Attempts have been made at reducing restenosis following
vascular intervention procedures by, for example, administering
pharmacologic agents and placement of endovascular stents. However,
although stents are reported to partially reduce restenosis
(Serruys, et al., N. Engl. J. Med., 331:489-495 (1994)), restenosis
and in-stent restenosis remain a significant problem. Therefore, a
need exists for new methods for inhibiting stenosis and
restenosis.
SUMMARY OF THE INVENTION
[0004] The invention relates to a method of inhibiting stenosis or
restenosis of a blood vessel following vascular injury. In one
embodiment the method comprises administering to a subject in need
thereof, a therapeutically effective amount of a first therapeutic
agent which inhibits the adhesion and/or recruitment of neutrophils
to a site of vascular injury, and a therapeutically effective
amount of a second therapeutic agent which inhibits the adhesion
and/or recruitment of mononuclear cells to a site of vascular
injury. In a certain embodiment, the method is a method of
inhibiting stenosis or restenosis following vascular injury which
occurs during or is caused by a therapeutic or diagnostic vascular
intervention procedure (e.g., angiography, angioplasty, vascular
by-pass surgery, vascular grafting, endarterectomy, atherectomy,
endovascular stenting, insertion of prosthetic valve and
transplantation of organs, tissues or cells). The first and second
therapeutic agents can independently be an antagonist of a cellular
adhesion molecule or an antagonist of chemokine receptor function,
for example. In certain embodiments, the first therapeutic agent
binds to an integrin (e.g., a .beta.2 integrin) and inhibits
integrin-mediated cellular adhesion. Preferably, the first
therapeutic agent binds CD18 and inhibits binding of one or more
ligands (e.g., ICAM-1, ICAM-2, ICAM-3, fibrinogen, C3bi, Factor X)
to a CD18 containing integrin. In additional embodiments, the
second therapeutic agent is a chemokine receptor antagonist.
Preferably, the second therapeutic agent can bind CCR2 and inhibit
the binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4, MCP-5)
to the receptor. In preferred embodiments, the first and second
therapeutic agents are antibodies or antigen-binding fragments
thereof.
[0005] In a more particular embodiment, the method is a method of
inhibiting stenosis or restenosis in a subject following
percutaneous transluminal coronary angioplasty (PTCA). In another
particular embodiment, the method is a method of inhibiting
stenosis or restenosis in a subject following a vascular
intervention procedure which includes placement of a stent. In
another embodiment, the method of inhibiting stenosis or restenosis
in a subject following vascular injury comprises administering to a
subject in need thereof, an effective amount of an agent which
inhibits recruitment and/or adhesion of neutrophils and mononuclear
cells to a site of vascular injury.
[0006] The invention further relates to an agent that inhibits
recruitment and/or adhesion of neutrophils or mononuclear cells to
sites of vascular injury (e.g. cellular adhesion molecule
antagonists (e.g., anti-CD18 antibodies), antagonists of chemokine
receptor function (e.g., anti-CCR2 antibodies)) for use in therapy
(including prophylaxis) or diagnosis, for example, as described
herein, and to the use of such an antagonist for the manufacture of
a medicament for the inhibition of stenosis or restenosis. The
invention also relates to a medicament for the inhibition of
stenosis or restenosis (e.g., following a vascular intervention
procedure (e.g., angioplasty, percutanious transluminal coronary
angioplasty) wherein said medicament comprises an agent that
inhibits recruitment and/or adhesion of neutrophils or mononuclear
cells to sites of vascular injury (e.g. cellular adhesion molecules
antagonists (e.g., anti-CD18 antibody), antagonist of chemokine
receptor function (e.g., anti-CCR2 antibody)).
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1A is a graph showing the concentrations of mAb 1D9
(CCR2) or mAb S-S.1 (Control) detected in serum of animals treated
with mAb 1D9 or mAb S-S. 1 at predetermined time points.
[0008] FIG. 1B is a graph showing the concentrations of mAb 1B4
(CD18) or mAb S-S.1 (Control) detected in serum of animals treated
with mAb 1B4 or mAb S-S.1 at predetermined time points.
[0009] FIG. 2A is a graph showing the amount of unbound CCR2
present on the surface of monocytes of animals treated with nAb 1D9
over time. Free CCR2 was detected by staining blood cells with FITC
conjugated anti-mouse IgG or with mAb 1D9 and then with FITC
conjugated anti-mouse IgG. The mean channel fluorescence (MCF) was
determined for each sample by flow cytometry and the difference in
MCF, which indicates the degree to which CCR2 was not saturated,
was determined.
[0010] FIG. 2B is a graph showing the amount of unbound CD18
present on the surface of neutrophils of animals treated with mAb
1B4 over time. Free CD18 was detected by staining blood cells with
FITC conjugated anti-mouse IgG or with mAb 1B4 and then with FITC
conjugated anti-mouse IgG. The mean channel fluorescence (MCF) was
determined for each sample by flow cytometry and the difference in
MCF, which indicates the degree to which CD18 was not saturated,
was determined.
[0011] FIG. 2C is a graph showing the amount of unbound CD18
present on the surface of monocytes in animals treated with mAb 1B4
over time. Free CD18 was detected by staining blood cells with FITC
conjugated anti-mouse IgG or with mAb 1B4 and then with FITC
conjugated anti-mouse IgG. The mean channel fluorescence (MCF) was
determined for each sample by flow cytometry and the difference in
MCF, which indicates the degree to which CD18 was not saturated,
was determined.
[0012] FIG. 3A is a graph showing the total white blood cell count
in the peripheral blood in animals treated with mAb S-S.1 or mAb 1
D9 at predetermined time points.
[0013] FIG. 3B is a graph showing the total neutrophil cell count
in the peripheral blood in animals treated with mAb S-S. 1 or mAb 1
D9 at predetermined time points.
[0014] FIG. 3C is a graph showing the total lymphocyte cell count
in the peripheral blood in animals treated with mAb S-S. 1 or mAb
1D9 at predetermined time points.
[0015] FIG. 3D is a graph showing the total monocyte cell count in
the peripheral blood in animals treated with mAb S-S. 1 or mAb 1 D9
at predetermined time points.
[0016] FIG. 3E is a graph showing the total white blood cell count
in the peripheral blood in animals treated with mAb S-S.1 or mAb
1B4 at predetermined time points.
[0017] FIG. 3F is a graph showing the total neutrophil count in the
peripheral blood in animals treated with mAb S-S. 1 or mAb 1B4 at
predetermined time points.
[0018] FIG. 3G is a graph showing the total lymphocyte count in the
peripheral blood in animals treated with mAb S-S.1 or mAb 1B4 at
predetermined time points.
[0019] FIG. 3H is a graph showing the total monocyte cell count in
the peripheral blood in animals treated with mAb S-S.1 or mAb 1B4
at predetermined time points.
[0020] FIG. 4A is a graph showing the titer of anti-1D9 (1D9
(CCR2)) antibody or anti-S-S.1 antibody (control) in the serum of
animals treated with mAb 1 D9 or mAb S-S. 1.
[0021] FIG. 4B is a graph showing the titer of anti-1B4 (1B4
(CD18)) antibody or anti-S-S.1 antibody (control) in the serum of
animals treated with mAb 1B4 or mAb S-S. 1.
[0022] FIG. 5A is a graph showing the luminal diameter of iliac
arteries of animals treated with mAb 1D9 or mAb S-S.1 (control).
Measurements were taken before angioplasty (pre), at the time of
stenting (deploy), about 10 minutes after placement of the stent
(post) and 29 days after the procedure (followup).
[0023] FIG. 5B is a histogram showing the late luminal loss at the
site of angioplasty in animals treated with mAb S-S.1 (control) or
mAb 1D9.
[0024] FIG. 5C is a histogram showing the restenosis index (late
luminal loss (LLL)/actual luminal gain after stent deployment
(ALG)) in animals treated with mAb S-S.1 (control) or mAb 1D9.
[0025] FIG. 5D is a graph showing the luminal diameter of iliac
arteries of animals treated with mAb 1B4 or mAb S-S. 1 (control).
Measurements were taken before angioplasty (pre), at time of
stenting (deploy), about 10 minutes after placement of the stent
(post) and 29 days after the procedure (followup).
[0026] FIG. 5E is a histogram showing the late luminal loss at the
site of angioplasty in animals treated with mAb S-S. 1 (control) or
mAb 1B4.
[0027] FIG. 5F is a histogram showing the restenosis index (late
luminal loss (LLL)/actual luminal gain after stent deployment
(ALG)) in animals treated with mAb S-S.1 (control) or mAb 1B4.
[0028] FIG. 6A is a histogram showing the intimal area (mm.sup.2)
measured in cross sections of vessels injured by balloon only or by
balloon and stent in animals treated with mAb S-S.1 (control) or
mAb 1D9.
[0029] FIG. 6B is a histogram showing the intima:media ratio
calculated from measurements on cross sections of vessels injured
by balloon only or by balloon and stent in animals treated with mAb
S-S. 1 (control) or mAb 1D9.
[0030] FIG. 6C is a histogram showing the intimal area (mm.sup.2)
measured in cross sections of vessels injured by balloon only or by
balloon and stent in animals treated with mAb S-S. 1 (control) or
mAb 1B4.
[0031] FIG. 6D is a histogram showing the intima:media ratio
calculated from measurements on cross sections of vessels injured
by balloon only or by balloon and stent in animals treated with mAb
S-S. 1 (control) or mAb 1B4.
[0032] FIGS. 7A and 7B are photomicrographs of cross sections of
vessels that underwent balloon injury and stent deployment in
animals treated with mAb S-S.1 (control, FIG. 7A) or mAb 1D9 (FIG.
7B).
[0033] FIGS. 8A and 8B are photomicrographs of cross sections of
vessels that underwent balloon injury and stent deployment in
animals treated with mAb S-S. 1 (control, FIG. 8A) or mAb 1B4 (FIG.
8B).
[0034] FIG. 9 shows the amino acid sequence (SEQ ID NO:1) of rat
mAb YFC51.1 light chain variable region. The signal sequence
consists of residues 1-20.
[0035] FIG. 10 shows the amino acid sequences of complementarity
determining regions 1, 2 and 3 (CDR1 (SEQ ID NO:2), CDR2 (SEQ ID
NO:3) and CDR3 (SEQ ID NO:4)) of the light chain of rat mAb
YFC51.1.
[0036] FIG. 11 shows the amino acid sequence (SEQ ID NO:5) of rat
mAb YFC51.1 heavy chain variable region. The signal sequence
consists of residues 1-19.
[0037] FIG. 12 shows the amino acid sequence of CDR1 (SEQ ID NO:6),
CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO:8) of the heavy chain of
rat mAb YFC51.1.
[0038] FIG. 13 shows the amino acid sequence (SEQ ID NO:9) of the
heavy chain variable region of LDP-01, a humanized YFC51.1. The
signal sequence consists of amino acid residues 1-19.
[0039] FIG. 14 shows the amino acid sequence (SEQ ID NO:10) of the
light chain variable region of LDP-01, a humanized YFC51.1. The
signal sequence consists of residues 1-19.
[0040] FIG. 15 shows the amino acid sequence (SEQ ID NO:11) of the
light chain variable region of murine mAb 1D9. CDR 1 consists of
amino acid residues 24-39, CDR 2 consists of amino acid residues
55-61, CDR 3 consists of amino acid residues 94-102.
[0041] FIG. 16 shows the amino acid sequence (SEQ ID NO:12) of the
heavy chain variable region of murine mAb 1D9. CDR 1 consists of
amino acid residues 31-35, CDR 2 consists of amino acid residues
50-68, CDR 3 consists of amino acid residues 101-106.
[0042] FIG. 17 shows the amino acid sequences of the light chain
variable region (V.kappa.) of murine mAb 1D9 (SEQ ID NO:11), the
light chain variable region (V.kappa.) of human antibody HF-21/28
(SEQ ID NO:13) and the variable regions of several humanized 1D9
light chains (1D9RK.sub.A V.kappa., SEQ ID NO: 14; 1D9RK.sub.B
V.kappa., SEQ ID NO: 15; 1D9RK.sub.C V.kappa., SEQ ID NO: 16;
1D9RK.sub.D V.kappa., SEQ ID NO: 17; 1D9RK.sub.E V.kappa., SEQ ID
NO: 18). Where the amino acid residues of the murine 1D9 light
chain variable region (SEQ ID NO:11) and the human HF-21/28 light
chain variable region (SEQ ID NO: 13; Kabat database ID number
005056, and Chastagner et al., Gene. 101(2):305-6 (1991), the
teachings of both of which are incorporated herein by reference in
their entirety) sequences match, a dot [.] is shown. Where no amino
acid is present at a specific residue position a dash [-] is shown.
Where an amino acid in the HF-21/28 frame work region (FR) is
changed in a humanized 1D9 variable region, it is highlighted in
bold. The CDRs (CDR1, CDR2 and CDR3) are indicated by [==L1==],
[==L2==] and [==L3==]. The numbering used is according to Kabat et
al., Sequences of proteins of immunological interest, Fifth
edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991).
[0043] FIG. 18 shows the amino acid sequences of heavy chain
variable region (V.sub.H) of murine mAb 1D9 (SEQ ID NO: 12), the
heavy chain variable region of human antibody 4B4'CL (SEQ ID NO:
19; Kabat data base ID number 000490, and Sanz et al., Journal of
Immunology. 142:883 (1989), the teachings of both of which are
incorporated herein by reference in their entirety), and the
variable regions of several humanized 1D9 heavy chains (1D9RH.sub.A
V.sub.H, SEQ ID NO: 20; 1D9RH.sub.B V.sub.H, SEQ ID NO: 21;
1D9RH.sub.C V.sub.H, SEQ ID NO: 22; 1D9RH.sub.D V.sub.H, SEQ ID NO:
23). Where the amino acid residues of the murine 1D9 heavy chain
variable region (SEQ ID NO: 12) and the human 4B4'CL heavy chain
variable region (SEQ ID NO: 19) sequences match, a dot [.] is
shown. Where no amino acid is present at a specific residue
position a dash [-] is shown. Where an amino acid in the 4B4'CL
heavy chain variable region is changed in a humanized 1D9 heavy
chain variable region, it is highlighted in bold. The CDRs (CDR1,
CDR2 and CDR3) are indicated by [==H1==], [==H2==] and [==H3==],
while [----------] denotes part of the H1 structure loop. The
numbering used is according to Kabat et al., Sequences of proteins
of immunological interest, Fifth edition, U.S. Department of Health
and Human Services, U.S. Government Printing Office (1991).
