U.S. patent application number 09/905848 was filed with the patent office on 2002-01-31 for anti-ccr2 antibodies and methods of use therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to LaRosa, Gregory J..
Application Number | 20020012664 09/905848 |
Document ID | / |
Family ID | 22398750 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012664 |
Kind Code |
A1 |
LaRosa, Gregory J. |
January 31, 2002 |
Anti-CCR2 antibodies and methods of use therefor
Abstract
The present invention relates to an antibody or functional
fragment thereof which binds to a mammalian (e.g., human)
CC-chemokine receptor 2 (CCR2) or a portion of the receptor and
blocks binding of a ligand to the receptor. The invention further
relates to a method of inhibiting the interaction of a cell bearing
mammalian CCR2 with a ligand thereof, and to use of the antibodies
and fragments in therapeutic, prophylactic and diagnostic
methods.
Inventors: |
LaRosa, Gregory J.; (West
Roxbury, MA) |
Correspondence
Address: |
Lisa M. Treannie, Esq.
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
22398750 |
Appl. No.: |
09/905848 |
Filed: |
July 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09905848 |
Jul 13, 2001 |
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09121781 |
Jul 23, 1998 |
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6312689 |
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Current U.S.
Class: |
424/130.1 ;
435/5; 435/7.9 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 37/02 20180101; A61P 37/08 20180101; A61P 9/14 20180101; A61P
17/02 20180101; A61P 21/00 20180101; A61P 19/00 20180101; A61P
41/00 20180101; A61P 27/16 20180101; A61P 9/00 20180101; A61P 35/00
20180101; A61P 5/48 20180101; C07K 2317/76 20130101; A61P 35/02
20180101; A61P 11/00 20180101; A61P 25/00 20180101; A61P 17/06
20180101; A61P 5/14 20180101; A61P 9/10 20180101; A61P 11/02
20180101; A61P 1/04 20180101; A61P 43/00 20180101; A61P 29/00
20180101; A61P 11/06 20180101; A61P 19/08 20180101; A61P 17/04
20180101; A61P 31/12 20180101; A61P 31/18 20180101; A61P 37/00
20180101; A61P 37/06 20180101; C07K 16/2866 20130101; A61P 13/12
20180101; A61P 21/04 20180101; A61K 2039/505 20130101; A61P 3/10
20180101; A61P 19/02 20180101 |
Class at
Publication: |
424/130.1 ;
435/7.9; 435/5 |
International
Class: |
A61K 039/395; G01N
033/53; G01N 033/542; C12Q 001/70 |
Claims
What is claimed is:
1. A method of detecting or identifying an agent which binds a
mammalian CC-chemokine receptor 2 or ligand-binding variant
thereof, comprising combining: a) an agent to be tested; b) an
antibody or antigen-binding fragment which binds to the amino
terminal domain of mammalian CC-chemokine receptor 2; and c) a
composition comprising a mammalian CC-chemokine receptor 2 or a
ligand-binding variant thereof, under conditions suitable for
binding of said antibody or antigen-binding fragment to said
mammalian CC-chemokine receptor 2 or ligand-binding variant
thereof, and detecting or measuring binding of said antibody or
antigen-binding fragment to said mammalian CC-chemokine receptor 2
or ligand-binding variant thereof.
2. A method according to claim 1, wherein the formation of a
complex between said antibody or antigen-binding fragment and said
mammalian CC-chemokine receptor 2 or ligand-binding variant is
monitored, and wherein a decrease in the amount of complex formed
relative to a suitable control is indicative that the agent binds
said receptor or ligand-binding variant thereof.
3. A method according to claim 1, wherein said composition
comprising a mammalian CC-chemokine receptor 2 or a ligand-binding
variant thereof is a cell bearing recombinant CC-chemokine receptor
2 or ligand-binding variant thereof.
4. A method according to claim 3, wherein said composition
comprising a mammalian CC-chemokine receptor 2 or a ligand-binding
variant thereof is a membrane fraction of said cell bearing
recombinant CC-chemokine receptor 2 or ligand-binding variant
thereof.
5. A method according to claim 1 wherein the antibody or
antigen-binding fragment is labeled with a label selected from the
group consisting of a radioisotope, spin label, antigen label,
enzyme label, fluorescent group and chemiluminescent group.
6. A method according to claim 1 wherein the agent is an antibody
or antigen-binding fragment having specificity for a mammalian
CC-chemokine receptor 2.
7. A method according to claim 1, wherein said mammalian
CC-chemokine receptor 2 or ligand-binding variant thereof is a
human CC-chemokine receptor 2 or ligand-binding variant
thereof.
8. A method according to claim 1, wherein said antibody or
antigen-binding fragment is a monoclonal antibody or
antigen-binding fragment thereof.
9. A method according to claim 1, wherein said antibody or
antigen-binding fragment is a chimeric antibody or antigen-binding
fragment thereof.
10. A method according to claim 1, wherein said antibody or
antigen-binding fragment is a human antibody or antigen-binding
fragment thereof.
11. A method according to claim 1, wherein said antibody or
antigen-binding fragment is a humanized antibody or antigen-binding
fragment thereof.
12. A method according to claim 11, wherein said humanized antibody
or antigen-binding fragment thereof comprises one or more
antigen-binding regions of monoclonal antibody 1D9.
13. A method according to claim 11, wherein said humanized antibody
comprises one or more complementarity-determining regions of
monoclonal antibody 1D9.
14. A method according to claim 13, wherein said humanized antibody
comprises six complementarity-determining regions of monoclonal
antibody 1D9.
15. A method according to claim 11, wherein said humanized antibody
or antigen-binding fragment thereof comprises one or more
antigen-binding regions of monoclonal antibody 8G2.
16. A method according to claim 11, wherein said humanized antibody
comprises one or more complementarity-determining regions of
monoclonal antibody 8G2.
17. A method according to claim 16, wherein said humanized antibody
comprises six complementarity-determining regions of monoclonal
antibody 8G2.
18. A method according to claim 1, wherein said antigen-binding
fragment is selected from the group consisting of an Fv fragment,
an Fab fragment, an Fab' fragment and an F(ab').sub.2 fragment.
19. A method according to claim 1, wherein said antibody or
antigen-binding fragment thereof is an antibody or fragment having
the epitopic specificity of monoclonal antibody 1D9.
20. A method according to claim 1, wherein said antibody or
antigen-binding fragment thereof is an antibody or fragment having
the epitopic specificity of monoclonal antibody 8G2.
21. A method according to claim 1, wherein said antibody or
antigen-binding fragment thereof is an antibody or fragment which
can compete with monoclonal antibody 1D9 for binding to mammalian
CC-chemokine receptor 2.
22. A method according to claim 1, wherein said antibody or
antigen-binding fragment thereof is an antibody or fragment which
can compete with monoclonal antibody 8G2 for binding to mammalian
CC-chemokine receptor 2.
23. A method according to claim 1, wherein said antibody or
antigen-binding fragment thereof binds a portion of the
amino-terminal domain which is from about amino acid 1 to about
amino acid 30 of said receptor.
24. A method of detecting or identifying an agent which binds a
mammalian CC-chemokine receptor 2 or ligand-binding variant
thereof, comprising combining: a) an agent to be tested; b)
monoclonal antibody 1D9 or an antigen-binding fragment thereof, and
c) a composition comprising a mammalian CC-chemokine receptor 2 or
a ligand-binding variant thereof, under conditions suitable for
binding of said monoclonal antibody 1D9 or antigen-binding fragment
to said mammalian CC-chemokine receptor 2 or ligand-binding variant
thereof, and detecting or measuring binding of said monoclonal
antibody 1D9 or antigen-binding fragment to said mammalian
CC-chemokine receptor 2 or ligand-binding variant thereof.
25. A method of detecting or identifying an agent which binds a
mammalian CC-chemokine receptor 2 or ligand-binding variant
thereof, comprising combining: a) an agent to be tested; b)
monoclonal antibody 8G2 or an antigen-binding fragment thereof; and
c) a composition comprising a mammalian CC-chemokine receptor 2 or
a ligand-binding variant thereof, under conditions suitable for
binding of said monoclonal antibody 8G2 or antigen-binding fragment
to said mammalian CC-chemokine receptor 2 or ligand-binding variant
thereof, and detecting or measuring binding of said monoclonal
antibody 8G2 or antigen-binding fragment to said mammalian
CC-chemokine receptor 2 or ligand-binding variant thereof.
Description
RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/121,781, filed Jul. 23, 1998, the entire teachings of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Over the past several years a growing family of leukocyte
chemoattractant/activating factors, termed chemokines, has been
described (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648
(1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873 (1994);
Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994)). Members of
this family are produced and secreted by many cell types in
response to early inflammatory mediators such as IL-1.beta. or
TNF.alpha.. The chemokine superfamily comprises two main branches:
the .alpha.-chemokines (or CXC chemokines) and the
.beta.-chemokines (CC chemokines). The .alpha.-chemokine branch
includes proteins such as IL-8, neutrophil activating peptide-2
(NAP-2), melanoma growth stimulatory activity (MGSA/gro or
GRO.alpha.), and ENA-78, each of which have attracting and
activating effects predominantly on neutrophils. The members of the
.beta.-chemokine branch affect other cell types such as monocytes,
lymphocytes, basophils, and eosinophils (Oppenheim, J. J. et al.,
Annu. Rev. Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv.
Imunol., 55:97-179 (1994); Miller and Krangel, Crit. Rev. Immunol.,
12:17-46 (1992); Jose, P. J., et al., J. Exp. Med.,
179:881-118(1994); Ponath, P. D., et al., J. Clin. Invest.,
97:604-612 (1996)), and include proteins such as monocyte
chemotactic proteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES,
and macrophage inflammatory proteins (MIP-1.alpha., MIP-1.beta.).
Recently a new class of membrane-bound chemokines designated CX3C
chemokines has been identified (Bazan, J. F., et al., Nature
385:640-644 (1997)). Chemokines can mediate a range of
pro-inflammatory effects on leukocytes, such as triggering of
chemotaxis, degranulation, synthesis of lipid mediators, and
integrin activation (Oppenheim, J. J. et al., Annu. Rev. Immunol.,
9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55:97-179
(1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol.,
12:17-46 (1992)). Lately, certain .beta.-chemokines have been shown
to suppress HIV-1 infection of human T cell lines in vitro (Cocchi,
F., et al., Science (Wash. D.C.), 270:1811-1815 (1995)).
[0003] Chemokines bind to 7 transmembrane spanning (7TMS) G
protein-coupled receptors (Murphy, P. M., Annu. Rev. Immunol.,
12:593-633 (1994)). Some known receptors for the CC or .beta.
chemokines include CCR1, which binds MIP-1.alpha. and RANTES
(Neote, K., et al., Cell, 72:415-425 (1993); Gao, J. L., J. Exp.
Med., 177:1421-1427 (1993)); CCR2, which binds chemokines including
MCP-1, MCP-2, MCP-3 and MCP-4 (Charo, I. F., et al., Proc. Natl.
Acad. Sci. USA, 91:2752-2756 (1994); Myers, S. J., et al., J. Biol.
Chem., 270:5786-5792 (1995); Gong et al., J. Biol. Chem.
272:11682-11685 (1997); Garcia-Zepeda et al., J. Immunol.
157:5613-5626 (1996)); CCR3, which binds chemokines including
eotaxin, RANTES and MCP-3 (Ponath, P. D., et al., J. Exp. Med.,
183:2437-2448 (1996)); CCR4, which has been found to signal in
response to MCP-1, MIP-1.alpha., and RANTES (Power, C. A., et al.,
J. Biol. Chem., 270:19495-19500 (1995)); and CCR5, which has been
shown to signal in response to MIP-1.alpha., MIP-1.beta. and RANTES
(Boring, L., et al., J. Biol. Chem., 271 (13):7551-7558 (1996);
Raport, C. J., J. Biol. Chem., 271:17161-17166 (1996); and Samson,
M. et al., Biochemistry, 35:3362-3367 (1996)).
[0004] CCR2 is expressed on the surface of several leukocyte
subsets, and appears to be expressed in two slightly different
forms (CCR2a and CCR2b) due to alternative splicing of the mRNA
encoding the carboxy-terminal region (Charo et al., Proc. Natl.
Acad. Sci. USA 91:2752-2756 (1994)). MCP-1 acts upon monocytes,
lymphocytes and basophils, inducing chemotaxis, granule release,
respiratory burst and histamine and cytokine release. Studies have
suggested that MCP-1 is implicated in the pathology of diseases
such as rheumatoid arthritis, atherosclerosis, granulomatous
diseases and multiple sclerosis (Koch, J. Clin. Invest. 90:772-79
(1992); Hosaka et al., Clin. Exp. Immunol. 97:451-457 (1994);
Schwartz et al., Am. J. Cardiol. 71(6):9B-14B (1993); Schimmer et
al., J. Immunol. 160:1466-1471 (1998); Flory et al., Lab. Invest.
69:396-404 (1993); Gong et al., J. Exp. Med. 186:131-137 (1997)).
Additionally, CCR2 can act as a co-receptor for HIV (Connor et al.,
J. Exp. Med. 185:621-628 (1997)). Thus, CCR2 receptor antagonists
may represent a new class of important therapeutic agents.
SUMMARY OF THE INVENTION
[0005] The present invention relates to an antibody
(immunoglobulin) or functional fragment thereof (e.g., an
antigen-binding fragment) which binds to a mammalian CC-chemokine
receptor 2 (also referred to as CCR2, CKR-2, MCP-1RA or MCP-1RB) or
portion of the receptor (anti-CCR2). In one embodiment, the
antibody of the present invention or fragment thereof has
specificity for human or rhesus CCR2 or a portion thereof. In
another embodiment, the antibody or fragment of the invention
blocks binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to
the receptor and inhibits function associated with binding of the
ligand to the receptor (e.g., leukocyte trafficking). For example,
as described herein, antibodies and fragments thereof of the
present invention which bind human or rhesus CCR2 or a portion
thereof, can block binding of a chemokine (e.g., MCP-1, MCP-2,
MCP-3, MCP-4) to the receptor and inhibit function associated with
binding of the chemokine to the receptor. In one embodiment, the
antibody is monoclonal antibody (mAb) LS132.1D9 (1D9) or an
antibody which can compete with 1D9 for binding to human CCR2 or a
portion of human CCR2. Functional fragments of the foregoing
antibodies are also envisioned.
[0006] In another embodiment, the antibody or functional fragment
of the present invention binds human CCR2 or a portion thereof, and
inhibits human immunodeficiency virus (HIV) binding to the
receptor, thereby inhibiting function associated with binding of
HIV to the receptor (e.g., HIV antigen release and infectivity). In
one embodiment, the antibody is monoclonal antibody 1D9 or an
antibody which can compete with 1D9 for binding to human CCR2 or a
portion of human CCR2.
[0007] The present invention also relates to an antibody or
functional fragment thereof (e.g., an antigen-binding fragment)
which binds to a mammalian CCR2 or portion of the receptor and
provides increased fluorescent staining intensity of CCR2 or
compositions comprising CCR2 relative to other anti-CCR2
antibodies. In one embodiment, the antibody is monoclonal antibody
1D9 or LS 132.8G2 (8G2) or an antibody which can compete with 1D9
or 8G2 for binding to human CCR2 or a portion of human CCR2.