DETAILED DESCRIPTION OF THE INVENTION
[0044] The invention relates to a method of inhibiting stenosis or
restenosis of a blood vessel following vascular injury, wherein the
recruitment and/or adhesion of neutrophils and the adhesion and/or
recruitment of mononuclear cells to a site of vascular injury is
inhibited. As used herein "mononuclear cell" refers to monocytes,
tissue macrophages and lymphocytes (e.g., T cells, B cells). Both
neutrophils and mononuclear cells play a role in the
pathophysiological response to vascular injury which leads to
stenosis or restenosis. However, these cells participate to varying
degrees in the process of vascular repair following different types
of vascular injury, for example, balloon injury or "deep injury"
produced by balloon angioplasty and placement of a stent.
[0045] As described herein a study in which the efficacy of murine
mAb 1D9 or murine mAb 1B4 (also referred to as mAb 1B4) in a model
of restenosis in Cynomolgus monkeys was conducted. Murine mAb 1D9
binds human and cynomolgus monkey CC-chemokine receptor 2 (CCR2)
and inhibits the binding of ligand (e.g., MCP-1, MCP-2, MCP-3,
MCP-4, MCP-5) to the receptor. CCR2 is expressed on mononuclear
cells (monocytes, activated T cells) and limited amounts on
basophils, but is not expressed on neutrophils. Murine mAb 1B4
binds human and cynomolgus monkey CD18, which is the common .beta.
chain component of members of the .beta.2 integrin family (e.g.,
CD11a/CD18 (LFA-1, .alpha..sub.1.beta..sub.2), CD11b/CD18 (Mac-1,
CR3, Mo1, .alpha..sub.M.beta..sub.2), CD11c/CD18 (p150,95,
.alpha..sub.X.beta..sub.2), CD11d/CD18). Murine mAb 1B4 can inhibit
the binding of ligands (e.g., ICAM-1) to .beta.2 integrins, and
thereby inhibit .beta.2 integrin mediated cellular adhesion. CD18
is expressed primarily on neutrophils and to a lesser extent on
mononuclear cells (monocytes and lymphocytes). Therefore, the study
of mAb 1D9, which can inhibit the recruitment and/or activation of
mononuclear cells to a site of vascular injury, and 1B4, which can
inhibit the recruitment and/or adhesion of neutrophils to a site of
vascular injury, in the model of restenosis provided an opportunity
to distinguish the pathological contribution of neutrophils and
mononuclear cells in vascular restenosis.
[0046] As described herein two types of vascular injury were
produced in iliac arteries of cynomolgus monkeys by performing
balloon angioplasty and deploying a stent in a portion of the area
where the balloon was inflated. Thus, segments of the artery were
injured by balloon only or by balloon plus stent. The results of
the study revealed that administration of anti-CCR2 mAb 1D9
inhibited neointimal hyperplasia within the segments of iliac
arteries injured by balloon plus stent, but not within the segment
injured by balloon only. In contrast, administration of anti-CD18
mAb 1B4 inhibited neointimal hyperplasia within segments of iliac
arteries injured by balloon plus stent and in segments injured by
balloon alone. The results of the study indicate that mononuclear
cells are important contributors to neointimal hyperplasia in
response to injury by balloon plus stent but not by balloon alone,
and that neutrophils provide an important (and perhaps predominant)
contribution to neointimal hyperplasia in response to both types of
injury.
[0047] The results of the study further indicate that simultaneous
inhibition of neutrophil and mononuclear cell participation in the
response to vascular injury or inhibition of neutrophil
participation followed by inhibition of mononuclear cell
participation can provide superior therapy for inhibiting stenosis
or restenosis following vascular injury. For example,
administration of an (i.e., one or more) agent which results in
inhibition of recruitment and/or adhesion of neutrophils and
mononuclear cells to a site of vascular injury can provide an
efficacious method of inhibiting stenosis or restenosis (e.g.,
in-stent restenosis).
[0048] In one aspect, the invention is a method of inhibiting
stenosis or restenosis following vascular injury comprising
administering to a subject in need thereof an effective amount of a
(i.e., one or more) suitable therapeutic agent which inhibits the
recruitment and/or adhesion of neutrophils and mononuclear cells to
a site of vascular injury.
[0049] Therapeutic Agents
[0050] Therapeutic agents which are suitable for administration in
accordance with the therapeutic methods described herein can
inhibit the recruitment and/or adhesion of neutrophils and/or
mononuclear cells to a site of vascular injury. Suitable
therapeutic agents can, for example, inhibit the activity (e.g.,
binding activity, signaling activity) of a cell surface molecule
through which cellular adhesion, chemotaxis and/or homing are
mediated. For example, antagonists of cellular adhesion molecules
(e.g., integrins (e.g., .beta.1, .beta.2, .beta.3, .beta.4,
.beta.5, .beta.6, .beta.7, .beta.8 integrins), selectins (e.g.,
E-selectin, P-selectin, L-selectin), cadherins (e.g., E-, P-,
N-cadherins) and immunoglobulin superfamily adhesion molecules
(e.g., LFA-2, LFA-3, CD44)) and antagonists of cytokine receptors
(e.g., antagonists of chemokine receptor function) can be used. In
addition, agents which bind to ligands of cellular adhesion
molecules or cytokines or chemokines and inhibit the binding of
ligand to receptors expressed on neutrophils and/or mononuclear
cells can be used.
[0051] As used herein, the term "cellular adhesion molecule
antagonist" refers to an agent (e.g., a molecule, a compound) which
can inhibit a function of a cellular adhesion molecule (e.g., a P2
integrin). For example, an antagonist of the .beta.2 integrin
CD11b/CD18 (Mac-1) can inhibit the binding of one or more ligands
(e.g., ICAM-1, fibrinogen, C3bi) to the integrin. Accordingly,
cellular adhesion mediated by integrin-ligand interactions can be
inhibited.
[0052] As used herein, the term "antagonist of chemokine receptor
function" refers to an agent (e.g., a molecule, a compound) which
can inhibit a (i.e., one or more) function of a chemokine receptor
(e.g., CC-chemokine receptor (e.g., CC-chemokine receptor 1 (CCR1),
CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9), CXC-chemokine
receptor (e.g., CXC-chemokine receptor 1 (IL-8R-1), CXCR2
(IL-8R-2), CXCR3, CXCR4), CX3C-chemokine receptor (e.g., CX3CR1)).
For example, an antagonist of CC-chemokine receptor 2 (CCR2)
function can inhibit the binding of one or more ligands (e.g.,
MCP-1, MCP-2, MCP-3, MCP-4, MCP-5) to CCR2 and/or inhibit signal
transduction mediated through CCR2 (e.g., GDP/GTP exchange by CCR2
associated G proteins, intracellular calcium flux). Accordingly,
CCR2-mediated processes and cellular responses (e.g.,
proliferation, migration, chemotactic responses, secretion or
degranulation) can be inhibited with an antagonist of CCR2
function. Preferred chemokine receptor antagonists for
administration in accordance with the method of the invention can
inhibit one or more functions of CCR2. As used herein,
"CC-chemokine receptor 2" ("CCR2") refers to CC-chemokine receptor
2a and/or CC-chemokine receptor 2b.
[0053] Preferably, the agent to be administered (e.g., cellular
adhesion molecule antagonist, antagonist of chemokine receptor
function) is a compound which is, for example, a small organic
molecule, natural product, protein (e.g., antibody, chemokine,
cytokine), peptide or peptidomimetic. Several types of molecules
that can be used to antagonize one or more functions of chemokine
receptors or cell adhesion molecules (e.g., integrins) are known in
the art, including small organic molecules, proteins, such as
antibodies (e.g., polyclonal sera, monoclonal, chimeric, humanized,
human) and antigen-binding fragments thereof (e.g., Fab, Fab',
F(ab').sub.2, Fv); and peptides.
[0054] Agents which can inhibit the recruitment and/or adhesion of
neutrophils and/or mononuclear cells to a site of vascular injury
can be identified, for example, by screening libraries or
collections of molecules, such as, the Chemical Repository of the
National Cancer Institute, as described herein or using other
suitable methods. Agents thus identified can be used in the
therapeutic methods described herein.
[0055] Another source of agents which can inhibit the recruitment
and/or adhesion of neutrophils and/or mononuclear cells to a site
of vascular injury (e.g., cellular adhesion molecule antagonist,
antagonist of chemokine receptor function) are combinatorial
libraries which can comprise many structurally distinct molecular
species. Combinatorial libraries can be used to identify lead
compounds or to optimize a previously identified lead. Such
libraries can be manufactured by well-known methods of
combinatorial chemistry and screened by suitable methods, such as
the methods described herein.
[0056] The term "natural product", as used herein, refers to a
compound which can be found in nature, for example, naturally
occurring metabolites of marine organisms (e.g., tunicates, algae),
plants or other organisms which possess biological activity, e.g.,
can antagonize chemokine receptor function. For example,
lactacystin, paclitaxel and cyclosporin A are natural products
which can be used as anti-proliferative or immunosuppressive
agents.
[0057] Natural products can be isolated and identified using
suitable methods. For example, a suitable biological source (e.g.,
vegetation) can be homogenized (e.g., by grinding) in a suitable
buffer and clarified by centrifugation, thereby producing an
extract. The resulting extract can be assayed for biological
activity, such as the capacity to antagonize a cellular adhesion
molecule or a chemokine receptor using, for example, the assays
described herein. Extracts which contain a desired activity can be
further processed to isolate active agent (e.g., cellular adhesion
molecule antagonist, antagonist of chemokine receptor function)
using suitable methods, such as, fractionation (e.g., column
chromatography (e.g., ion exchange, reverse phase, affinity), phase
partitioning, fractional crystallization) and assaying for
biological activity (e.g., antagonism of CCR2 activity). Once
isolated the structure of a natural product can be determined
(e.g., by nuclear magnetic resonance (NMR)) and those of skill in
the art can devise a synthetic scheme for synthesizing the natural
product. Thus, a natural product can be isolated (e.g.,
substantially purified) from nature or can be fully or partially
synthetic. A natural product can be modified (e.g., derivatized) to
optimize its therapeutic potential. Thus, the term "natural
product", as used herein, includes those compounds which are
produced using standard medicinal chemistry techniques to optimize
the therapeutic potential of a compound which can be isolated from
nature.
[0058] The term "peptide", as used herein, refers to a compound
consisting of from about two to about ninety amino acid residues
wherein the amino group of one amino acid is linked to the carboxyl
group of another amino acid by a peptide bond. A peptide can be,
for example, derived or removed from a native protein by enzymatic
or chemical cleavage, or can be prepared using conventional peptide
synthesis techniques (e.g., solid phase synthesis) or molecular
biology techniques (see Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1989)). A "peptide" can comprise any suitable L- and/or
D-amino acid, for example, common .alpha.-amino acids (e.g.,
alanine, glycine, valine), non-.alpha.-amino acids (e.g.,
.beta.-alanine, 4-aminobutyric acid, 6-aminocaproic acid,
sarcosine, statine), and unusual amino acids (e.g., citrulline,
homocitruline, homoserine, norleucine, norvaline, ornithine). The
amino, carboxyl and/or other functional groups on a peptide can be
free (e.g., unmodified) or protected with a suitable protecting
group. Suitable protecting groups for amino and carboxyl groups,
and methods for adding or removing protecting groups are known in
the art and are disclosed in, for example, Green and Wuts,
"Protecting Groups in Organic Synthesis", John Wiley and Sons,
1991. The functional groups of a peptide can also be derivatized
(e.g., alkylated) using art-known methods.
[0059] Peptides can be synthesized and assembled into libraries
comprising a few to many discrete molecular species. Such libraries
can be prepared using well-known methods of combinatorial
chemistry, and can be screened as described herein or using other
suitable methods to determine if the library comprises peptides
with a desired biological activity (e.g., cellular adhesion
molecule antagonist, antagonist of chemokine receptor function).
Such peptide antagonists can then be isolated using suitable
methods.
[0060] The term "peptidomimetic", as used herein, refers to
molecules which are not polypeptides, but which mimic aspects of
their structures. For example, polysaccharides can be prepared that
have the same functional groups as peptides. Peptidomimetics can be
designed, for example, by establishing the three dimensional
structure of a peptide agent in the environment in which it is
bound or will bind to a target molecule (e.g., cellular adhesion
molecule, chemokine receptor). The peptidomimetic comprises at
least two components, the binding moiety or moieties and the
backbone or supporting structure.
[0061] The binding moieties are the chemical atoms or groups which
will react or form a complex (e.g., through hydrophobic or ionic
interactions) with a target molecule (e.g., cellular adhesion
molecule, chemokine receptor), for example, with the amino acid(s)
at or near the ligand binding site. For example, the binding
moieties in a peptidomimetic can be the same as those in a peptide
antagonist of a cellular adhesion molecule (e.g., an integrin) or
chemokine receptor. The binding moieties can be an atom or chemical
group which reacts with the receptor in the same or similar manner
as the binding moiety in the peptide antagonist. For example, when
it is desirable to inhibit integrin-mediated adhesion a
peptidomimetic which resembles an RGD-containing peptide can be
prepared. Examples of binding moieties suitable for use in
designing a peptidomimetic for a basic amino acid in a peptide are
nitrogen containing groups, such as amines, ammoniums, guanidines
and amides or phosphoniums. Examples of binding moieties suitable
for use in designing a peptidomimetic for an acidic amino acid can
be, for example, carboxyl, lower alkyl carboxylic acid ester,
sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous
acid or ester thereof.
[0062] The supporting structure is the chemical entity that, when
bound to the binding moiety or moieties, provides the three
dimensional configuration of the peptidomimetic. The supporting
structure can be organic or inorganic. Examples of organic
supporting structures include polysaccharides, polymers or
oligomers of organic synthetic polymers (such as, polyvinyl alcohol
or polylactide). It is preferred that the supporting structure
possess substantially the same size and dimensions as the peptide
backbone or supporting structure. This can be determined by
calculating or measuring the size of the atoms and bonds of the
peptide and peptidomimetic. In one embodiment, the nitrogen of the
peptide bond can be substituted with oxygen or sulfur, thereby
forming a polyester backbone. In another embodiment, the carbonyl
can be substituted with a sulfonyl group or sulfinyl group, thereby
forming a polyamide (e.g., a polysulfonamide). Reverse amides of
the peptide can be made (e.g., substituting one or more --CONH--
groups for a --NHCO-- group). In yet another embodiment, the
peptide backbone can be substituted with a polysilane backbone.
[0063] These compounds can be manufactured by known methods. For
example, a polyester peptidomimetic can be prepared by substituting
a hydroxyl group for the corresponding .alpha.-amino group on amino
acids, thereby preparing a hydroxyacid and sequentially esterifying
the hydroxyacids, optionally blocking the basic and acidic side
chains to minimize side reactions. Determining an appropriate
chemical synthesis route can generally be readily identified upon
determining the chemical structure.
[0064] Peptidomimetics can be synthesized and assembled into
libraries comprising a few to many discrete molecular species. Such
libraries can be prepared using well-known methods of combinatorial
chemistry, and can be screened as described herein to determine if
the library comprises one or more peptidomimetics which antagonize
a cellular adhesion molecule or a chemokine receptor, for example.