[0008] The present invention further relates to a method of
inhibiting the interaction of a cell bearing mammalian (e.g.,
human, non-human primate or murine) CCR2 with a ligand thereof,
comprising contacting the cell with an effective amount of an
antibody or functional fragment thereof which binds to a mammalian
CCR2 or a portion of CCR2. Suitable cells include granulocytes,
leukocytes, such as monocytes, macrophages, basophils and
eosinophils, mast cells, and lymphocytes including T cells (e.g.,
CD8+ cells, CD4+ cells, CD25+ cells, CD45RO+ cells), and other
cells expressing CCR2 such as a recombinant cell expressing CCR2
(e.g., transfected cells). In a particular embodiment, the antibody
is 1D9 or an antibody which can compete with 1D9 for binding to
human CCR2 or a portion of human CCR2.
[0009] Another embodiment of the invention relates to a method of
inhibiting the interaction of a cell bearing mammalian CCR2 with a
chemokine, comprising contacting said cell with an effective amount
of an antibody or functional fragment thereof which binds to CCR2
or a portion of said receptor. In one embodiment of the method, the
antibody or functional fragment thereof is any one or more of 1D9,
an antigen-binding fragment of 1D9 or an antibody or fragment
thereof having an epitopic specificity which is the same as or
similar to that of 1D9. Furthermore, the invention relates to a
method of inhibiting a function associated with binding of a
chemokine to CCR2, comprising administering an effective amount of
an antibody or functional fragment thereof which binds to a
mammalian CCR2 protein or a portion of said receptor. In one aspect
of the method, the antibody or functional fragment thereof is any
one or more of 1D9, an antigen-binding fragment of 1D9 or an
antibody or fragment thereof having an epitopic specificity which
is the same as or similar to that of 1D9.
[0010] Another aspect of the invention is a method of identifying
expression of a mammalian CCR2 or portion of the receptor by a
cell. According to the method, a composition comprising a cell or
fraction thereof (e.g., a membrane fraction) is contacted with an
antibody or functional fragment thereof (e.g., 1D9 or 8G2) which
binds to a mammalian CCR2 protein or portion of the receptor under
conditions appropriate for binding of the antibody thereto, and the
formation of a complex between said antibody or fragment and said
protein or portion thereof is detected. Detection of the complex,
directly or indirectly, indicates the presence of the receptor on
the cell. The present invention also relates to a kit for use in
detecting the presence of CCR2 or a portion thereof in a biological
sample, comprising an antibody or functional fragment thereof which
binds to a mammalian CC-chemokine receptor 2 or a portion of said
receptor, and one or more ancillary reagents suitable for detecting
the presence of a complex between said antibody or fragment and
said protein or portion thereof.
[0011] Also encompassed by the present invention are methods of
identifying additional ligands or other substances which bind a
mammalian CCR2 protein, including inhibitors and/or promoters of
mammalian CCR2 function. For example, agents having the same or a
similar binding specificity as that of an antibody of the present
invention or functional fragment thereof can be identified by a
competition assay with said antibody or fragment. Thus, the present
invention also encompasses methods of identifying ligands or other
substances which bind the CCR2 receptor, including inhibitors
(e.g., antagonists) or promoters (e.g., agonists) of receptor
function. In one embodiment, cells which naturally express CCR2
receptor protein or suitable host cells which have been engineered
to express a CCR2 receptor or variant encoded by a nucleic acid
introduced into said cells are used in an assay to identify and
assess the efficacy of ligands, inhibitors or promoters of receptor
function. Such cells are also useful in assessing the function of
the expressed receptor protein or polypeptide.
[0012] Thus, the invention also relates to a method of detecting or
identifying an agent which binds a mammalian CCR2 or ligand binding
variant thereof, comprising combining an agent to be tested, an
antibody or antigen-binding fragment of the present invention
(e.g., monoclonal antibody 1D9, an antibody having an epitopic
specificity which is the same as or similar to that of 1D9,
antigen-binding fragments of 1D9, monoclonal antibody 8G2, an
antibody having an epitopic specificity which is the same as or
similar to that of 8G2, and antigen-binding fragments of 8G2) and a
composition comprising a mammalian CCR2 protein or a ligand binding
variant thereof. The foregoing components can be combined under
conditions suitable for binding of the antibody or antigen-binding
fragment to mammalian CCR2 protein or a ligand binding variant
thereof, and binding of the antibody or fragment to the mammalian
CCR2 protein or ligand binding variant is detected or measured,
either directly or indirectly, according to methods described
herein or other suitable methods. A decrease in the amount of
complex formed relative to a suitable control (e.g., in the absence
of the agent to be tested) is indicative that the agent binds said
receptor or variant. The composition comprising a mammalian CCR2
protein or a ligand binding variant thereof can be a membrane
fraction of a cell bearing recombinant CCR2 protein or ligand
binding variant thereof. The antibody or fragment thereof can be
labeled with a label such as a radioisotope, spin label, antigen
label, enzyme label, fluorescent group and chemiluminescent group.
These and similar assays can be used to detect agents, including
ligands (e.g., chemokines which interact with CCR2) or other
substances, including inhibitors or promoters of receptor function,
which can bind CCR2 and compete with the antibodies described
herein for binding to the receptor.
[0013] According to the present invention, ligands, inhibitors or
promoters of receptor function can be identified in a suitable
assay, and further assessed for therapeutic effect. Inhibitors of
receptor function can be used to inhibit (reduce or prevent)
receptor activity, and ligands and/or promoters can be used to
induce (trigger or enhance) normal receptor function where
indicated. The present invention also provides a method of treating
inflammatory diseases, autoimmune diseases, atherosclerosis, and
graft rejection, or HIV infection, comprising administering an
inhibitor of receptor function (e.g., chemokine binding or HIV
binding) to an individual (e.g., a mammal, such as a human). The
present invention further provides a method of stimulating receptor
function by administering a novel ligand or promoter to an
individual, providing a new approach to selective stimulation of
leukocyte function, which is useful, for example, in the treatment
of infectious diseases and cancer.
[0014] Another aspect of the invention relates to a method of
inhibiting HIV infection of a cell which expresses a mammalian CCR2
or portion thereof, comprising contacting the cell with an
effective amount of an antibody or functional fragment thereof
which binds to a mammalian CCR2 or portion of the receptor and
inhibits HIV binding and infection. In a particular embodiment of
the invention, the antibody or functional fragment thereof is any
of 1D9, an antibody having an epitopic specificity which is the
same as or similar to that of 1D9, an antibody which can compete
with 1D9 for binding to human CCR2, and antigen-binding fragments
thereof.
[0015] Also encompassed by the present invention is a method of
inhibiting (e.g., treating) HIV in a patient, comprising
administering to the patient an effective amount of an antibody or
functional fragment thereof which binds to a mammalian CCR2 or a
portion of said receptor and inhibits HIV binding to the CCR2
receptor. The anti-CCR2 antibody or fragment can be administered
alone or in combination with one or more additional therapeutic
agents, e.g., one or more antibodies which bind a co-receptor for
HIV infection and inhibit binding to said co-receptor, such as an
anti-CCR3, anti-CCR5, and/or anti-CXCR4 antibody.
[0016] Another aspect of the invention also relates to a method of
preventing or inhibiting HIV infection in an individual, comprising
administering to the individual an effective amount of an antibody
or functional fragment thereof which binds to CCR2 and inhibits HIV
binding to CCR2. According to the method, preventing HIV infection
includes treatment in order to prevent (reduce or eliminate)
infection of new cells in an infected individual or in order to
prevent infection in an individual who may be, may have been or has
been exposed to HIV. For example, individuals such as an HIV
infected individual, a fetus of an HIV infected female, or a health
care worker can be treated according to the method of the present
invention.
[0017] The present invention also encompasses a method of
inhibiting leukocyte trafficking in a patient, comprising
administering to the patient an effective amount of an antibody or
functional fragment thereof which binds to a mammalian CCR2 or
portion of said receptor and inhibits function associated with
binding of a ligand to the receptor.