Such peptidomimetic antagonists can then be isolated by suitable
methods.
[0065] In one embodiment, the agent (e.g., antagonist of chemokine
function, cell adhesion molecule antagonists) is an antibody or
antigen-binding fragment thereof. In certain embodiments, the
antibody or antigen binding fragment can have binding specificity
for an integrin (e.g., a .beta.2 integrin (e.g., CD11a/CD18 (LFA-1,
.beta..sub.1.beta..sub.2), CD11b/CD18 (Mac-1, CR3, Mo1,
.alpha..sub.M.beta..sub.2), CD11c/CD18 (p150,95,
.alpha..sub.X.beta.2), CD11d/CD18) or a chemokine receptor (e.g.,
CCR2). The antibody can be polyclonal or monoclonal, and the term
"antibody" is intended to encompass both polyclonal and monoclonal
antibodies. The terms polyclonal and monoclonal refer to the degree
of homogeneity of an antibody preparation, and are not intended to
be limited to particular methods of production. The term "antibody"
as used herein also encompasses functional fragments of antibodies,
including fragments of human, chimeric, humanized, primatized,
veneered or single chain antibodies. Functional fragments include
antigen-binding fragments which bind to a .beta.2 integrin or
chemokine receptor, for example. For example, antibody fragments
capable of binding to CCR2 or portions thereof, including, but not
limited to Fv, Fab, Fab' and F(ab').sub.2 fragments can be
administered in accordance with the therapeutic methods of the
invention. Such fragments can be produced by enzymatic cleavage or
by recombinant techniques. For example, papain or pepsin cleavage
can generate Fab or F(ab').sub.2 fragments, respectively. Other
proteases with the requisite substrate specificity can also be used
to generate Fab or F(ab').sub.2 fragments. Antibodies can also be
produced in a variety of truncated forms using antibody genes in
which one or more stop codons has been introduced upstream of the
natural stop site. For example, a chimeric gene encoding a
F(ab').sub.2 heavy chain portion can be designed to include DNA
sequences encoding the CH.sub.1 domain and hinge region of the
heavy chain. Single chain antibodies, and human, chimeric,
humanized or primatized (CDR-grafted), or veneered antibodies, as
well as chimeric, CDR-grafted or veneered single chain antibodies,
comprising portions derived from different species, and the like
are also encompassed by the present invention and the term
"antibody". The various portions of these antibodies can be joined
together chemically by conventional techniques, or can be prepared
as a contiguous protein using genetic engineering techniques. For
example, nucleic acids encoding a chimeric or humanized chain can
be expressed to produce a contiguous protein. See, e.g., Cabilly et
al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,
European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO
86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276
B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No.
0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; and
Padlan, E. A. et al., EP 0 519 596 A1. See also, Newman, R. et al.,
BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody,
and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al.,
Science, 242: 423-426 (1988)) regarding single chain
antibodies.
[0066] Humanized antibodies can be produced using synthetic or
recombinant DNA technology using standard methods or other suitable
techniques. Nucleic acid (e.g., cDNA) sequences coding for
humanized variable regions can also be constructed using PCR
mutagenesis methods to alter DNA sequences encoding a human or
humanized chain, such as a DNA template from a previously humanized
variable region (see e.g., Kamman, M., et al., Nucl. Acids Res.,
17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856
(1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):
2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101:
297-302 (1991)). Using these or other suitable methods, variants
can also be readily produced. In one embodiment, cloned variable
regions can be mutated, and sequences encoding variants with the
desired specificity can be selected (e.g., from a phage library;
see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et
al., WO 93/06213, published Apr. 1, 1993). As used herein, an
antigen-binding fragment of a humanized immunoglobulin heavy or
light chain is intended to mean a fragment which binds to an
antigen when paired with a complementary chain. That is, an
antigen-binding fragment of a humanized light chain will bind to an
antigen when paired with a heavy chain (e.g., murine, chimeric,
humanized) comprising a variable region, and an antigen-binding
fragment of a humanized heavy chain will bind to an antigen when
paired with a light chain (e.g., murine, chimeric, humanized)
comprising a variable region. Antibodies (e.g., human, humanized
and chimeric antibodies) can comprise a constant region (e.g., a
human constant region) derived from the K or A light chains, and/or
the .gamma. (e.g., .gamma.1, .gamma.2, .gamma.3, .gamma.4), .mu.,
.alpha. (e.g., .alpha.1, .alpha.2), .delta. or .epsilon. heavy
chains of human antibodies, including allelic variants. A
particular constant region (e.g., IgG1), variant or portions
thereof can be selected in order to tailor effector function. For
example, an mutated constant region (variant) can be incorporated
into a fusion protein to minimize binding to Fc receptors and/or
ability to fix complement (see e.g., see e.g., Winter et al., WO
88/07089 (published Sep. 22, 1988), GB 2,209,757 B, U.S. Pat. No.
5,624,821, and U.S. Pat. No. 5,648,260; Morrison et al., WO
89/07142; Morgan et al., WO 94/29351 (published Dec. 22,
1994)).
[0067] Antibodies which specifically bind to a desired mammalian
(e.g., human) protein (e.g., cell adhesion protein, chemokine
receptor) can be raised against an appropriate immunogen, such as
isolated and/or recombinant human CCR2 or portions thereof
(including synthetic molecules, such as synthetic peptides).
Antibodies which specifically bind a desired protein can also be
raised by immunizing a suitable host (e.g., mouse) with cells that
naturally expresses said protein. (see e.g., U.S. Pat. No.
5,440,020, the entire teachings of which are incorporated herein by
reference). In addition, cells expressing recombinant protein such
as transfected cells, can be used as immunogens or in a screen for
antibody which binds said protein (See e.g., Chuntharapai et al.,
J. Immunol., 152: 1783-1789 (1994); Chuntharapai et al., U.S. Pat.
No. 5,440,021).
[0068] Preparation of immunizing antigen, and polyclonal and
monoclonal antibody production can be performed using any suitable
technique. A variety of methods have been described (see e.g.,
Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:
511-519 (1976); Milstein et al., Nature 266: 550-552 (1977);
Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane,
1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In
Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.
M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter
11, (1991)). When monoclonal antibodies are desired, a hybridoma is
generally produced by fusing a suitable immortal cell line (e.g., a
myeloma cell line such as SP2/0 or P3X63Ag8.653) with antibody
producing cells. The antibody producing cells, preferably those
obtained from the spleen or lymph nodes, can be obtained from
animals immunized with the antigen of interest. The fused cells
(hybridomas) can be isolated using selective culture conditions,
and cloned by limiting dilution. Cells which produce antibodies
with the desired specificity can be selected by a suitable assay
(e.g., ELISA).
[0069] Other suitable methods of producing or isolating antibodies
of the requisite specificity can be used, including, for example,
methods which select recombinant antibody from a library (e.g., a
phage display library). Transgenic animals capable of producing a
repertoire of human antibodies (e.g., Xenomouse.RTM. (Abgenix,
Fremont, Calif.)) can be produced using suitable methods (see e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555
(1993); Jakobovits et al., Nature, 362: 255-258 (1993); Lonberg et
al., U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No.
5,545,807; Lonberg et al., WO97/13852).
[0070] In a preferred embodiment, the antibody or antigen-binding
fragment thereof has specificity for a mammalian CD18 (e.g., human
CD18), the common .beta. chain of the .beta.2 integrins, and can
inhibit cellular adhesion mediated through binding of a ligand
(i.e., one or more ligands (e.g., ICAM-1, ICAM-2, fibrinogen)) to a
.beta.2 integrin. Antibodies which bind CD18 and inhibit
CD18-mediated cellular adhesion include, for example, humanized mAb
1B4 (also referred to as humanized mAb 1B4) (EP 0 438 312 A2), mAb
60.3 (Kling, D. et al., Arterioscler. Thromb. 12:997-1007 (1992)),
mAb R15.7 (Guszman, L. A., et al. Coronary Artery Dis., 6:693-701
(1995); Golino, P. et al. Thromb. Haemost., 77:783-788 (1997)), rat
mAb YFC51.1, LDP-01 a humanized YFC51.1 (U.S. Pat. Nos. 5,985,279
and 5,997,867, the entire teachings of each of the preceding U.S.
patents are incorporated herein by reference). Other antibodies
which can be administered in accordance with the invention include
antibodies which bind to Mac-1 and inhibit Mac-1 mediated cellular
adhesion, for example mAb M1/70 (Rogers, C. et al., Proc. Natl.
Acad. Sci., U.S.A., 95:10134-10139 (1998)) and mAb 7E3 or c7E3 Fab
(Simon, D. I. et al., Arterioscler Thromb Vasc Biol., 17:528-535
(1997)).
[0071] Other preferred antibodies bind mammalian CCR2 (e.g., human
CCR2) and inhibit the binding of a ligand (e.g., MCP-1, MCP-2,
MCP-3, MCP-4, MCP-5) to the receptor. Murine monoclonal antibodies
designated 1D9 (also referred to as LS132.1D9 or 1D9-2-121-3-6) and
8G2 (also referred to as LS132.8G2), which bind CCR2 and inhibit
the binding of ligand to the receptor, were produced as described
herein. Hybridoma cell lines producing the antibodies were
deposited on Jul. 17, 1998, on behalf of LeukoSite, Inc., 215 First
Street, Cambridge, Mass. 02142, U.S.A., (now Millennium
Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, Mass. 02139,
U.S.A.) at the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110, U.S.A., under Accession Nos.
HB-12549 (1D9) and HB-12550 (8G2). These antibodies and, for
example, chimeric or humanized version of the antibodies can be
administered in accordance with the method of the invention. An
antibody which binds CCR2 and inhibits the binding of a ligand
(e.g., MCP-1, MCP-2, MCP-3, MCP-4, MCP-5) to the receptor can
comprise a humanized 1D9 light chain comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or a
humanized 1D9 heavy chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21,
SEQ ID NO: 22, and SEQ ID NO: 23. In certain embodiments, an
antibody which binds CCR2 and inhibits the binding of a ligand to
the receptor can comprise a humanized chain (e.g., a humanized 1D9
light chain comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18, or a humanized 1D9 heavy chain
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ
ID NO: 23) and a complementary chain (heavy or light as
appropriate) which is, for example, human, nonhuman (e.g., rodent
(e.g., murine), primate) humanized or chimeric. A complementary
light or heavy chain is one which is capable of associating with a
selected heavy or light chain, respectively, resulting in an
antibody or antigen-binding fragment which binds CCR2 and inhibits
the binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4, MCP-5)
to the receptor. Antigen-binding fragments of such antibodies
(e.g., Fab fragments, F(ab').sub.2 fragments, Fab' fragments, Fv
fragments) can also be administered in accordance with the method
of the invention.
[0072] In certain embodiments, a humanized antibody which binds
CCR2 and inhibits the binding of a ligand (e.g., MCP-1, MCP-2,
MCP-3, MCP-4, MCP-5) to the receptor is administered. In particular
embodiments, the humanized antibody can comprise a light chain
comprising the amino acid sequence of SEQ ID NO: 14 and a heavy
chain comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ
ID NO: 23. In other embodiments, the humanized antibody can
comprise a light chain comprising the amino acid sequence of SEQ ID
NO: 15 and a heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID
NO: 22 and SEQ ID NO: 23. In other embodiments, the humanized
antibody which binds CCR2 and inhibits the binding of a ligand to
the receptor can comprise a light chain comprising the amino acid
sequence of SEQ ID NO: 16 and a heavy chain comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: 20,
SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23. In other
embodiments, the humanized antibody can comprise a light chain
comprising the amino acid sequence of SEQ ID NO: 17 and a heavy
chain comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ
ID NO: 23. In further embodiments, the humanized antibody can
comprise a light chain comprising the amino acid sequence of SEQ ID
NO: 18 and a heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID
NO: 22 and SEQ ID NO: 23.
[0073] In additional embodiments, the humanized antibody which
binds CCR2 and inhibits the binding of a ligand (e.g., MCP-1,
MCP-2, MCP-3, MCP-4, MCP-5) to the receptor can comprise a heavy
chain comprising the amino acid sequence of SEQ ID NO: 20 and a
light chain comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18. In other embodiments, the
humanized antibody can comprise a heavy chain comprising the amino
acid sequence of SEQ ID NO: 21 and a light chain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:
18. In other embodiments, the humanized antibody can comprise a
heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and
a light chain comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18. In further embodiments, the
humanized antibody which binds CCR2 and inhibits the binding of a
ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4, MCP-5) to the receptor
can comprise a heavy chain comprising the amino acid sequence of
SEQ ID NO: 23 and a light chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
[0074] In additional embodiments, the antibody which binds CCR2 and
inhibits the binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4,
MCP-5) to the receptor can comprise a light chain comprising the
variable region of murine antibody 1D9 (SEQ ID NO:11) and a
complementary heavy chain, for example, a heavy chain comprising a
variable region having an amino acid sequence selected from the
group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and
SEQ ID NO: 23. In further embodiments, the antibody which binds
CCR2 and inhibits the binding of a ligand (e.g., MCP-1, MCP-2,
MCP-3, MCP-4, MCP-5) to the receptor can comprise a heavy chain
comprising the variable region of murine antibody 1D9 (SEQ ID
NO:12) and a complementary light chain, for example, a light chain
comprising a variable region having an amino acid sequence selected
from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
[0075] A preferred antibody or antigen-binding fragment thereof
that can be administered to inhibit stenosis or restenosis in
accordance with the invention can be a humanized 1D9 antibody or
antigen binding fragment thereof, comprising a light chain
comprising the amino acid sequence of SEQ ID NO: 14 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 20.
[0076] Additional antibodies including human, humanized and
chimeric antibodies and the like, having binding specificity for a
cellular adhesion molecule (e.g., integrin (e.g., CD18), selectin,
cadherin, immunoglobulin adhesion molecule) or chemokine receptor
(e.g., CCR2) can be prepared using the methods described herein or
other suitable methods.
[0077] Assessment of Activity of Agents
[0078] The activity of agents (e.g., cell adhesion molecule
antagonists, chemokine receptor antagonists) can be assessed using
any suitable assay. For example, antagonists of chemokine receptor
function can be identified in a suitable binding or chemotaxis
assay. In one example, antagonists of CCR2 function can be
identified in a competitive binding assay where a reduction in the
binding of a ligand of CCR2 (in the presence of an agent (e.g.,
antibody)), as compared to binding of the ligand in the absence of
the agent, is detected or measured. A composition comprising an
isolated and/or recombinant mammalian CCR2 or functional variant
thereof can be contacted with the ligand and agent simultaneously,
or one after the other, in either order. A reduction in the extent
of binding of the ligand in the presence of the antibody, is
indicative of inhibition of binding by the antibody. For example,
binding of the ligand could be decreased or abolished.
[0079] Direct inhibition of the binding of a ligand (e.g., a
chemokine such as MCP-1) to a mammalian CCR2 or ligand-binding
variant thereof by an agent (e.g., antibody) can be monitored. For
example, the ability of an agent to inhibit the binding of
.sup.125I-labeled MCP-1, .sup.125I-labeled MCP-2, .sup.125I-labeled
MCP-3 or .sup.125I-labeled MCP-4 to mammalian CCR2 can be
monitored. Such an assay can be conducted using suitable cells
bearing CCR2 or a ligand-binding variant thereof, such as isolated
blood cells (e.g., T cells, PBMC) or a suitable cell line naturally
expressing CCR2, or a cell line containing nucleic acid encoding a
mammalian CCR2 (e.g., a cell line expressing recombinant CCR2), or
a membrane fraction from said cells, for instance.
[0080] Other methods of identifying antagonists of CCR2 function
are available, such as other suitable binding assays, or methods
which monitor events which are triggered upon binding of ligand to
receptor, including signaling function and/or stimulation of a
cellular response (e.g., leukocyte trafficking, leukocyte
chemotaxis). It will be understood that the agents which inhibit
other cytokine receptors (e.g., other chemokine receptors), can be
identified by suitable modification of the described assays. For
example, agents which antagonize CC-chemokine receptor 1 (CCR1) can
be identified in assays using a composition comprising CCR1 such as
THP-1 cell membranes and a labeled CCR1 ligand (e.g., RANTES).
[0081] Cell adhesion molecule antagonists can be identified using a
suitable binding assay. For example, cellular adherence can be
monitored by methods known in the art or other suitable methods. In
one suitable assay, an agent to be tested can be combined with (a)
non adherent cells which express a cellular adhesion molecule
(e.g., an integrin), and (b) a composition comprising a ligand
(e.g., a substrate such as a culture well coated with a ligand, a
culture well containing adherent cells which express a ligand of
the cellular adhesion molecule), and maintained under conditions
suitable for ligand-receptor mediated adhesion. Labeling of cells
with a fluorescent dye provides a convenient means of detecting
adherent cells. Nonadherent cells can be removed (e.g., by washing)
and the number of adherent cells determined. A reduction in the
number of adherent cells in wells containing a test agent (e.g.,
antibody) in comparison to suitable control wells (e.g., wells that
do not contain a test agent) indicates that the agent is an
antagonist of the cellular adhesion molecule.
[0082] Therapeutic Methods
[0083] The invention provides a method of inhibiting (e.g.,
reducing the severity of or preventing) stenosis or restenosis
following a vascular injury in a subject, such as a human. The
injury can occur during and/or be caused by a diagnostic or
therapeutic vascular intervention procedure, such as, angiography,
angioplasty (e.g., performed by balloon, atherectomy, laser
angioplasty or other suitable methods (with or without rotablation
and/or stent placement)), endarterectomy, coronary artery by-pass
surgery, stent placement (e.g., endovascular stent, coronary
stent), and/or other vascular intervention procedures (e.g.,
vascular surgery, vascular graft, deployment of a peripheral stent,
insertion of a prosthetic valve or vessel (e.g., in autologous,
non-autologous or synthetic vessel graft), transplantation of
organs, tissues or cells, intravascular brachytherapy). In a
particular aspect, the method can be used to inhibit stenosis or
restenosis following a coronary artery intervention procedure, such
as percutaneous transluminal coronary angioplasty (PTCA), or a
vascular intervention procedure which includes placement of a stent
(e.g., PTCA plus endovascular stent placement).
[0084] In one aspect, the method of inhibiting stenosis or
restenosis following a vascular injury comprises administering to a
subject in need thereof an effective amount of an (i.e., one or
more) agent which inhibits the recruitment and/or adhesion of
neutrophils or mononuclear cells to a site of vascular injury. The
method includes therapeutic or prophylactic treatment. According to
the method, the stenosis or restenosis can be prevented or reduced
(inhibited) in whole or in part.
[0085] In one embodiment, a single agent which inhibits the
recruitment and/or adhesion of neutrophils or mononuclear cells to
a site of vascular injury is administered. The agent can be, for
example, an antibody which binds to a cellular adhesion molecule
and thereby prevents adhesion of neutrophils and mononuclear cells
to a site of vascular injury. In particular embodiments, the agent
is an antibody which binds to an integrin (e.g., .beta.2 integrin)
and inhibits integrin mediated adhesion. In other embodiments, the
agent is an antibody which binds to a chemokine receptor (e.g.,
CCR2) and inhibits binding of ligand (e.g., MCP-1, MCP-2, MCP-3,
MCP-4, MCP-5) to the receptor, thereby inhibiting recruitment
and/or adhesion of neutrophils and mononuclear cells to a site of
vascular injury.
[0086] In a preferred aspect, the method of inhibiting stenosis or
restenosis following vascular injury in a subject in need thereof,
comprises administering to the subject a first agent which inhibits
the adhesion and/or recruitment of neutrophils to a site of
vascular injury, and a second agent which inhibits adhesion and/or
recruitment of mononuclear cells to a site of vascular injury. In
certain embodiments, the first agent is a cellular adhesion
molecule antagonist. In particular embodiments, the first agent can
inhibit integrin-mediated adhesion of neutrophils to a site of
vascular injury. In more particular embodiments, the first agent
can inhibit P2 integrin-mediated neutrophil adhesion to a site
vascular injury. For example, the first agent can inhibit
neutrophil adhesion mediated by CD11a/CD18 (LFA-1,
.alpha..sub.1.beta..sub.2), CD11b/CD18 (Mac-1, CR3, Mol,
.alpha..sub.M.beta..sub.2), CD11c/CD18 (p150,95,
.alpha..sub.x.beta..sub.2) and/or CD11d/CD18. In a preferred
embodiment the first agent is an antibody which binds CD18 and
thereby inhibits .beta.2-integrin-mediated adhesion of neutrophils
to a site of vascular injury. Preferred anti-CD18 antibodies for
administration to humans include humanized YFC51.1 antibodies (see
U.S. Pat. Nos. 5,985,279 and 5,997,867), such as LDP-01 (humanized
YFC51.1 which comprises a human .gamma.1 heavy chain constant
region having two mutations (Leu.sup.235.fwdarw.Ala.sup.235 and
Gly.sup.237.fwdarw.Ala.sup.237) which reduce binding to Fc.gamma.
receptors).
[0087] The second agent administered in accordance with the method
can be a cell adhesion molecule antagonist, such as a peptide,
small molecule or antibody which inhibits the adhesion of
mononuclear cells to sites of vascular injury. The second agent can
also be an antagonist of chemokine receptor function. In certain
embodiments, the second agent is an antagonist of a CC-chemokine
receptor. In particular embodiments, the second agent is an
antagonist of CC-chemokine receptor 2 (CCR2). Preferred antagonists
of chemokine receptor function include small organic molecules and
antibodies or antigen-binding fragments thereof that bind CCR2 and
inhibit the binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4,
MCP-5) to the receptor. The murine monoclonal antibodies designated
1D9 and 8G2, and humanized, human or chimeric antibodies which have
the same or similar epitopic specificity as mAb 1D9 or mAb 8G2 or
which bind to human CCR2 and inhibit the binding of a ligand to the
receptor are particularly preferred.
[0088] The methods described herein can also be used to treat a
subject having an inflammatory disease or condition mediated by
early neutrophil activity and later mononuclear cell activity. For
example the methods described herein can be used to treat a subject
having mastitis (mammary gland), vaginitis, cholecystitis,
cholangitis or pericholangitis (bile duct and surrounding tissue of
the liver), chronic bronchitis, chronic sinusitis, asthma, and
graft versus host disease (e.g., in the gastrointestinal tract).
Chronic inflammatory diseases of the lung which result in
interstitial fibrosis, such as interstitial lung diseases (ILD)
(e.g., chronic obstructive pulmonary disease, idiopathic pulmonary
fibrosis, or ILD associated with rheumatoid arthritis, or other
autoimmune conditions), hypersensitivity pneumonitis, collagen
diseases, sarcoidosis, and other idiopathic conditions can be
amenable to treatment. Pancreatitis and insulin-dependent diabetes
mellitus are other diseases which can be treated using the present
method.
[0089] The methods of the invention can also be used to treat
inflammatory bowel disease (IBD), such as ulcerative colitis,
Crohn's disease, ileitis, Celiac disease, nontropical Sprue,
enteritis, enteropathy associated with seronegative arthropathies,
microscopic or collagenous colitis, eosinophilic gastroenteritis,
or pouchitis resulting after proctocolectomy, and ileoanal
anastomosis.
[0090] Additional diseases or conditions, including chronic
diseases, of humans or other species which can be treated in
accordance with the method of the invention, include, but are not
limited to:
[0091] inflammatory or allergic diseases and conditions, including
systemic anaphylaxis or hypersensitivity responses, drug allergies
(e.g., to penicillin, cephalosporins), insect sting allergies;
psoriasis and inflammatory dermatoses such as dermatitis, eczema,
atopic dermatitis, allergic contact dermatitis, urticaria;
vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity
vasculitis); spondyloarthropathies; scleroderma; respiratory
allergic diseases such as asthma, allergic rhinitis;
[0092] autoimmune diseases, such as arthritis (e.g., rheumatoid
arthritis, psoriatic arthritis), multiple sclerosis, systemic lupus
erythematosus, myasthenia gravis, juvenile onset diabetes,
glomerulonephritis and other nephritides, autoimmune thyroiditis,
Behcet's disease;
[0093] graft rejection (e.g., in transplantation), including
allograft rejection or graft-versus-host disease;
[0094] other diseases or conditions in which undesirable
inflammatory responses are to be inhibited can be treated,
including, but not limited to, atherosclerosis, restenosis,
myositis (including polymyositis, dermatomyositis).
[0095] Modes of Administration
[0096] A "subject" is preferably a human, but can also be a mammal
in need of veterinary treatment, e.g., domestic animals (e.g.,
dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl,
pigs, horses, and the like) and laboratory animals (e.g., rats,
mice, guinea pigs, and the like).
[0097] An "effective amount" of an agent (e.g., antagonist of
chemokine receptor (e.g., CCR2) function, cellular adhesion
molecule (e.g., .beta.2 integrin) antagonist) is an amount
sufficient to achieve a desired therapeutic and/or prophylactic
effect, such as an amount sufficient to inhibit (i.e., reduce or
prevent) recruitment and/or adhesion of neutrophils and/or
mononuclear cells to sites of vascular injury, and thereby inhibit
stenosis or restenosis. For example, an effective amount of a
cellular adhesion molecule antagonist is an amount sufficient to
inhibit binding of neutrophils and/or mononuclear cells to a site
of vascular injury. An effective amount of an antagonist of
chemokine receptor (e.g., CCR2) function is an amount sufficient to
inhibit a (i.e., one or more) function of the receptor (e.g.,
ligand-induced cellular migration, ligand-induced integrin
activation, ligand-induced transient increase in the concentration
of intracellular free calcium [Ca.sup.2+].sub.i and/or
ligand-induced secretion (e.g. degranulation) of proinflammatory
mediators), and thereby inhibit recruitment and/or adhesion of
neutrophils and/or mononuclear cells to a site of vascular
injury.
[0098] If desired, the agent(s) which inhibit recruitment and/or
activation of neutrophils and/or mononuclear cells to a site of
vascular injury can be co-administered with one or more addition
therapeutic agents, for example, a fibrinolytic agent (e.g.,
Retavase), a thrombolytic agent, such as a plasminogen activator
(e.g., tissue plasminogen activator, urokinase, streptokinase,
recombinant plasminogen activator), anticoagulant (e.g., heparin,
hirulog, hirudin, aspirin), or a coumarin anticoagulant (e.g.,
warfarin, ethyledine dicoumarol), a .beta.-adrenergic blocker
(e.g., alprenolol, acebutolol, propanolol), calcium channel blocker
(e.g., nifedipine, diltiazem, cinnarizine, bencyclane), gpIIb/IIIa
antagonists (e.g., c7E3 Fab (ReoPro.RTM., abciximab, Centocor,
Inc., Malvern, Pa.)), vasodilator (e.g., nitroglycerin,
amotriphene, erythritol, prenylamine) or an agent which stimulates
the production of nitric oxide (see, for example, Singh et al.,
U.S. Pat. No. 5,811,437).
[0099] The amount of agent (e.g., cellular adhesion molecule
antagonist, antagonist of chemokine receptor function, additional
therapeutic agent) administered to the individual will depend on
the characteristics of the individual, such as general health, age,
sex, body weight and tolerance to drugs as well as the degree,
severity and type of vascular injury and desired therapeutic
effect. The skilled artisan will be able to determine appropriate
dosages which can be dependent on these and other factors.
Typically, an effective amount can range from about 0.01 mg per day
to about 100 mg per day for an adult. Preferably, the dosage ranges
from about 1 mg per day to about 100 mg per day or from about 1 mg
per day to about 10 mg per day. Antibodies and antigen-binding
fragments thereof, particularly human, humanized and chimeric
antibodies and antigen-binding fragments thereof can often be
administered with less frequency than other types of therapeutics.
For example, an effective amount of an antibody or antigen-binding
fragment thereof can range from about 0.01 mg/kg to about 5 or 10
mg/kg administered daily, weekly, biweekly or monthly.
[0100] The agent (e.g., cellular adhesion molecule antagonist,
antagonist of chemokine receptor function, additional therapeutic
agent) can be administered by any suitable route, including, for
example, orally (e.g., in capsules, suspensions or tablets) or by
parenteral administration. Parenteral administration can include,
for example, intramuscular, intravenous, intraarterial,
intraarticular, intrathecal, subcutaneous, or intraperitoneal
administration. The agent (e.g., cellular adhesion molecule
antagonist, antagonist of chemokine receptor function, additional
therapeutic agent) can also be administered orally (e.g., dietary),
transdermally, topically, by inhalation (e.g., intrabronchial,
intranasal, oral inhalation or intranasal drops) or rectally.
Administration can be local (e.g., at the site of vascular injury)
or systemic as indicated. The agent can be administered in a single
dose, continuous infusion, or in multiple doses and/or infusions
(e.g., a bolus dose followed by continuous infusion). The preferred
mode of administration can vary depending upon the particular agent
(e.g., cellular adhesion molecule antagonist, antagonist of
chemokine receptor function, additional therapeutic agent) chosen,
however, oral or parenteral administration is generally
preferred.