[0018] The present invention also relates to a method of inhibiting
or treating CCR2-mediated disorders, such as inflammatory
disorders, comprising administering to a patient an effective
amount of an antibody or functional fragment thereof which binds to
a mammalian CCR2 or portion of said receptor and inhibits
CCR2-mediated function.
[0019] The present invention further relates to an antibody or
fragment thereof as described herein (e.g., monoclonal antibody 1D9
or an antigen-binding fragment thereof) for use in therapy
(including prophylaxis) or diagnosis, and to the use of such an
antibody or fragment for the manufacture of a medicament for the
treatment of a CCR2-mediated disorder, or other disease or
inflammatory condition as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-1O are fluorescence activated cell scanning (FACS)
histogram profiles illustrating that mAbs 1D9 and 8G2 stain CCR2
transfectants but not CCR5 or CCR1 transfectants. L1/2 (also
referred to herein as L1.2) murine pre-B lymphoma host cells were
transfected with CCR2, CCR5 and CCR1 as indicated, and stained with
antibodies with different receptor specificities. Staining was
analyzed by flow cytometry.
[0021] FIGS. 2A-2L are FACS dot plots showing expression of CCR2 on
most monocytes, a subpopulation of lymphocytes and a small subset
of granulocytes. Whole blood cells were stained with one of three
anti-CCR2 mAbs (5A11, generated using a peptide consisting of the
first 32 amino acids of the CCR2 amino-terminus as an immunogen,
and 1D9 and 8G2 generated as described herein using CCR2b L1/2 cell
transfectants as the immunogen). Staining was analyzed by flow
cytometry, and the lymphocyte, granulocyte and monocyte populations
were gated using the forward and side light scatter. The X-axis
represents forward light scatter (a measure of cell size), and the
Y-axis fluorescence intensity of staining for CCR2. The level of
negative control staining is indicated by a line.
[0022] FIGS. 3A-3I are FACS dot plots showing that mAb 1D9 stains
an IgE positive population in peripheral blood (basophils) using
two-color staining for IgE and CCR2. Whole blood cells were first
stained with either a negative control antibody (anti-Flag),
anti-CCR2 antibody 1D9, or an anti-CXCR1 antibody, as indicated,
and detected by an anti-mouse-FITC conjugate. A second staining was
done using either PBS or a biotinylated antibody specific for IgE
or CD16, as indicated, and detected with a
streptavidin-phycoerythrin. Staining was analyzed by flow
cytometry.
[0023] FIG. 4 illustrates that mAb 1D9 inhibits [.sup.125I]MCP-1
binding to THP-1 cell membranes. 3.0 .mu.g of THP-1 membrane
protein was incubated with 0.1 nM [.sup.125I]MCP-1 in the presence
of various concentrations of 1D9 or the isotype-matched anti-CXCR3
antibody 1C6. The amount of bound tracer was determined by
separation of free from bound by filtration and scintillation
counting. The data was analyzed to determine the IC.sub.50 value by
non-linear regression using a 4-parameter logistic equation with
KaleidaGraph software.
[0024] FIG. 5 illustrates that mAb 1D9 inhibits [.sup.125I]MCP-1
binding to fresh human PBMC. Freshly isolated peripheral blood
mononuclear cells (500,000) were incubated with 0.1 nM
[.sup.125I]MCP-1 in the presence of various concentrations of 1D9
or the isotype-matched anti-CXCR3 antibody 1C6. The amount of bound
tracer was determined by separation of free from bound by
filtration and scintillation counting. The data was analyzed to
determine the IC.sub.50 value as for FIG. 4.
[0025] FIGS. 6A and 6B are graphs demonstrating that mAb 1D9
inhibits MCP-1-induced chemotaxis, but not RANTES-induced
chemotaxis, of fresh PBMC. FIG. 6A shows the results of chemotaxis
assays of PBMC to 10 nM MCP-1 with no antibody, or 0.1 or 10
.mu.g/ml of 1D9 or nonspecific murine IgG2a. The spontaneous
nonspecific migration is also indicated. FIG. 6B shows the results
of chemotaxis assays of PBMC to 10 nM RANTES with no antibody, 10
.mu.g/ml 1D9 or 10 .mu.g/ml nonspecific murine IgG2a. The
spontaneous nonspecific migration in the absence of RANTES is also
indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to an antibody (anti-CCR2) or
functional fragment thereof which binds mammalian CC-chemokine
receptor 2 (CCR2, CKR-2, MCP-1RA or MCP-1RB) or a portion of CCR2.
In one embodiment, the antibody has specificity for human or rhesus
CCR2 or portion thereof. In one embodiment, the antibodies
(immunoglobulins) are raised against an isolated and/or recombinant
mammalian CCR2 or portion thereof (e.g., peptide) or against a host
cell which expresses mammalian CCR2. In a preferred embodiment, the
antibodies specifically bind human CCR2 receptor(s) (e.g., CCR2a
and/or CCR2b) or a portion thereof, and in a particularly preferred
embodiment the antibodies have specificity for a naturally
occurring or endogenous human CCR2. As used herein, "CC-chemokine
receptor 2" ("CCR2") refers to CC-chemokine receptor 2a and/or
CC-chemokine receptor 2b. Antibodies or functional fragments
thereof which can inhibit one or more functions characteristic of a
mammalian CCR2, such as a binding activity (e.g., ligand, inhibitor
and/or promoter binding), a signaling activity (e.g., activation of
a mammalian G protein, induction of a rapid and transient increase
in the concentration of cytosolic free calcium [Ca.sup.2+]I),
and/or stimulation of a cellular response (e.g., stimulation of
chemotaxis, exocytosis or inflammatory mediator release by
leukocytes, integrin activation) are also encompassed by the
present invention, such as an antibody which can inhibit binding of
a ligand (i.e., one or more ligands) to CCR2 and/or one or more
functions mediated by CCR2 in response to a ligand. For example, in
one aspect, the antibodies or functional fragments thereof can
inhibit (reduce or prevent) the interaction of receptor with a
natural ligand, such as MCP-1, MCP-2, MCP-3 and/or MCP-4. In
another aspect, an antibody or functional fragment thereof that
binds to CCR2 can inhibit binding of MCP-1, MCP-2, MCP-3 and/or
MCP-4 and/or HIV to mammalian CCR2 (e.g., human CCR2, non-human
primate CCR2, murine CCR2). The antibodies or functional fragments
thereof of the present invention can inhibit functions mediated by
human CCR2, including leukocyte trafficking, HIV entry into a cell,
T cell activation, inflammatory mediator release and/or leukocyte
degranulation. Preferably, the antibodies or fragments can bind
CCR2 with an affinity of at least about 0.1.times.10.sup.-9 M,
preferably at least about 1.times.10.sup.-9 M, and more preferably
at least about 3.times.10.sup.-9 M. In a particular embodiment,
antibodies or functional fragments thereof demonstrate inhibition
of chemokine-induced (e.g., MCP-1-induced) chemotaxis of cells
(e.g., PBMC) at less than about 150 .mu.g/ml, preferably less than
about 100 .mu.g/ml, more preferably less than about 50 .mu.g/ml,
and even more preferably less than about 20 .mu.g/ml.
[0027] In a further embodiment of the invention, the antibodies or
functional fragments thereof of the invention can inhibit binding
of a CCR2 ligand (e.g., a chemokine) to CCR2 with an IC.sub.50 of
less than about 1.0 .mu.g/ml, preferably less than about 0.05
.mu.g/ml, and more preferably less than about 0.005 .mu.g/ml.
[0028] Murine monoclonal antibodies specific for CCR2, designated
1D9 and 8G2, were produced as described herein. In a preferred
embodiment, the antibodies of the present invention bind human
CCR2, and have an epitopic specificity which is the same as or
similar to that of murine 1D9 or 8G2 antibody described herein.