[0101] Preferably, the timing of administration of an effective
amount of the agent(s) is selected to provide for inhibition of
recruitment and/or activation of neutrophils and mononuclear cells
at the time of vascular injury. It is also preferred that the agent
which inhibits neutrophil recruitment and/or adhesion be
administered in an amount and with a frequence which is sufficient
to inhibit neutrophil recruitment and/or adhesion to a site of
vascular injury for about one week following vascular injury. The
agent which inhibits mononuclear cell recruitment and/or adhesion
is preferably administered in an amount and with a frequency which
is sufficient to inhibit mononuclear cell recruitment and/or
adhesion to a site of vascular injury for a period of at least
about two weeks to about 1 year following vascular injury. In some
instances it can be desirable to administer an agent which inhibits
neutrophil recruitment and/or adhesion prior to or subsequent to
administration of an agent which inhibits mononuclear cell
recruitment and/or adhesion. For example, in one embodiment an
agent which inhibits neutrophil recruitment and/or adhesion to a
site of vascular injury is administered to a subject after an agent
which inhibits mononuclear cell recruitment and/or activation to a
site of vascular invury. The skilled artisan will be able to
determine appropriate dosage and timing for administration of the
agents based upon the particular agents selected, characteristics
of the subject and other factors.
[0102] For example, where a subject is scheduled to undergo a
vascular intervention procedure (e.g., PTCA), a first agent which
inhibits recruitment and/or adhesion of neutrophils to a site of
vascular injury and a second agent which inhibits recruitment
and/or adhesion of mononuclear cells to a site of vascular injury
can be administered prior to the procedure and/or periprocedurally.
The first agent and the second agent can be administered as a
single dose or repeatedly, if necessary to maintain inhibition of
recruitment and/or adhesion of neutrophils and mononuclear cells at
a site of vascular injury for about one week following the vascular
intervention procedure. At that time, administration of the first
agent can be discontinued and the second agent can be administered
as necessary to maintain inhibition of recruitment and/or adhesion
of mononuclear cells at a site of vascular injury for a period of
at least about two weeks to about 1 year following the vascular
intervention procedure.
[0103] The agent (e.g., cellular adhesion molecule antagonist,
antagonist of chemokine receptor function, additional therapeutic
agent) can be administered as a neutral compound or as a salt.
Salts of compounds containing an amine or other basic group can be
obtained, for example, by reacting with a suitable organic or
inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic
acid, perchloric acid and the like. Compounds with a quaternary
ammonium group also contain a counteranion such as chloride,
bromide, iodide, acetate, perchlorate and the like. Salts of
compounds containing a carboxylic acid or other acidic functional
group can be prepared by reacting with a suitable base, for
example, a hydroxide base. Salts of acidic functional groups
contain a countercation such as sodium, potassium and the like.
[0104] The agent(s) (e.g., cellular adhesion molecule antagonist,
antagonist of chemokine receptor function, as described herein) can
be administered to the subject as part of a pharmaceutical or
physiological composition for inhibiting stenosis or restenosis.
Such a composition can comprise an (i.e., one or more) agent (e.g.,
cellular adhesion molecule antagonist, antagonist of chemokine
receptor function, additional therapeutic agent) and a
physiologically acceptable carrier. Pharmaceutical compositions can
further comprise one or more additional therapeutic agents (e.g.,
anticoagulant, thrombolytic agent). Alternatively, an agent (e.g.,
cellular adhesion molecule antagonist, antagonist of chemokine
receptor function, as described herein) and an additional
therapeutic agent can be components of separate pharmaceutical
compositions which can be mixed together prior to administration or
administered separately. Formulation will vary according to the
route of administration selected (e.g., solution, emulsion,
capsule). Suitable pharmaceutical carriers can contain inert
ingredients which do not interact with the agent(s) (e.g., cellular
adhesion molecule antagonist, antagonist of chemokine receptor
function, additional therapeutic agent). Standard pharmaceutical
formulation techniques can be employed, such as those described in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa. Suitable physiological carriers for parenteral
administration include, for example, sterile water, physiological
saline, bacteriostatic saline (saline containing about 0.9% mg/ml
benzyl alcohol), phosphate-buffered saline, Hank's solution,
Ringer's-lactate and the like. Methods for encapsulating
compositions (such as in a coating of hard gelatin or cyclodextran)
are known in the art (Baker, et al., "Controlled Release of
Biological Active Agents", John Wiley and Sons, 1986).
EXAMPLE
[0105] The effects of murine monoclonal antibodies which bind human
integrin CD18 or human chemokine receptor CCR2 in a model of
restenosis in cynomolgus monkeys was evaluated.
[0106] Study Design
[0107] Cynomolgus monkeys were randomized on the basis of body
weight to groups to receive treatment with either an irrelevant
murine monoclonal antibody (mAb) as an IgG2a isotype control
(S-S.1), an anti-human CCR2 mAb (1D9) or an anti-human CD18 mAb
(1B4). Animals were administered a loading dose of mAb
intravenously (IV) on Day -1, followed by daily SC injections on
Days 1-13. On Day 1, all animals underwent bilateral balloon
angioplasty-induced iliac artery endothelial denudation, followed
by intravascular stent placement, as a model of restenosis. Animals
were euthanized at the end of the test period to allow perfusion
fixation and collection of the iliac arteries and other tissue
samples (see Table A).
[0108] Efficacy of treatment was evaluated by use of quantitative
angiography at the time of stent placement and at the end of the
study, and by immunohistologic and morphometric evaluation of iliac
artery tissue. Blood samples were collected periodically for assay
of serum mAb levels (pharmacokinetics), leukocyte mAb binding
(pharmacodynamics), anti-mAb antiglobulin response
(immunogenicity), and for hematology and serum chemistry (safety).
Safety was further evaluated by recording vital signs during
infusion and body weights, clinical observations and injection site
observations during the test period. Other tissue samples were not
evaluated unless warranted (see Table B).
1TABLE A Study Design Treatment Group No. No. Test Dose and Dose
Model of (Description) Animals Materials Regimen Restenosis
Euthanasia 1 5 S-S.1 5 mg/kg, IV, in 30 mL Bilateral Day 29 (IgG2a
over 30 min balloon control) on Day - 1; angioplasty- 2 5 1D9 1
mg/kg, SC, induced iliacartery (anti-CCR2) in 3 mL endothelial 3 5
1B4 on Days 1-13 denudation and (anti-CD18) intravascular stent
placement on Day 1 There is no Day 0; Day - 1 precedes Day 1. Day 1
was not the same calendar day for all animals. Day - 1 treatment
was via peripheral vein. Day 1-13 SC treatment was given in the
intrascapular area. Day 1 treatment was prior to
angioplasty/stenting. Doses were based on Day -1 body weight and
were maintained throughout the treatment period. IV =
intravascular; SC = subcutaneous; M = male.
[0109]
2TABLE B Study Procedures PK IMG Sera PD Anti- mAb mAb mAb Safety
Efficacy Study conc. binding Abs Hem SC Clinical Injection Vital
Quantitative Day Blood Collection BW Obs. Site Obs. Signs Nx
Angiography BL X X X X X X -1 X(pre) X(pre) X(pre) X X X Day -1 X
X(post) X(post) (pre) (pre) (pre and 1 X X X post) Day 1 X 8 X X X
X X X then (pre and 15 X X X X X X daily post) then 22 X X X X X X
daily 29 X X X X X X X X Vol. 1.0 1.0 1.0 0.5 1.0 (mL) Anti- None
Heparin None EDTA None coagulant Notes a (3.sup.rd) b (1.sup.st) a
(3.sup.rd) c d (2.sup.nd) (4.sup.th) a = Prioritize as 3.sup.rd
sample(s). Freeze (-70.degree. C.) in 100 .mu.L aliquots. b =
Prioritize as 1.sup.st sample. c = Prioritize as 2.sup.nd sample
collected. Use microtainers. d = Prioritize as 4.sup.th sample.
Freeze (-70.degree. C.) residual sera in single aliquot. Procedures
(except vital signs, angiography and Nx) were done prior to
treatment, unless otherwise specified. Abbreviations: Abs =
antibodies; BL = baseline; BW = body weight; Hem = hematology; IMG
= immunogenicity; mAb = monoclonal antibody; Nx = euthanasia,
perfusion and tissue collection; PD = pharmacodynamics; PK =
pharmacokinetics; pre/post = pre- and post-infusion; SC = serum
chemistry; X = was performed.
[0110] Disease Model
[0111] Atherosclerosis is a disease in humans in which lipid-rich
fibro-inflammatory plaques accumulate within the wall of the
coronary vessels, encroaching upon and narrowing ("stenosing") the
lumen, thus limiting oxygenated blood supply to cardiac tissue and
resulting in acute myocardial pain and/or infarction. Current
medical practice to address compromised coronary vessels involves
mechanical dilatation of the vessel with a balloon catheter via
percutaneous transluminal coronary angioplasty (PCTA), often
followed by placement of an intravascular stent to maintain luminal
diameter..sup.1 In a significant number of patients, late(r)
restenosis limits the effectiveness of this procedure..sup.2
Neointimal hyperplasia, vascular smooth muscle cell (VSMC)
proliferation and infiltrative leukocytes characterize the area of
restenosis. Possible mechanisms involved in this process include
platelet aggregation (thrombosis), endothelial cell activation and
VSMC proliferation and migration. A variety of animal models of
atherosclerosis and/or restenosis have been developed, in species
such as mice, rats, rabbits, pigs, and nonhuman primates
(cynomolgus monkeys and baboons). The model of neointimal
hyperplasia used in this study, balloon angioplasty-induced
endothelial denudation followed by stent placement, has been
previously used in rabbits to elucidate some of the mechanisms
involved in restenosis..sup.4
[0112] Test Materials
[0113] 1D9 is a murine IgG2a mAb that recognizes CCR2 on monocytes
of humans and nonhuman primates. 1B4 is a murine IgG2a mAb that
recognizes CD18 on human, nonhuman primate and rabbit neutrophils.
1B4 was produced using a commercially available cell line that
makes the antibody (ATCC Accession No. HB-10164). S-S. 1 is a
murine IgG2a mAb directed against sheep red blood cells. S-S. 1 was
produced using a commercially available cell line that makes the
antibody (ATCC Accession No. TIB-111) and is being used as an
irrelevant isotype-matched control antibody.
[0114] Dose and Dose Regimen
[0115] The dose and dose regimen were selected because they were
anticipated to result in peak and trough sera mAb concentrations in
excess of those required to maintain continuous saturation of CCR2
or CD18 on leukocytes through at least Day 14. It was recognized
that neutralizing monkey anti-mouse mAb antiglobulin (MAMA)
responses would develop in these animals and that these responses
may have affected sera or cell-bound mAb levels and thus PK, PD
and/or efficacy endpoints.
[0116] Vital Sign Monitoring
[0117] These mAbs, as with many other antibodies, have the
potential to induce a "first-dose effect" related to cytokine
release during initial infusion, or to precipitate ADCC
(antibody-dependent cell-mediated cytotoxicity) or
complement-mediated cell lysis. These effects can result in
transient adverse physiologic changes, such as hypotension and
bronchoconstriction, which are usually not life threatening.
Monitoring vital signs allowed detection of such changes.
[0118] Test System
[0119] The murine anti-human CCR2 mAb and murine anti-human CD18
mAb also bind Cynomolgus monkey CCR2 and CD18, respectively.
[0120] Number of Animals
[0121] The number of animals used in this study was sufficient for
evaluation of the results. Although 4 animals/group has previously
been sufficient to allow detection of efficacy in a rabbit
model,.sup.4 it was considered appropriate to use 5 animals/group
in this study because of potentially greater variability in degree
of vascular injury and response thereto in monkeys.
[0122] Test Materials and Formulation
[0123] Characterization
[0124] The mAb solutions were biochemically characterized prior to
use (see Table C).
[0125] Stability
[0126] Samples of the test articles were retrieved from the test
site at completion of dosing and characterized biochemically. No
significant changes in the samples, relative to the original
characterization, were detected.
[0127] Dose Formulation Methods
[0128] On the day(s) of use, an appropriate number of vials of the
frozen mAb solutions were brought to room temperature and
appropriate volumes diluted in vehicle (saline) as necessary to
provide uniform total volumes for IV (30 mL in a 60 cc syringe) or
SC (3 mL in a 3 cc syringe) administration to all animals. The date
of thawing was recorded on the vial(s). Unused (thawed, opened)
bulk mAb solutions were refrigerated (2-8.degree. C.) for use on
subsequent day(s).
[0129] Dose Formulation Samples
[0130] No dose formulation samples were collected.
[0131] Disposition
[0132] Residual diluted dose formulations were discarded.
3TABLE C Test Materials Storage Physical Lot Identification Conc.
Conditions Description Supplier Manufacturer No. Biohazards Saline
N/A Ambient Clear liquid Primedica TBD TBD None; (Vehicle for Use
dilution) standard S-S.1 4.2 mg/mL -70.degree. C. until Clear to
LeukoSite LeukoSite TBD precautions (Irrelevant thawed, then
slightly IgG2a control 2-8 .degree. C. cloudy mAb).sup.a solution
1D9 6.8 mg/mL Therapeutic LS132- (Anti-CCR2 Antibody 1D9 mAb)
Center Batch 2 1B4 5 mg/mL LeukoSite TBD (Anti-CD18 mAb).sup.b a =
Cell line obtained from ATCC; No. TIB-111 also referred to as
S-S.1. b = Cell line obtained from ATCC; No. HB-10164. TBD =
documented in study file; ATCC: American Type Culture
Collection
[0133] Test System
[0134] Animals
[0135] Species: Macaca fascicularis
[0136] Common name: Cynomolgus monkey
[0137] Number of Animals: 15
[0138] Age and Gender: Young-adult males
[0139] Weight at Initiation of Treatment: .about.4 kg
[0140] Source and Selection
[0141] Animals were obtained from a source approved by the Testing
Facility. Animals were selected from those available at the time of
the study and appeared to be in good health, as determined by a
veterinarian. All animals completed a period of quarantine, and
each animal was identified by a unique number. All animals used in
the study were euthanized at the end of the study.
[0142] Animal Care
[0143] The Testing Facility was accredited by the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC) and
licensed by the United States Department of Agriculture (USDA) to
conduct research in laboratory animals in compliance with the
Animal Welfare Act, USDA regulations and National Research Council
(NRC) guidelines..sup.3,4,5 Animal activities described herein were
subject to review and approval by the Institutional Animal Care and
Use Committee (IACUC) of the Testing Facility.
[0144] Animal husbandry, diet, water and environmental conditions
were performed in compliance with NRC guidelines".sup.7 and Testing
Facility standard operating procedures (SOPs).
[0145] Methods
[0146] Randomization
[0147] Animals considered suitable for the study were randomized to
treatment groups by body weight and assigned unique consecutive
identification numbers within each group. The order in which
animals were assigned to undergo procedures was rotated among
groups on the basis of identification numbers to minimize
procedural bias.
[0148] Acclimation to Physical Restraint
[0149] Animals were acclimated to the rope-and-collar method of
physical restraint and to restraint in a primate chair prior to
initiation of treatment.