Antibodies with an epitopic specificity which is the same as or
similar to that of murine 1D9 monoclonal antibody can be identified
by their ability to compete with murine 1D9 monoclonal antibody for
binding to human CCR2 (e.g., to cells bearing human CCR2, such as
transfectants bearing CCR2, CD8+ cells, CD4+ cells, CDR45RO+ cells,
CD25+ cells, monocytes, dendritic cells, macrophages and
basophils). Similarly, antibodies with an epitopic specificity
which is the same as or similar to that of murine 8G2 monoclonal
antibody can be identified by their ability to compete with murine
8G2 monoclonal antibody for binding to human CCR2. Using receptor
chimeras (Rucker et al., Cell 87:437-446 (1996)), the binding site
of mAbs 1D9 and 8G2 has been mapped to the amino-terminal domain of
human CC-chemokine receptor 2, specifically to an epitope
comprising from about amino acid 1 to about amino acid 30 of the
protein. Using these or other suitable techniques, antibodies
having an epitopic specificity which is the same as or similar to
that of an antibody of the present invention can be identified.
mAbs 1D9 and 8G2 have epitopic specificity for the amino-terminal
domain of the CCR2 receptor, e.g., from about amino acid number 1
to about amino acid number 30 of the receptor protein. Thus, the
invention pertains to an antibody or functional portion thereof
which binds to the amino-terminal domain or portion thereof of
mammalian CC-chemokine receptor 2, and particularly to an epitope
comprising from about amino acid 1 to about amino acid 30 of
mammalian CC-chemokine receptor 2.
[0029] The invention also relates to a bispecific antibody, or
functional fragment thereof (e.g., F(ab').sub.2), which has the
same or similar epitopic specificity as at least two of the
antibodies described herein (see, e.g., U.S. Pat. No. 5,141,736
(Iwasa et al.), U.S. Pat. Nos. 4,444,878, 5,292,668, 5,523,210 (all
to Paulus et al.) and U.S. Pat. No. 5,496,549 (Yamazaki et al.).
For example, a bispecific antibody of the present invention can
have the same or similar epitopic specificity as mAb 1D9 and 8G2,
e.g., binds the amino terminal domain, or portion thereof, of
mammalian CCR2 protein.
[0030] Hybridoma cell lines producing antibodies according to the
present invention were deposited on Jul. 17, 1998, on behalf of
LeukoSite, Inc., 215 First Street, Cambridge, Mass. 02142, 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). The present invention also
pertains to the hybridoma cell lines deposited under ATCC Accession
No. HB-12549 and ATCC Accession No. HB-12550, as well as to the
monoclonal antibodies produced by the hybridoma cell lines
deposited under ATCC Accession Nos. HB-12549 and HB-12550.
[0031] The antibodies of the present invention can be polyclonal or
monoclonal, and the term "antibody" is intended to encompass both
polyclonal and monoclonal antibodies. Furthermore, it is understood
that methods described herein which utilize 8G2 can also utilize
functional fragments (e.g., antigen-binding fragments) of 8G2,
antibodies which have the same or similar epitopic specificity as
8G2, and combinations thereof, optionally in combination with
antibodies or fragments having an epitopic specificity which is not
the same as or similar to 8G2; similarly, methods described as
utilizing 1D9 can also utilize functional fragments of 1D9,
antibodies which have the same or similar epitopic specificity as
1D9, and combinations thereof, optionally in combination with
antibodies or fragments having an epitopic specificity which is not
the same as or similar to 1D9. Antibodies of the present invention
can be raised against an appropriate immunogen, such as isolated
and/or recombinant mammalian CCR2 protein or portion thereof, or
synthetic molecules, such as synthetic peptides. In a preferred
embodiment, cells which express receptor, such as transfected
cells, can be used as immunogens or in a screen for antibody which
binds receptor.
[0032] The antibodies of the present invention, and fragments
thereof, are useful in therapeutic, diagnostic and research
applications as described herein. The present invention encompasses
an antibody or functional portion thereof of the present invention
(e.g., mAb 1D9 or 8G2, or antigen-binding fragments thereof) for
use in therapy (including prophylaxis) or diagnosis (e.g., of
particular diseases or conditions as described herein), and use of
such antibodies or functional portions thereof for the manufacture
of a medicament for use in treatment of diseases or conditions as
described herein.
[0033] Preparation of immunizing antigen, and polyclonal and
monoclonal antibody production can be performed as described
herein, or using other suitable techniques. 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)). Generally, a hybridoma can be
produced by fusing a suitable immortal cell line (e.g., a myeloma
cell line such as SP2/0) with antibody producing cells. The
antibody producing cell, preferably those of the spleen or lymph
nodes, are 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 binding properties
can be selected by a suitable assay (e.g., ELISA).
[0034] Other suitable methods of producing or isolating antibodies
which bind CCR2, including human or artificial antibodies, can be
used, including, for example, methods which select recombinant
antibody (e.g., single chain Fv or Fab) from a library, or which
rely upon immunization of transgenic animals (e.g., mice) capable
of producing a repertoire of human antibodies (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).
[0035] Single chain antibodies, and chimeric, humanized or
primatized (CDR-grafted) antibodies, as well as chimeric or
CDR-grafted single chain antibodies, and the like, comprising
portions derived from different species, 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; and Queen et
al., U.S. Pat. Nos. 5,585,089, 5,698,761 and 5,698,762. 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.
[0036] In addition, functional fragments of antibodies, including
fragments of chimeric, humanized, primatized or single chain
antibodies, can also be produced. Functional fragments of the
foregoing antibodies retain at least one binding function and/or
modulation function of the full-length antibody from which they are
derived. Preferred functional fragments retain an antigen-binding
function of a corresponding full-length antibody (e.g., the ability
to bind a mammalian CCR2). Particularly preferred functional
fragments retain the ability to inhibit one or more functions
characteristic of a mammalian CCR2, such as a binding activity, a
signaling activity, and/or stimulation of a cellular response. For
example, in one embodiment, a functional fragment can inhibit the
interaction of CCR2 with one or more of its ligands (e.g., MCP-1,
MCP-2, MCP-3 and/or MCP-4) and/or can inhibit one or more
receptor-mediated functions, such as leukocyte trafficking, HIV
entry into cells, T cell activation, inflammatory mediator release
and/or leukocyte degranulation.
[0037] For example, antibody fragments capable of binding to a
mammalian CCR2 receptor or portion thereof, including, but not
limited to, Fv, Fab, Fab' and F(ab').sub.2 fragments are
encompassed by the invention. Such fragments can be produced by
enzymatic cleavage or by recombinant techniques, for example. For
instance, papain or pepsin cleavage can generate Fab or
F(ab').sub.2 fragments, respectively. 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.
[0038] The term "humanized immunoglobulin" as used herein refers to
an immunoglobulin comprising portions of immunoglobulins of
different origin, wherein at least one portion is of human origin.
Accordingly, the present invention relates to a humanized
immunoglobulin which binds mammalian CCR2 (e.g., human CCRI, murine
CCR2), said immunoglobulin comprising an antigen-binding region of
nonhuman origin (e.g., rodent) and at least a portion of an
immunoglobulin of human origin (e.g., a human framework region, a
human constant region or portion thereof). For example, the
humanized antibody can comprise portions derived from an
immunoglobulin of nonhuman origin with the requisite specificity,
such as a mouse, and from immunoglobulin sequences of human origin
(e.g., a chimeric immunoglobulin), joined together chemically by
conventional techniques (e.g., synthetic) or prepared as a
contiguous polypeptide using genetic engineering techniques (e.g.,
DNA encoding the protein portions of the chimeric antibody can be
expressed to produce a contiguous polypeptide chain). Another
example of a humanized immunoglobulin of the present invention is
an immunoglobulin containing one or more immunoglobulin chains
comprising a CDR of nonhuman origin (e.g., one or more CDRs derived
from an antibody of nonhuman origin) and a framework region derived
from a light and/or heavy chain of human origin (e.g., CDR-grafted
antibodies with or without framework changes). In one embodiment,
the humanized immunoglobulin can compete with murine 1D9 or 8G2
monoclonal antibody for binding to human CCR2. In a preferred
embodiment, the antigen-binding region of the humanized
immunoglobulin (a) is derived from 1D9 monoclonal antibody (e.g.,
as in a humanized immunoglobulin comprising CDR1, CDR2 and CDR3 of
the 1D9 light chain and CDR1, CDR2 and CDR3 of the 1D9 heavy chain)
or (b) is derived from 8G2 monoclonal antibody (e.g., as in a
humanized immunoglobulin comprising CDR1, CDR2 and CDR3 of the 8G2
light chain and CDR1, CDR2 and CDR3 of the 8G2 heavy chain).
Chimeric or CDR-grafted single chain antibodies are also
encompassed by the term humanized immunoglobulin.
[0039] Such humanized immunoglobulins can be produced using
synthetic and/or recombinant nucleic acids to prepare genes (e.g.,
cDNA) encoding the desired humanized chain. For example, nucleic
acid (e.g., DNA) sequences coding for humanized variable regions
can 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 mutagenized, 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)).
[0040] Anti-idiotypic antibodies are also provided. Anti-idiotypic
antibodies recognize antigenic determinants associated with the
antigen-binding site of another antibody. Anti-idiotypic antibodies
can be prepared against second antibody by immunizing an animal of
the same species, and preferably of the same strain, as the animal
used to produce the second antibody. See e.g., U.S. Pat. No.
4,699,880.
[0041] The present invention also pertains to the hybridoma cell
lines deposited under ATCC Accession Nos. HB-12549 and HB-12550, as
well as to the monoclonal antibodies produced by the hybridoma cell
lines deposited under ATCC Accession Nos. HB-12549 and HB-12550.
The cell lines of the present invention have uses other than for
the production of the monoclonal antibodies. For example, the cell
lines of the present invention can be fused with other cells (such
as suitably drug-marked human myeloma, mouse myeloma, human-mouse
heteromyeloma or human lymphoblastoid cells) to produce additional
hybridomas, and thus provide for the transfer of the genes encoding
the monoclonal antibodies. In addition, the cell lines can be used
as a source of nucleic acids encoding the anti-CCR2 immunoglobulin
chains, which can be isolated and expressed (e.g., upon transfer to
other cells using any suitable technique (see e.g., Cabilly et al.,
U.S. Pat. No. 4,816,567; Winter, U.S. Pat. No. 5,225,539)). For
instance, clones comprising a rearranged anti-CCR2 light or heavy
chain can be isolated (e.g., by PCR) or cDNA libraries can be
prepared from mRNA isolated from the cell lines, and cDNA clones
encoding an anti-CCR2 immunoglobulin chain can be isolated. Thus,
nucleic acids encoding the heavy and/or light chains of the
antibodies or portions thereof can be obtained and used in
accordance with recombinant DNA techniques for the production of
the specific immunoglobulin, immunoglobulin chain, or variants
thereof (e.g., humanized immunoglobulins) in a variety of host
cells or in an in vitro translation system. For example, the
nucleic acids, including cDNAs, or derivatives thereof encoding
variants such as a humanized immunoglobulin or immunoglobulin
chain, can be placed into suitable prokaryotic or eukaryotic
vectors (e.g., expression vectors) and introduced into a suitable
host cell by an appropriate method (e.g., transformation,
transfection, electroporation, infection), such that the nucleic
acid is operably linked to one or more expression control elements
(e.g., in the vector or integrated into the host cell genome). For
production, host cells can be maintained under conditions suitable
for expression (e.g., in the presence of inducer, suitable media
supplemented with appropriate salts, growth factors, antibiotic,
nutritional supplements, etc.), whereby the encoded polypeptide is
produced. If desired, the encoded protein can be recovered and/or
isolated (e.g., from the host cells, medium, milk). It will be
appreciated that the method of production encompasses expression in
a host cell of a transgenic animal (see e.g., WO 92/03918, GenPharm
International, published Mar. 19, 1992).
[0042] As described herein, antibodies and functional fragments
thereof of the present invention can block (inhibit) binding of a
ligand to CCR2 and/or inhibit function associated with binding of
the ligand to the CCR2. As discussed below various methods can be
used to assess inhibition of binding of a ligand to CCR2 and/or
function associated with binding of the ligand to the receptor.
[0043] Binding Assays
[0044] As used herein "mammalian CCR2 protein" refers to naturally
occurring or endogenous mammalian CCR2 proteins and to proteins
having an amino acid sequence which is the same as that of a
naturally occurring or endogenous corresponding mammalian CCR2
protein (e.g., recombinant proteins). Accordingly, as defined
herein, the term includes mature receptor protein, polymorphic or
allelic variants, and other isoforms of a mammalian CCR2 (e.g.,
produced by alternative splicing or other cellular processes), and
modified or unmodified forms of the foregoing (e.g., glycosylated,
unglycosylated). Mammalian CCR2 proteins can be isolated and/or
recombinant proteins (including synthetically produced proteins).
Naturally occurring or endogenous mammalian CCR2 proteins include
wild type proteins such as mature CCR2, polymorphic or allelic
variants and other isoforms which occur naturally in mammals (e.g.,
humans, non-human primates), such as the CCR2a and CCR2b forms of
the receptor protein which are produced by alternative splicing of
the carboxy-terminus of the protein. Such proteins can be recovered
or isolated from a source which naturally produces mammalian CCR2,
for example. These proteins and mammalian CCR2 proteins having the
same amino acid sequence as a naturally occurring or endogenous
corresponding mammalian CCR2, are referred to by the name of the
corresponding mammal. For example, where the corresponding mammal
is a human, the protein is designated as a human CCR2 protein
(e.g., a recombinant human CCR2 produced in a suitable host
cell).
[0045] "Functional variants" of mammalian CCR2 proteins include
functional fragments, functional mutant proteins, and/or functional
fusion proteins (e.g., produced via mutagenesis and/or recombinant
techniques). Generally, fragments or portions of mammalian CCR2
proteins include those having a deletion (i.e., one or more
deletions) of an amino acid (i.e., one or more amino acids)
relative to the mature mammalian CCR2 protein (such as N-terminal,
C-terminal or internal deletions). Fragments or portions in which
only contiguous amino acids have been deleted or in which
non-contiguous amino acids have been deleted relative to mature
mammalian CCR2 protein are also envisioned.
[0046] Generally, mutants of mammalian CCR2 proteins include
natural or artificial variants of a mammalian CCR2 protein
differing by the addition, deletion and/or substitution of one or
more contiguous or non-contiguous amino acid residues (e.g.,
receptor chimeras). Such mutations can be in a conserved region or
nonconserved region (compared to other CXC and/or CC chemokine
receptors), extracellular, cytoplasmic, or transmembrane region,
for example.
[0047] Generally, fusion proteins encompass polypeptides comprising
a mammalian CCR2 (e.g., human CCR2) as a first moiety, linked via a
peptide cond to a second moiety not occurring in the mammalian CCR2
as found in nature. Thus, the second moiety can be an amino acid,
oligopeptide or polypeptide. The first moiety can be in an
N-terminal location, C-terminal location or internal to the fusion
protein. In one embodiment, the fusion protein comprises an
affinity ligand (e.g., an enzyme, an antigen, epitope tage) as the
first moiety, and a second moiety comprising a linker sequence and
human CCR2 or a portion thereof.