[0150] Tranquilization
[0151] Animals were tranquilized (ketamine HCl, 5-10 mg/kg, IM, to
effect) as necessary to facilitate handling, blood collection or
other technical procedures.
[0152] Fasting
[0153] All food was withheld overnight prior to tranquilization or
anesthesia. Water was not be withheld.
[0154] Dose Calculation
[0155] Doses were calculated based on Day -1 body weight. The doses
were maintained throughout the treatment period.
[0156] Dose Administration
[0157] All treatments were administered using in-line or
syringe-tip low protein-binding filters. IV treatments were
administered while animals were restrained in a primate chair, via
a percutaneous catheter placed in a peripheral vein, using a
clinical grade infusion pump. SC treatment were given in the
intrascapular area, using a 23-guage needle.
[0158] Blood Collection
[0159] Blood samples were collected from tranquilized animals via
direct venipuncture of a femoral vein. Blood collection was
alternated between left and right femoral veins when possible.
Considerable efforts were made to minimize local vascular trauma or
bleeding. It was acceptable to not collect individual samples if
difficulty in collecting them suggested the likelihood of inducing
local vascular trauma (e.g. hematoma formation,
arteriopuncture).
[0160] Concurrent Therapy
[0161] Concurrent therapy in accordance with accepted veterinary
practices was utilized if deemed necessary by a veterinarian.
[0162] Animal Observation
[0163] Body weights were recorded approximately weekly (see Table
B). Cage side observations for moribundity and mortality were
performed twice daily.
[0164] Clinical Observations
[0165] Clinical observations for evidence of treatment-related
effects were performed beginning prior to and approximately 1 hour
after treatment on Day -1, and daily thereafter. On days of SC
treatment clinical observations were performed prior to
treatment.
[0166] Injection Site Observations
[0167] The SC injection site (interscapular area) was observed
beginning prior to injection on Day 1, and daily thereafter. The
site was subjectively scored for swelling and/or erythema (0=none,
1=mild, 2=moderate, 3=marked).
[0168] Vital Sign Monitoring During Infusion
[0169] During IV treatment vital signs (heart rate, respiratory
rate, rectal body temperature and indirect blood pressure) were
monitored intermittently for indications of adverse reactions.
Representative values for these parameters were recorded prior to,
at .about.10 minute intervals during, and at the end of
infusion.
[0170] If adverse reactions occurred, treatment may have been
interrupted or discontinued. A Testing Facility veterinarian
determined the appropriate therapy, if any, in consultation with
the Study Director and/or study Sponsor's Representative.
[0171] Angioplasty and Stenting Procedures
[0172] Anticoagulant Therapy
[0173] Animals received aspirin (.about.40 mg, orally) daily to
provide anticoagulant function and minimize stent thrombosis
beginning on Day -3.
[0174] Antibiotic Therapy
[0175] Animals received a single prophylactic injection of
benzathine/procaine penicillin-G (42,000 IU/kg, IM) on Day 1 prior
to angioplasty.
[0176] Anesthesia
[0177] Animals were pre-anesthetized (ketamine HCl, 10 mg/kg, IM;
atropine SO.sub.4, 0.04 mg/kg, IM) then intubated and maintained in
anesthesia with isoflurane inhalant anesthetic gas.
[0178] Preparation
[0179] Animals were positioned on a procedure table in dorsal
recumbency. The bladder was catheterized to prevent urine
accumulation. Sites for vascular access were clipped and prepared
for aseptic surgery. A catheter was placed in a peripheral vein to
facilitate maintenance fluid administration (lactated Ringer's
solution, 5-10 mL/kg/hr).
[0180] Heparinization
[0181] Heparin (100 U/kg, IV, initially) was administered prior to
angioplasty to provide anticoagulation. Activated clotting time
(ACT) was monitored periodically and additional heparin was
administered as necessary to maintain ACT values >250 seconds
for the duration of the angioplasty procedure.
[0182] Instrumentation
[0183] The right carotid artery was surgically exposed and a 6Fr
percutaneous vascular introducer sheath (e.g. CP-07711, ARROW
International, Reading, Pa. 19605) was placed to facilitate
interventional catheter placement.
[0184] Utilizing fluoroscopic guidance, a 6Fr guide catheter was
passed antegrade to the level at which the distal abdominal aorta
bifurcates into the right and left iliac arteries. A radiopaque
0.014-inch guide wire (e.g. 22225M, Advanced Cardiovascular
Systems, Inc., Temecula, Calif. 92591) was used to facilitate
passage of the guide catheter or other catheters as necessary.
Radiopaque contrast media (e.g. Omnipaque.TM., iohexol injection,
Nycomed, Princeton, N.J. 75039) was used as necessary to facilitate
fluoroscopy.
[0185] Videotaping of Angiography
[0186] The fluoroscopic procedures were videotaped for each animal
to facilitate measurements for quantitative angiography.
Information identifying the study number, study day, animal number
and procedure were also recorded on the videotape.
[0187] Pre-angioplasty Angiography
[0188] Prior to angioplasty, nitroglycerine (50 .mu.g, IA) was
administered to induce arterial dilatation. Radiopaque contrast
media was administered to facilitate angiography.
[0189] Endothelial Denudation via Balloon Angioplasty
[0190] An 80 cm, 3Fr Fogarty balloon embolectomy catheter (e.g.
120803F, Baxter Healthcare Corp., Irvine, Calif. 92714) with a
balloon appropriately sized for the vessel was passed via the guide
catheter into the right iliac artery, to a level about 4 cm distal
to the aortic bifurcation. The balloon was then inflated with 0.6
cc air and withdrawn inflated over an about 3 cm section of artery
to facilitate endothelial denudation. Balloon angioplasty was
performed three times. This procedure was then repeated in the
contralateral (left) iliac artery and the balloon embolectomy
catheter was withdrawn. In some cases the left iliac artery was
denuded first, followed by the right.
[0191] Stent Placement
[0192] An appropriate-sized dilation catheter (Ninja.TM. PTCA
dilation catheter with SLX.TM. coating, Cordis Corp., Miami Fla.
33102) fitted with a balloon-expandable 7-mm stent (e.g., one half
of a 15-mm long stent (e.g. CS15-030, Palmaz-Schatz.RTM. crown
balloon-expandable stent, Cordis Corp., Miami Fla. 33102)) was then
passed into the right iliac artery to the level of the midpoint of
endothelial denudation. The balloon was inflated to the appropriate
inflation pressure required to expand the stent sufficiently to
provide a balloon/stent:artery ratio of 1.1-1.2 (typically 6 Atm
for 2.5, 3.0 or 3.5 mm catheters). The balloon was deflated and the
catheter was withdrawn. This procedure was repeated in the
contralateral (left) iliac artery. In some cases the left iliac
artery was stented first, followed by the right.
[0193] Post-Angioplasty Angiography
[0194] Approximately 10 min after placement of the second stent,
nitroglycerine (50 .mu.g, IA) was administered to induce arterial
dilatation for quantitative angiography of both arteries.
Radiopaque contrast media was administered to facilitate
angiography.
[0195] Recovery
[0196] The vascular introducer sheath was removed and the carotid
artery was ligated. The incision was closed with appropriate
suture. The animals recovered from anesthesia and were returned to
their cages.
[0197] Analgesia
[0198] Animals received a single injection of buprenorphine (0.01
mg/kg, IM) after completion of the procedures.
[0199] Follow-up Angiography
[0200] Anesthesia
[0201] Prior to euthanasia and arterial tissue collection (see
Paragraph VIII.L) animals were pre-anesthetized (ketamine HCl, 10
mg/kg, IM; atropine SO.sub.4, 0.04 mg/kg, IM) then intubated and
maintained in anesthesia with isoflurane inhalant anesthetic
gas.
[0202] Preparation
[0203] Animals were positioned on a procedure table in dorsal
recumbency. A catheter was placed in the peripheral vein. The
incision site was clipped and washed; strict asepsis was not
required for this terminal procedure.
[0204] Method
[0205] Heparin (150 U/kg, IV) was administered. Radiopaque contrast
media was used as necessary to facilitate fluoroscopy. The left
carotid artery was surgically exposed and a 6Fr percutaneous
vascular introducer sheath placed. Utilizing fluoroscopic guidance,
a 6Fr guide catheter was passed antegrade to the level at which the
distal abdominal aorta bifurcates into the right and left iliac
arteries. Nitroglycerine (50 .mu.g, IA) was administered.
Radiopaque contrast media was administered to facilitate
angiography. The fluoroscopic procedures were videotaped for each
animal to facilitate measurements for quantitative angiography.
[0206] Arterial Tissue Collection
[0207] Euthanasia
[0208] Animals were already anesthetized for follow-up angiography.
Animals were euthanized in accordance with American Veterinary
Medical Association (AVMA) guidelines.sup.3 by deep anesthesia
(sodium pentobarbital, 35 mg/kg, IV), followed by
exsanguination.
[0209] Perfusion
[0210] A midline laparotomy incision was made and a cannula was
placed in the descending abdominal aorta and advanced to the level
of the bifurcation. The iliac arteries were flushed with 100 mL
lactated Ringer's solution, followed by perfusion with 0.4%
paraformaldeyde (PFA) for about 5 min at 100 mmHg pressure.
[0211] Arterial Tissue Removal
[0212] Right and left iliac arteries were separately excised, with
the proximal ends identified (e.g. by ligature), and immersed in
0.4% PFA.
[0213] Limited Gross Necropsy
[0214] Animals underwent a limited necropsy, defined as evaluation
of the external body and abdominal and thoracic cavities.
[0215] Limited Organ/Tissue Collection
[0216] Representative samples from specified organs and tissues
(see Table D) were collected and fixed in 10% neutral-buffered
formalin for histopathologic evaluation or embedded and frozen in
OCT for immunohistology.
4TABLE D Limited Organ/Tissue Collection Injection sites
(interscapular area) Brain (cerebrum) Adrenal glands Heart Bone
marrow (sternum) Ileum (ileocecocolic junction) Eyes.sup.a Kidney
Heart Liver Kidneys Lung Large intestine (cecum, colon) Lymph node
(iliac, inguinal) Liver Sciatic nerve Lung Spinal cord Lymph nodes
(axillary, inguinal, Spleen mesenteric) Thymus Small intestine
(duodenum, jejunum, ileum) Spleen Thymus Thyroid gland (with
parathyroid) .sup.a= Eyes were fixed in Davidson's fixative. b =
All cell counts were reported as absolute values only. Other cell
types (e.g. precursor cells) if observed were counted. Other
morphologic features (e.g. RBC staining characteristics) if
present, were documented.
[0217] Sample Processing
[0218] Blood Samples
[0219] Hematology
[0220] Blood samples were analyzed (see Table E) using a hematology
analyzer. Blood smear differential were performed by manual
microscopy.
[0221] Serum Chemistry
[0222] Serum samples were analyzed using a chemistry analyzer (see
Table F).
5TABLE E Hematology Parameters Total leukocyte count (WBC) Blood
smear evaluation and Erythrocyte count (RBC) differential:.sup.b
Hemoglobin concentration (HGB) Segmented neutrophil count
Hematocrit value (HCT).sup.a (APLY) Mean corpuscular volume
(MCV).sup.a Band neutrophil count (ABND) Mean corpuscular
hemoglobin (MCH).sup.a Lymphocyte count (ALYM) Mean corpuscular
hemoglobin Monocyte count (AMNO) concentration (MCHC).sup.a
Eosinophil count (AEOS) Platelet count (PLT) Basophil count (ABSO)
Nucleated RBC count (ANRC) .sup.a= Calculated value. .sup.b= All
cell counts were reported as absolute values only. Other cell types
(e.g. precursor cells), if observed, were counted. Other
morphologic features (e.g. RBC staining characteristics) if
present, were documented.
[0223]
6TABLE F Serum Chemistry Parameters Glucose (GLU) Sodium (NA) Blood
urea nitrogen (BUN) Potassium (K) Creatinine (CRE) Chloride (CL)
Total protein (TPR) Total cholesterol (CHOL) Albumin (ALB) Total
bilirubin (TBIL) Globulin (GLOB)a Triglycerides (TRG)
Albumin/Globulin ratio Alanine aminotransferase (ALT) (A/G)a
Aspartate aminotransferase (AST) Calcium (CAL) Alkaline phosphatase
(ALK) Phosphorus (PHOS) Gama glutamyl transferase (GGT) a =
Calculated value.
[0224] Samples for Additional Analyses
[0225] Blood samples for pharmakodynamic assays and sera samples
for pharmacokinetic and immunogenicity assays were obtained.
[0226] Pharmacokinetics
[0227] Serum therapeutic 1B4 or 1D9 monoclonal antibody (mAb)
levels were determined by enzyme-linked immunosorbent assay (ELISA)
for murine IgG.
[0228] Briefly, 96-well plates (NUNC #4-39454) were coated with 100
.mu.l goat-anti-mouse IgG+IgM antibody (Jackson Immunoresearch
#115-005-068) at 2.5 .mu.g/ml in carbonate buffer pH 9.3 overnight
at 4.degree. C. Plates were subsequently washed 3 times with PBS
0.5% Tween-20 and blocked with 300 .mu.l PBS/1% BSA for 60 minutes
at 37.degree. C. Following 3 additional washes with PBS-Tween,
serum samples were diluted 1:100 in PBS/1% BSA and 100 .mu.l
aliquots were added to duplicate wells in the plate. The antibody
standard (MOPC-21, Sigma) was diluted to 50 ng/ml and 100 .mu.l
aliquots were added to the plate. Subsequently, all samples were
diluted 2-fold across the plate and incubated at room temperature
for 2 hours. The plate was subsequently washed again with PBS/0.5%
Tween-20 and 100 .mu.l of peroxidase-conjugated goat anti-mouse
IgG+IgM (Jackson Immunoresearch #115-035-068) was added at a
concentration of 375 ng/ml and incubated for 2 hours at room
temperature. Following additional washes with PBS-Tween, plates
were developed with o-phenylenediamine (OPD, Sigma) in citric acid
buffer pH 5.0, and analyzed on a 96-well fluorescent plate reader
(Dynatech MR4000) at 492 nm. The dilutions of the antibody standard
was used to construct a standard curve, and the serum antibody
concentration was automatically derived from the standard curve and
dilution factor data provided using Biolinx 2.22 software.
[0229] Pharmacodynamics
[0230] Target Saturation
[0231] Saturation of 1B4 target (CD18) or 1D9 target (CCR2) on
appropriate leukocyte subsets (neutrophils and monocytes for CD18
and monocytes for CCR2) was determined by flow cytometry
assays.
[0232] Determination of Saturation of Circulating Leukocytes with
1D9 (anti-CCR2)
[0233] Blood was collected in heparin from the test animals at
specified intervals prior to and after the administration of 1D9.
Samples of whole blood were stained ("spiked") with supersaturating
amounts of 1D9 or nothing. The blood samples were washed in buffer
and stained with FITC conjugated goat-anti-mouse IgG. After daily
standardization of the flow cytometer with FITC-labeled beads, to
ensure equivalent day-to-day sensitivity to FITC, the blood was
lysed (red blood cells were lysed) using ammonium chloride lysing
solution and the fluorescence of lymphocyte, monocyte and
granulocyte populations was determined. The degree of saturation of
CCR2 on monocytes by the administered 1D9 was determined by the
difference between the mean channel fluorescence (MCF) of the
sample with no added 1D9 and the sample with the added spike of
1D9. In practice, CCR2 on the surface of the cells which were not
coated with the 1D9 delivered in vivo was stained by the
exogenously added 1D9 and the mean channel fluorescence of the
unspiked sample was dimmer than the mean channel fluorescence of
the spiked sample. The difference in staining intensity is a
reflection of free (unsaturated) CCR2 on the cell surface.
[0234] Determination of Saturation of Circulating Leukocytes with
1B4 (anti-CD18)
[0235] Blood was collected in heparin from the test animals at
specified intervals prior to and after the administration of 1B4.
Samples of whole blood were stained ("spiked") with supersaturating
amounts of 1B4 or nothing. The blood samples were washed in buffer
and stained with FITC conjugated goat-anti-mouse IgG. After daily
standardization of the flow cytometer with FITC-labeled beads, to
ensure equivalent day-to-day sensitivity to FITC, the blood was
lysed (red blood cells were lysed) using ammonium chloride lysing
solution and the fluorescence of lymphocyte, monocyte and
granulocyte populations was determined. The degree of saturation of
CD18 on either neutrophils or monocytes by the administered 1B4 was
determined by the difference between the mean channel fluorescence
(MCF) of the sample with no added 1B4 and the sample with the added
spike of 1B4. In practice, free CD18 on the surface of the cells
which were not coated with the 1B4 delivered in vivo was stained by
the exogenously added 1B4 and the mean channel fluorescence of the
unspiked sample was dimmer than the mean channel fluorescence of
the spiked sample. The difference in staining intensity was a
reflection of free (unsaturated) CD18 on the cell surface.
[0236] Determination of Saturation of Circulating Leukocytes with
S-S. 1 (Irrelevant Isotype Control Antibody, also Referred to as
TIB-111)
[0237] S-S. 1 is a non-cell binding irrelevant murine antibody.
Assays to determine potential "saturation" of leukocyte antigens
with this mAb were performed as above, with the understanding that
a positive result (cell staining) was unlikely to be seen and that
there would consistently be no difference in mean channel
fluorescence between unspiked and spiked samples over time.
[0238] Peripheral Blood Leukocyte Dynamics
[0239] The effect of mAb administration on leukocyte dynamics
(trafficking, margination/demargination) was identified indirectly
by evaluating the numbers of leukocytes in circulation, as compared
to prior to treatment. Inhibition of leukocyte adhesion and/or
chemotaxis would be expected to prevent normal trafficking and to
result in elevated circulating cell numbers. Routine hematology was
performed to determine the total numbers of peripheral blood
leukocytes, as well as the number of neutrophils, lymphocytes and
monocytes.
[0240] Immunogenicity
[0241] Measurement of Antibody Responses to 1D9 (anti-CCR2)
[0242] Sera samples were collected at specified times and stored
frozen until completion of the study. Anti-1D9 antibodies were
detected using two assays.
[0243] The first assay was designed to detect both anti-idiotype
and anti-isotype antibodies. This assay was performed by coating
the wells of a microtiter plate with 1D9 and blocking unused
protein binding sites with BSA. The sera were then diluted
appropriately and several dilutions were added to duplicate wells
of the plate. Antibodies in the sera were allowed to bind for 2
hours at 37 degrees C., and then the wells were shaken out and
washed 3 times in PBS with Tween 20. Monkey anti-1D9 antibodies
were detected with HRP-conjugated goat anti-human IgG (absorbed
against mouse proteins). After 2 hours, unbound detection antibody
was washed away in three washes of the plate with PBS Tween. Bound
complexes were detected by the addition of o-phenylenediamine to
produce a yellow color. Color was read at 490 nm on an ELISA plate
reader. Titers were determined by calculating the inverse of the
dilution of the sera which produced an optical density equivalent
to the optical density produced by a specific dilution of a
commercial HRP-conjugated goat anti-mouse IgG (absorbed against
human serum proteins).
[0244] The second assay was used to assess the proportion of the
response which reacted with the 1D9 idiotype compared with the
response to mouse IgG2a. This was a competitive ELISA in which the
sera from a peak antibody response sample were diluted to produce
an optical density between 0.6-1.0. The diluted sera were added to
triplicate wells of an ELISA plate coated with 1D9 as above. The
sera was added alone, mixed with 5 .mu.g of commercial mouse IgG2a,
or mixed with 5 .mu.g of 1D9. The ELISA was carried out as above
and monkey antibody bound to the 1D9 on the plate was detected
using HRP-anti-human IgG, as above. By comparing the optical
density of signals produced by the uncompeted sera with those
produced by sera spiked with mouse IgG2a or 1D9 it was possible to
assess the specificity of the anti-1D9 antibodies which developed
in animals treated with 1D9.
[0245] Measurement of Antibody Responses to 1B4 (CD18)
[0246] Sera samples were collected at specified times and stored
frozen until completion of the study. Anti-1B4 antibodies were
detected using two assays.
[0247] The first assay was designed to detect both anti-idiotype
and anti-isotype antibodies. This assay was performed by coating
the wells of a microtiter plate with 1B4 and blocking unused
protein binding sites with BSA. The sera was then diluted
appropriately and several dilutions were added to duplicate wells
of the plate. Antibodies in the sera were allowed to bind for 2
hours at 37 degrees C., and then the wells were shaken out and
washed 3 times in PBS with Tween 20. Monkey anti-1B4 antibodies
were detected with HRP-conjugated goat anti-human IgG (absorbed
against mouse proteins). After 2 hours, unbound detection antibody
was washed away in three washes of the plate with PBS Tween. Bound
complexes were detected by the addition of o-phenylenediamine to
produce a yellow color. Color was read at 490 nm on an ELISA plate
reader. Titers were determined by calculating the inverse of the
dilution of the sera which produced an optical density equivalent
to the optical density produced by a specific dilution of a
commercial HRP-conjugated goat anti-mouse IgG (absorbed against
human serum proteins).
[0248] The second assay was used to assess the proportion of the
response which reacted with the 1B4 idiotype compared with the
response to mouse IgG2a. This was a competitive ELISA in which the
sera from a peak antibody response sample was diluted to produce an
optical density between 0.6-1.0. The diluted sera was added to
triplicate wells of an ELISA plate coated with 1B4 as above. The
sera was added alone, mixed with 5 .mu.g of commercial mouse IgG2a,
or mixed with 5 .mu.g of 1B4. The ELISA was carried out as above
and monkey antibody bound to the 1B4 on the plate was detected
using HRP-anti-human IgG, as above. By comparing the optical
density of signals produced by the uncompeted sera with those
produced by sera spiked with mouse IgG2a or 1B4 it was possible to
assess the specificity of the anti-1B4 antibodies which developed
in animals treated with 1B4.
[0249] Measurement of Antibody Responses to S-S. 1 (Irrelevant
Isotype Control Antibody)
[0250] Anti-S-S. 1 antibodies were detected using two assays, as
described above.
[0251] Quantitative Angiography Calculations:
[0252] Control of Bias
[0253] At the time of angioplasty and stenting, angiography
measurements were performed. The measurement were taken in a
non-blinded fashion to determine the diameter of each artery and to
select the appropriate size balloon dilation catheter and inflation
pressure for expansion of the stents, thus providing the desired
balloon/stent:artery ratio. Non-blinded measurements were performed
at follow-up. For the purpose of evaluating treatment effect(s),
videorecorded images were replayed on a larger video screen and
evaluated in a blinded fashion by an independent observer.
[0254] Angiography Measurements
[0255] Blinded angiography measurements were performed by measuring
the fluoroscopy images directly from the video screen at the
mid-stent area with digital calipers. For both iliac arteries, the
following parameters were measured (in mm):
[0256] Angioplasty/stenting
[0257] Actual guide catheter o.d. (actual measurement) (a)
[0258] Observed guide catheter o.d. (observed on video screen as
magnified image) (b)
[0259] Pre-angioplasty luminal i.d. (x)
[0260] Post-angioplasty in-stent inflated balloon o.d. (y)
[0261] Post-angioplasty/stent in-stent luminal i.d. (x')
[0262] Follow-up
[0263] Actual follow-up guide catheter o.d. (c)
[0264] Observed follow-up guide catheter o.d. (d)
[0265] Follow-up in-stent luminal i.d. (x")
[0266] Restenosis Calculations
[0267] The following calculations were performed:
[0268] Angioplasty/stenting
[0269] Magnification correction factor 1 (MCF1)=[b]+[a]
[0270] Balloon/stent:artery ratio=[y:x]=1.1-1.2, ideally
[0271] Acute luminal gain (ALG; in mm)=[(x')(MCF1)]-[(x)(MCF1)]
[0272] Follow-up
[0273] Magnification correction factor 2 (MF2)=[d]+[c]
[0274] Late luminal loss (LLL; in mm)=[(x')(MCF1)]-[(x")(MCF2)]
[0275] Arterial Tissue Analysis
[0276] Control of bias
[0277] Arterial tissue samples were randomly assigned accession or
identification numbers that did not indicate group or animal
number. The person(s) evaluating arterial tissue samples for
effect(s) of treatment were blinded to the identity of the
samples.
[0278] Tissue Processing
[0279] The non-stented (balloon-injured) proximal and distal
arterial segments were separated from the stented segments, with
the proximal ends of each was identified and marked. Stented
arterial segments were embedded in methacrylate and multiple 5 mm
cross-sections were cut with a tungsten carbide knife. Non-stented
arterial segments were embedded in paraffin to preserve
antigenicity, but were not processed further unless warranted.
[0280] Stented sections were stained with verHoeff's tissue elastin
stain, hematoxylin and eosin (H+E), and various immunocytochemical
markers for cells incorporating BrdU or for cell types such as
smooth muscle cells, endothelial cells, and inflammatory cells.
[0281] Evaluation of Neointimal Hyperplasia
[0282] In-stent cross-sectional neointimal (on the luminal side of
the internal elastic membrane [IEL]) and medial (on the abluminal
side of the IEL) areas (mm.sup.2) were measured
histomorphometrically using computer-assisted digital
planimetry..sup.3 To minimize sampling error, 3 elastin-stained
in-stent cross-sections, one each from the proximal, middle and
distal portions of the right and left iliac arteries, were analyzed
morphometrically. The composite value for the left or right artery
was expressed as the mean value of the 3 measurements for each
artery.
[0283] Each cross-section was scored (0-3) for the deep
stent-induced arterial injury associated with each stent strut
(8-12/cross-section) and an average depth of injury score for each
cross-section was calculated..sup.19 These values were used to
evaluate whether the initial injury was comparable across
groups.
[0284] Statistical Analysis
[0285] Analysis of efficacy data by T-test between treated and
control groups was performed and these values are reported.
[0286] Results
[0287] Safety
[0288] There were no treatment-related effects on vital signs
during infusion. There were no treatment-related effects on body
weight or clinical observations during the study. Individual
injection sites in one or more animals showed, mild, transient
erythema which was not considered an adverse reaction. There were
no adverse events associated with the catheterization incisions
(i.e., no impairment of wound healing and no indication of
bacterial infection). There were no adverse effects on clinical
pathology parameters. As expected, serum globulin levels were
elevated in treated and control animals. Leukocyte counts were
affected by 1B4 and 1D9 administration (see below). There were no
treatment-related gross lesions at necropsy.
[0289] Pharmacokinetics
[0290] Serum mAb levels (mean.+-.stdev), relative to control mAb,
are presented in FIGS. 1A and 1B.
[0291] Administration of 1D9 resulted in serum concentrations
>50 .mu.g/mL at the time of angioplasty and stent deployment
(Day 1) and maintenance of serum concentrations >1 .mu.g/mL
through Day 8. By Day 15, 1D9 levels were virtually undetectable,
despite continuation of dosing from Day .sup.-1 to 13.
[0292] Administration of 1B4 resulted in serum concentrations
>50 .mu.g/mL at the time of angioplasty and stent deployment
(Day 1) and maintenance of serum concentrations 1 .mu.g/mL through
Day 8. By Day 15, 1B4 levels were virtually undetectable, despite
continuation of dosing from Day .sup.-1 to 13.
[0293] Pharmacodynamics
[0294] Leukocyte Target Saturation
[0295] Leukocyte target saturation (mean.+-.stdev), relative to
control mAb, is presented in FIGS. 2A-2C.
[0296] Leukocyte counts were not affected by administration of I
D9.
[0297] Administration of 1B4 resulted in rapid saturation of
neutrophil and monocyte CD18 on Day .sup.-1 immediately after IV
infusion and maintenance of target saturation through Day 8. [Day
.sup.-8 levels were not available]. By Day 15, available CD18
binding sites on leukocytes (unsaturated targets) returned to
baseline levels.
[0298] Peripheral Blood Leukocyte Dynamics
[0299] Peripheral blood leukocyte counts (mean.+-.stdev), relative
to control mAb, are presented in FIGS. 3A-3H.
[0300] Administration of 1D9 resulted in altered monocyte dynamics
attributed to CCR2 saturation, as indicated by moderate monocytosis
on Days 8 and 15. Although not determined, these cell counts were
likely elevated at earlier timepoints as well. Other leukocytes
were not affected.
[0301] Administration of 1B4 resulted in altered leukocyte dynamics
attributed to CD18 saturation, as indicated by the pronounced
leukocytosis, neutrophilia, lymphocytosis and monocytosis on Day 8.
Although not determined, these cell counts were likely elevated at
earlier timepoints as well.
[0302] Immunogenicity
[0303] The anti-mAb antibody titers (mean.+-.stdev), relative to
control mAb, are presented in FIGS. 4A-4B.
[0304] Anti-globulin responses developed in all animals, detected
as early as Day 8. The majority of these responses were
anti-idiotype (directed against the variable region, specifically
the complementarity determining region), rather than anti-isotype
(directed against the constant region). The rapid increase in
potentially neutralizing anti-idiotype antibodies from Day 8 to Day
15 corresponds with the loss of circulating mAb levels, the loss of
leukocyte target saturation and the return of peripheral blood
leukocyte counts to baseline (normal) levels. These observations
are consistent with anti-mAb antibodies binding to the therapeutic
mAb and preventing (neutralizing) the activity. Further, these
observations suggest that effective sera/leukocyte levels of
therapeutic mAb were only maintained through Day 8.