[0048] A "functional fragment or portion", "functional mutant"
and/or "functional fusion protein" of a mammalian CCR2 protein
refers to an isolated and/or recombinant protein or polypeptide
which has at least one function characteristic of a mammalian CCR2
protein as described herein, such as a binding activity, a
signaling activity and/or ability to stimulate a cellular response.
Preferred functional variants can bind a ligand (i.e., one or more
ligands such as MCP-1, MCP-2, MCP-3 and/or MCP-4), and are referred
to herein as "ligand binding variants".
[0049] In one embodiment, a functional variant of mammalian CCR2
shares at least about 85% sequence identity with said mammalian
CCR2, preferably at least about 90% sequence identity, and more
preferably at least about 95% sequence identity with said mammalian
CCR2. The nucleic acid and amino acid sequences of human CCR2a and
CCR2b are described in U.S. Pat. No. 5,707,815. Sequence identity
can be determine using a suitable program, such as the Blastx
program (Version 1.4), using appropriate parameters, such as
default parameters. In one embodiment, parameters for Blastx search
are scoring matrix BLOSUM62, W=3. In another embodiment, a
functional variant comprises a nucleic acid sequence which is
different from the naturally-occurring nucleic acid molecule but
which, due to the degeneracy of the genetic code, encodes mammalian
CCR2 or a portion thereof.
[0050] A composition comprising an isolated and/or recombinant
mammalian CCR2 or functional variant thereof can be maintained
under conditions suitable for binding, the mammalian CCR2 or
variant is contacted with an antibody or fragment to be tested, and
binding is detected or measured directly or indirectly. In one
embodiment, cells which naturally express CCR2 or cells comprising
a recombinant nucleic acid sequence which encodes a mammalian CCR2
or variant thereof are used. The cells are maintained under
conditions appropriate for expression of receptor. The cells are
contacted with an antibody or fragment under conditions suitable
for binding (e.g., in a suitable binding buffer), and binding is
detected by standard techniques. To determine binding, the extent
of binding can be determined relative to a suitable control (e.g.,
compared with background determined in the absence of antibody,
compared with binding of a second antibody (i.e., a standard),
compared with binding of antibody to untransfected cells). A
cellular fraction, such as a membrane fraction, containing receptor
or liposomes comprising receptor can be used in lieu of whole
cells.
[0051] In one embodiment, the antibody is labeled with a suitable
label (e.g., fluorescent label, isotope label, antigen or epitope
label, enzyme label), and binding is determined by detection of the
label. In another embodiment, bound antibody can be detected by
labeled second antibody. Specificity of binding can be assessed by
competition or displacement, for example, using unlabeled antibody
or a ligand as competitor.
[0052] Binding inhibition assays can also be used to identify
antibodies or fragments thereof which bind CCR2 and inhibit binding
of another compound such as a ligand (e.g., MCP-1, MCP-2, MCP-3
and/or MCP-4) to CCR2 or a functional variant. For example, a
binding assay can be conducted in which a reduction in the binding
of a ligand of CCR2 (in the presence of an antibody), as compared
to binding of the ligand in the absence of the antibody, 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 antibody 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.
[0053] In one embodiment, direct inhibition of the binding of a
ligand (e.g., a chemokine such as MCP-1) to a mammalian CCR2 or
variant thereof by an antibody or fragment is monitored. For
example, the ability of an antibody 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 functional 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, or a membrane fraction from said cells, for
instance.
[0054] Other methods of identifying the presence of an antibody
which binds CCR2 are available, such as other suitable binding
assays, or methods which monitor events which are triggered by
receptor binding, including signaling function and/or stimulation
of a cellular response (e.g., leukocyte trafficking).
[0055] It will be understood that the inhibitory effect of
antibodies of the present invention can be assessed in a binding
inhibition assay. Competition between antibodies for receptor
binding can also be assessed in the method. Antibodies which are
identified in this manner can be further assessed to determine
whether, subsequent to binding, they act to inhibit other functions
of CCR2 and/or to assess their therapeutic utility.
[0056] Signaling Assays
[0057] The binding of a ligand or promoter, such as an agonist, to
CCR2 can result in signaling by this G protein-coupled receptor,
and the activity of G proteins as well as other intracellular
signaling molecules is stimulated. The induction of signaling
function by a compound (e.g., an antibody or fragment thereof) can
be monitored using any suitable method. Such an assay can be used
to identify antibody agonists of CCR2. The inhibitory activity of
an antibody or functional fragment thereof can be determined using
a ligand or promoter in the assay, and assessing the ability of the
antibody to inhibit the activity induced by ligand or promoter.
[0058] G protein activity, such as hydrolysis of GTP to GDP, or
later signaling events triggered by receptor binding, such as
induction of rapid and transient increase in the concentration of
intracellular (cytosolic) free calcium [Ca.sup.2+].sub.I, can be
assayed by methods known in the art or other suitable methods (see
e.g., Neote, K. et al., Cell, 72: 415-425 1993); Van Riper et al.,
J. Exp. Med., 177: 851-856 (1993); Dahinden, C. A. et al., J. Exp.
Med., 179: 751-756 (1994)).
[0059] For example, the functional assay of Sledziewski et al.
using hybrid G protein coupled receptors can be used to monitor the
ability a ligand or promoter to bind receptor and activate a G
protein (Sledziewski et al., U.S. Pat. No. 5,284,746, the teachings
of which are incorporated herein by reference).
[0060] Such assays can be performed in the presence of the antibody
or fragment thereof to be assessed, and the ability of the antibody
or fragment to inhibit the activity induced by the ligand or
promoter is determined using known methods and/or methods described
herein.
[0061] Chemotaxis and Assays of Cellular Stimulation
[0062] Chemotaxis assays can also be used to assess the ability of
an antibody or functional fragment thereof to block binding of a
ligand to mammalian CCR2 or functional variant thereof and/or
inhibit function associated with binding of the ligand to the
receptor. These assays are based on the functional migration of
cells in vitro or in vivo induced by a compound. Chemotaxis can be
assessed as described in the Examples, e.g., in an assay utilizing
a 96-well chemotaxis plate, or using other art-recognized methods
for assessing chemotaxis. For example, the use of an in vitro
transendothelial chemotaxis assay is described by Springer et al.
(Springer et al., WO 94/20142, published Sep. 15, 1994, the
teachings of which are incorporated herein by reference; see also
Berman et al., Immunol. Invest. 17: 625-677 (1988)). Migration
across endothelium into collagen gels has also been described
(Kavanaugh et al., J. Immunol., 146: 4149-4156 (1991)). Stable
transfectants of mouse L1l-2 pre-B cells or of other suitable host
cells capable of chemotaxis can be used in chemotaxis assays, for
example.
[0063] Generally, chemotaxis assays monitor the directional
movement or migration of a suitable cell (such as a leukocyte
(e.g., lymphocyte, eosinophil, basophil)) into or through a barrier
(e.g., endothelium, a filter), toward increased levels of a
compound, from a first surface of the barrier toward an opposite
second surface. Membranes or filters provide convenient barriers,
such that the directional movement or migration of a suitable cell
into or through a filter, toward increased levels of a compound,
from a first surface of the filter toward an opposite second
surface of the filter, is monitored. In some assays, the membrane
is coated with a substance to facilitate adhesion, such as ICAM-1,
fibronectin or collagen. Such assays provide an in vitro
approximation of leukocyte "homing".
[0064] For example, one can detect or measure inhibition of the
migration of cells in a suitable container (a containing means),
from a first chamber into or through a microporous membrane into a
second chamber which contains an antibody to be tested, and which
is divided from the first chamber by the membrane. A suitable
membrane, having a suitable pore size for monitoring specific
migration in response to compound, including, for example,
nitrocellulose, polycarbonate, is selected. For example, pore sizes
of about 3-8 microns, and preferably about 5-8 microns can be used.
Pore size can be uniform on a filter or within a range of suitable
pore sizes.