[0305] Efficacy
[0306] Quantitative Angiography
[0307] The blinded quantitative angiography results
(mean.+-.stdev), relative to control mAb, are presented in FIGS.
5A-5F.
[0308] Administration of 1D9 tended to decrease the late luminal
loss (LLL) (p=0.11) and the index (LLL/ALG) (p=0.07) as measured at
the mid-stent region of the iliac arteries, but this difference was
not significant.
[0309] Administration of 1B4 tended to decrease the late luminal
loss (LLL) (p=0.06) and significantly decreased the index (LLL/ALG)
(p<0.05) as measured at the mid-stent region of the iliac
arteries. Blockade of CD18 appeared to be more effective than
blockade of CCR2, as measured by angiography.
[0310] Histomorphometric Analysis
[0311] The blinded histomorphometric analysis results
(mean.+-.stdev), relative to control mAb, are presented in FIG.
6A-6D.
[0312] The blinded histomorphometric analysis results for intimal
area (mm.sup.2) and intima:media (I:M) ratio (mean.+-.stdev),
relative to control mAb, are presented in FIGS. 6A-6D. Severity
scores indicated that there was no difference between groups in the
degree of stent-mediated injury to the arteries, thus differences
between groups are attributable to treatment and not to differences
in degree of injury.
[0313] Administration of 1D9 inhibited neointimal hyperplasia
within the balloon+stent, but not the balloon-only, segments of the
iliac arteries (p=0.03 for intimal area, p=0.05 for I:M ratio).
Because CCR2 is present on mononuclear cells (monocytes and
activated T cells), but not neutrophils, these data suggest that
mononuclear cells are important contributors to balloon+stent, but
not balloon-only, neointimal hyperplasia. It does not exclude the
possibility that other cells, not expressing CCR2, are contributors
to balloon-only and balloon+stent neointimal hyperplasia. The
observation of effective reduction of balloon+stent neointimal
hyperplasia with anti-CCR2 inhibition may be relevant for
balloon+stent (in-stent) restenosis in humans.
[0314] Administration of 1B4 inhibited neointimal hyperplasia
within the balloon-only (p=0.02 for intimal area, p=0.01 for I:M
ratio) and balloon+stent (p<0.01 for both intimal area and I:M
ratio) segments of the iliac arteries. Because CD18 is present
primarily on neutrophils, and to a lesser extent on mononuclear
cells (monocytes and lymphocytes), these data suggest that
neutrophils are important (and perhaps predominant) contributors to
both balloon-only and balloon+stent neointimal hyperplasia. It does
not exclude the possibility that other cells (i.e., mononuclear
cells) expressing CD18 also are contributors to neointimal
hyperplasia with either injury. The observation of effective
reduction of balloon-only and balloon+stent neointimal hyperplasia
with anti-CD18 inhibition may be relevant for balloon-only and
balloon+stent (in-stent) restenosis in humans.
[0315] The results with 1D9 and 1B4 treatment demonstrate that CD18
blockade is effective in both balloon-only and balloon+stent
neointimal hyperplasia, while CCR2 blockade is effective in
balloon+stent injury only. CCR2 blockade appears slightly less
effective than CD18 blockade in balloon+stent injury (as was seen
with quantitative angiography). This is perhaps due to differences
in immunogenicity and subsequent neutralizing effects (i.e.,
duration of effective blockade relative to target cell
participation kinetics), or to the possibility that CCR2 blockade
does not affect one or more of the cell types contributing to this
lesion. Collectively, these results support the conclusion that
neutrophils are important contributors in both types of injury, and
that mononuclear cells are additional contributors to balloon+stent
injury, but not balloon-only injury. It is therefore likely that
simultaneous or sequential inhibition of both neutrophil and
mononuclear cell participation, such as by combination therapy with
an anti-CD18 and an anti-CCR2 agent, would be more effective for
in-stent restenosis than either agent alone.
REFERENCES CITED IN EXAMPLE
[0316] 1. Code of Federal Regulations (CFR). Title 21; Part 58,
Good Laboratory Practice Regulations: Final Rule. Washington (DC),
Office of the Federal Register. Dec. 22, 1978 (Revised Apr. 1,
1993).
[0317] 2. Holmes D R, Vlietstra R E, Smith H C, Vetrovec G W, Kent
K M, Cowley M J, Faxon D P, Gruntzig A R, Kelsey S F, Detre K M,
van Raden M J, Mock MB. Restenosis after percutaneous transluminal
angioplasty (PTCA): a report from the PCTS registry from the
National Heart, Lung and Blood Institute. Am J Cardiol 1984;
53:77C-81C.
[0318] 3. Serruys P W, Luitjen H E, Beatt K J, Geuskens R, de
Feyter P J, van den Brand M, Reiber J H, ten Katen H J, van Es G A,
Hugenholtz P G. Incidence of restenosis after successful coronary
angioplasty: a time-related phenomenon: a quantitative angiographic
study in 342 consecutive patients at 1, 2, 3, and 4 months.
Circulation 1988; 77:361-371.
[0319] 4. Rogers C, Edelman E R, Simon D I. A mAb to the
.beta.2-leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal
thickening after angioplasty or stent implantation in rabbits. Proc
Natl Acad Sci USA 1998; 95:10134-10139.
[0320] 5. Rogers C, Edelman E R: Endovascular stent design dictates
experimental restenosis and thrombosis. Circulation 1995;
91:2995-3001.
[0321] 6. Ponath P. Chemokine receptor antagonists: novel
therapeutics for inflammation and AIDS. Exp Opin Invest Drugs 1998;
7:1-18.
[0322] 7. Nelken N A, Coughlin S R, Gordon D, Wilcox J N. Monocyte
chemoattractant protein-1 in human atheromatous plaques. J Clin
Invest 1991; 88:1121-1127.
[0323] 8. Boring L, Gosling J, Cleary M, Charo I F. Decreased
lesion formation in CCR2.sup.-/- mice reveals a role for chemokines
in the initiation of atherosclerosis. Nature 1998; 394:894-897.
[0324] 9. Gu L, Okada Y, Clinton S K, Gerard C, Sukhova G K, Libby
P, Rollins B J. Absence of monocyte chemoattractant protein-1
reduces atherosclerosis in low density lipoprotein
receptor-deficient mice. Mol Cell 1998; 2:275-281.
[0325] 10. Furukawa Y, Matsumori A, Ohashi N, Shioi T, Ono K,
Harada A, Matsushima K, Sasayama S. Anti-monocyte chemoattractant
protein-1/monocyte chemotactant and activating factor antibody
inhibits neointimal hyperplasia in injured rat carotid arteries.
Circ Res 1999; 84:306-314.
[0326] 11. Kling D, Fingerle J, Harlan J M, Lobb R R, Lang F.
Mononuclear leukocytes invade rabbit arterial intima during
thickening formation via CD18- and VLA-4-dependent mechanisms and
stimulate smooth muscle migration. Circ Res 1995; 77:1121-8.
[0327] 12. Kling D, Fingerle J, Harlan J M. Inhibition of leukocyte
extravasation with a monoclonal antibody to CD18 during formation
of experimental intimal thickening in rabbit carotid arteries.
Arterioscler Thromb 1992; 12:997-1007.
[0328] 13. Golino P, Ambrosio G, Ragni M, Cirillo P, Esposito N,
Willerson J T, Rothlein R, Petrucci L, Condorelli M, Chiariello M,
Buja L M. Inhibition of leukocyte and platelet adhesion reduces
neointimal hyperplasia after arterial injury. Thromb Haemostasis
1997; 77:783-8.
[0329] 14. Guzman L A, Forudi F, Villa A E, Topol E J. Role of
leukocytes in neointimal formation after balloon angioplasty in the
rabbit atherosclerotic model. Coronary Art Dis 1995; 6:693-701.
[0330] 15. United States Code. Title 7 U.S.C. Sections 2131-22159
(The Animal Welfare Act as amended by P.L. 99-198), effective Dec.
23, 1986.
[0331] 16. Code of Federal Regulations (CFR). Title 9; Chapter 1,
Subchapter A (Animal Welfare Standards), Final Rule, Parts 1-3.
Washington (DC), Office of the Federal Register. Jan. 1, 1997.
[0332] 17. National Research Council, Institute of Laboratory
Animal Resources. Guide for the Care and Use of Laboratory Animals.
Washington (DC): National Academy Press, 1996.
[0333] 18. American Veterinary Medical Association. Report of the
American Veterinary Association (AVMA) panel on euthanasia. J Am
Vet Med Assoc 1993; 202:229-249.
[0334] 19. Schwartz R S, et al., Restenosis and Proportional
Neointimal Response to Coronary Artery Injury: Results in a Porcine
Model. J Am Coll Cardiol 1992; 19:267-274.
[0335] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
23 1 125 PRT Unknown SITE (1)...(125) YFC51.1 light chain variable
region with signal sequence 1 Met Arg Val Gln Val Gln Phe Leu Gly
Leu Leu Leu Leu Trp Thr Ser 1 5 10 15 Gly Ala Gln Cys Asp Val Gln
Met Thr Gln Ser Pro Ser Tyr Leu Ala 20 25 30 Ala Ser Pro Gly Glu
Ser Val Ser Ile Ser Cys Lys Ala Ser Lys Ser 35 40 45 Ile Ser Asn
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Glu Ala Asn 50 55 60 Lys
Leu Leu Val Tyr Tyr Gly Ser Thr Leu Arg Ser Gly Ile Pro Ser 65 70
75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Arg 85 90 95 Asn Leu Glu Pro Ala Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Tyr 100 105 110 Glu Arg Pro Leu Thr Phe Gly Ser Gly Thr Lys
Leu Glu 115 120 125 2 11 PRT Unknown SITE (1)...(11) CDR1 of
YFC51.1 light chain 2 Lys Ala Ser Lys Ser Ile Ser Asn Tyr Leu Ala 1
5 10 3 7 PRT Unknown SITE (1)...(7) CDR2 of YFC51.1 light chain 3
Tyr Gly Ser Thr Leu Arg Ser 1 5 4 9 PRT Unknown SITE (1)...(9) CDR3
of YFC51.1 light chain 4 Gln Gln Tyr Tyr Glu Arg Pro Leu Thr 1 5 5
139 PRT Unknown SITE (1)...(139) YFC51.1 heavy chain variable
region 5 Met Lys Cys Ser Trp Ile Asn Leu Phe Leu Met Ala Leu Ala
Ser Gly 1 5 10 15 Val Tyr Ala Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Arg Arg 20 25 30 Pro Gly Ser Ser Val Lys Leu Ser Cys Lys
Thr Ser Gly Tyr Ser Ile 35 40 45 Lys Asp Tyr Leu Leu His Trp Val
Lys His Arg Pro Glu Tyr Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile
Asp Pro Glu Asp Gly Glu Thr Lys Tyr Gly 65 70 75 80 Gln Lys Phe Gln
Ser Arg Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala
Tyr Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Thr 100 105 110
Tyr Phe Cys Thr Arg Gly Glu Tyr Arg Tyr Asn Ser Trp Phe Asp Tyr 115
120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 6 5 PRT
Unknown SITE (1)...(5) CDR1 of YFC51.1 heavy chain 6 Asp Tyr Leu
Leu His 1 5 7 17 PRT Unknown SITE (1)...(17) CDR2 of YFC51.1 heavy
chain 7 Trp Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Gly Gln Lys Phe
Gln 1 5 10 15 Ser 8 11 PRT Unknown SITE (1)...(11) CDR3 of YFC51.1
heavy chain 8 Gly Glu Tyr Arg Tyr Asn Ser Trp Phe Asp Tyr 1 5 10 9
139 PRT Artificial Sequence Humanized heavy chain variable region
with signal sequence 9 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Arg 20 25 30 Pro Ser Gln Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Phe Thr Phe 35 40 45 Thr Asp Tyr Leu Leu
His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu 50 55 60 Glu Trp Ile
Gly Trp Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Gly 65 70 75 80 Gln
Lys Phe Gln Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn 85 90
95 Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
100 105 110 Tyr Tyr Cys Ala Arg Gly Glu Tyr Arg Tyr Asn Ser Trp Phe
Asp Tyr 115 120 125 Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser 130
135 10 127 PRT Artificial Sequence Humanized light chain variable
region with signal sequence 10 Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile 35 40 45 Ser Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 50 55 60 Leu
Leu Ile Tyr Tyr Gly Ser Thr Leu Arg Ser Gly Val Pro Ser Arg 65 70
75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser 85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Glu 100 105 110 Arg Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 115 120 125 11 112 PRT Unknown SITE (1)...(112)
Murine mAb 1D9 light chain variable region 11 Asp Val Val Met Thr
Gln Thr Pro Leu Thr Leu Ser Val Thr Val Gly 1 5 10 15 His Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp
Gly Lys Thr Phe Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40
45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 110 12 117 PRT Unknown SITE
(1)...(117) Murine mAb 1D9 heavy chain variable region 12 Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Ala Tyr 20
25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Arg Ile Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Tyr Thr Ile Ser Arg Asp
Asp Ser Glu Ser Met 65 70 75 80 Leu Phe Leu Gln Met Asn Asn Leu Lys
Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Thr Phe Tyr Gly
Asn Gly Val Trp Gly Thr Gly Thr Thr 100 105 110 Val Thr Val Ser Ser
115 13 111 PRT Homo sapiens 13 Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr
Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg
Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
Gly 85 90 95 Thr His Trp Pro Phe Thr Phe Gly Gln Gly Thr Arg Leu
Glu Ile 100 105 110 14 112 PRT Artificial Sequence Humanized
sequence 14 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe Leu Asn Trp Phe Gln
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val
Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His
Phe Pro Tyr Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110
15 112 PRT Artificial Sequence Humanized sequence 15 Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln
Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25
30 Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 16 112 PRT Artificial
Sequence Humanized sequence 16 Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys
Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe
Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg
Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln
Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu
Glu Ile Lys 100 105 110 17 112 PRT Artificial Sequence Humanized
sequence 17 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Leu Gly 1 5 10 15 His Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val
Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His
Phe Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110
18 112 PRT Artificial Sequence Humanized sequence 18 Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 His
Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25
30 Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 19 119 PRT Homo sapiens
19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn
Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr
Asp Ser Leu Pro Pro His Arg Val Trp Gly Gln Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115 20 117 PRT Artificial Sequence Humanized
sequence 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Thr Lys Asn Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Thr Thr Phe Tyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 21 117 PRT Artificial Sequence Humanized
sequence 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser
Phe Asn Ala Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Thr Lys Asn Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Thr Thr Phe Tyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 22 117 PRT Artificial Sequence Humanized
sequence 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser
Phe Asn Ala Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Thr Lys Asn Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Tyr
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Thr Thr Phe Tyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 23 117 PRT Artificial Sequence Humanized
sequence 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser
Phe Asn Ala Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Thr Lys Asn Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Tyr
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Val Thr Phe Tyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115
* * * * *