[0065] To assess migration and inhibition of migration, the
distance of migration into the filter, the number of cells crossing
the filter that remain adherent to the second surface of the
filter, and/or the number of cells that accumulate in the second
chamber can be determined using standard techniques (e.g.,
microscopy). In one embodiment, the cells are labeled with a
detectable label (e.g., radioisotope, fluorescent label, antigen or
epitope label), and migration can be assessed in the presence and
absence of the antibody or fragment by determining the presence of
the label adherent to the membrane and/or present in the second
chamber using an appropriate method (e.g., by detecting
radioactivity, fluorescence, immunoassay). The extent of migration
induced by an antibody agonist can be determined relative to a
suitable control (e.g., compared to background migration determined
in the absence of the antibody, compared to the extent of migration
induced by a second compound (i.e., a standard), compared with
migration of untransfected cells induced by the antibody).
[0066] In one embodiment, particularly for T cells, monocytes or
cells expressing a mammalian CCR2, transendothelial migration can
be monitored. In this embodiment, transmigration through an
endothelial cell layer is assessed. To prepare the cell layer,
endothelial cells can be cultured on a microporous filter or
membrane, optionally coated with a substance such as collagen,
fibronectin, or other extracellular matrix proteins, to facilitate
the attachment of endothelial cells. Preferably, endothelial cells
are cultured until a confluent monolayer is formed. A variety of
mammalian endothelial cells can are available for monolayer
formation, including for example, vein, artery or microvascular
endothelium, such as human umbilical vein endothelial cells
(Clonetics Corp, San Diego, Calif.). To assay chemotaxis in
response to a particular mammalian receptor, endothelial cells of
the same mammal are preferred; however endothelial cells from a
heterologous mammalian species or genus can also be used.
[0067] Generally, the assay is performed by detecting the
directional migration of cells into or through a membrane or
filter, in a direction toward increased levels of a compound, from
a first surface of the filter toward an opposite second surface of
the filter, wherein the filter contains an endothelial cell layer
on a first surface. Directional migration occurs from the area
adjacent to the first surface, into or through the membrane,
towards a compound situated on the opposite side of the filter. The
concentration of compound present in the area adjacent to the
second surface, is greater than that in the area adjacent to the
first surface.
[0068] In one embodiment used to test for an antibody inhibitor, a
composition comprising cells capable of migration and expressing a
mammalian CCR2 receptor can be placed in the first chamber. A
composition comprising one or more ligands or promoters capable of
inducing chemotaxis of the cells in the first chamber (having
chemoattractant function) is placed in the second chamber.
Preferably shortly before the cells are placed in the first
chamber, or simultaneously with the cells, a composition comprising
the antibody to be tested is placed, preferably, in the first
chamber. Antibodies or functional fragments thereof which can bind
receptor and inhibit the induction of chemotaxis, by a ligand or
promoter, of the cells expressing a mammalian CCR2 in this assay
are inhibitors of receptor function (e.g., inhibitors of
stimulatory function). A reduction in the extent of migration
induced by the ligand or promoter in the presence of the antibody
or fragment is indicative of inhibitory activity. Separate binding
studies (see above) could be performed to determine whether
inhibition is a result of binding of the antibody to receptor or
occurs via a different mechanism.
[0069] In vivo assays which monitor leukocyte infiltration of a
tissue, in response to injection of a compound (e.g., chemokine or
antibody) in the tissue, are described below (see Models of
Inflammation). These models of in vivo homing measure the ability
of cells to respond to a ligand or promoter by emigration and
chemotaxis to a site of inflammation and to assess the ability of
an antibody or fragment thereof to block this emigration.
[0070] In addition to the methods described, the effects of an
antibody or fragment on the stimulatory function of CCR2 can be
assessed by monitoring cellular responses induced by active
receptor, using suitable host cells containing receptor.
[0071] Identification of Additional Ligands, Inhibitors and/or
Promoters of Mammalian CCR2 Function
[0072] The assays described above, which can be used to assess
binding and function of the antibodies and fragments of the present
invention, can be adapted to identify additional ligands or other
substances which bind a mammalian CCR2 or functional variant
thereof, as well as inhibitors and/or promoters of mammalian CCR2
function. For example, agents having the same or a similar binding
specificity as that of an antibody of the present invention or
functional portion thereof can be identified by a competition assay
with said antibody or portion thereof. Thus, the present invention
also encompasses methods of identifying ligands of the receptor or
other substances which bind a mammalian CCR2 protein, as well as
inhibitors (e.g., antagonists) or promoters (e.g., agonists) of
receptor function. In one embodiment, cells bearing a mammalian
CCR2 protein or functional variant thereof (e.g., leukocytes, cell
lines or suitable host cells which have been engineered to express
a mammalian CCR2 protein or functional variant encoded by a nucleic
acid introduced into said cells) are used in an assay to identify
and assess the efficacy of ligands or other substances which bind
receptor, including inhibitors or promoters of receptor function.
Such cells are also useful in assessing the function of the
expressed receptor protein or polypeptide.
[0073] According to the present invention, ligands and other
substances which bind receptor, inhibitors and promoters of
receptor function can be identified in a suitable assay, and
further assessed for therapeutic effect. Inhibitors of receptor
function can be used to inhibit (reduce or prevent) receptor
activity, and ligands and/or promoters can be used to induce
(trigger or enhance) normal receptor function where indicated.
Thus, the present invention provides a method of treating
inflammatory diseases, including autoimmune disease and graft
rejection, comprising administering an inhibitor of receptor
function to an individual (e.g., a mammal). The present invention
further provides a method of stimulating receptor function by
administering a novel ligand or promoter of receptor function to an
individual, providing a new approach to selective stimulation of
leukocyte function, which is useful, for example, in the treatment
of infectious diseases and cancer.
[0074] As used herein, a "ligand" of a mammalian CCR2 protein
refers to a particular class of substances which bind to a
mammalian CCR2 protein, including natural ligands and synthetic
and/or recombinant forms of natural ligands. Infectious agents
having a tropism for mammalian CCR2-positive cells (e.g., viruses
such as HIV) can also bind to a mammalian CCR2 protein. A natural
ligand of a selected mammalian receptor is of a mammalian origin
which is the same as that of the mammalian CCR2 protein (e.g., a
chemokine such as MCP-1, MCP-2, MCP-3 and/or MCP-4). In a preferred
embodiment, ligand binding of a mammalian CCR2 protein occurs with
high affinity.
[0075] As used herein, an "inhibitor" is a substance which inhibits
(decreases or prevents) at least one function characteristic of a
mammalian CCR2 protein (e.g., a human CCR2), such as a binding
activity (e.g., ligand binding, promoter binding, antibody
binding), a signaling activity (e.g., activation of a mammalian G
protein, induction of rapid and transient increase in the
concentration of cytosolic free calcium [Ca.sup.2+].sub.I), and/or
cellular response function (e.g., stimulation of chemotaxis,
exocytosis or inflammatory mediator release by leukocytes). An
inhibitor is also a substance which inhibits HIV entry into a cell.
The term inhibitor refers to substances including antagonists which
bind receptor (e.g., an antibody, a mutant of a natural ligand,
small molecular weight organic molecules, other competitive
inhibitors of ligand binding), and substances which inhibit
receptor function without binding thereto (e.g., an anti-idiotypic
antibody).
[0076] As used herein, a "promoter" is a substance which promotes
(induces, causes, enhances or increases) at least one function
characteristic of a mammalian CCR2 protein (e.g., a human CCR2),
such as a binding activity (e.g., ligand, inhibitor and/or promoter
binding), a signaling activity (e.g., activation of a mammalian G
protein, induction of rapid and transient increase in the
concentration of cytosolic free calcium [Ca.sup.2-].sub.I), and/or
a cellular response function (e.g., stimulation of chemotaxis,
exocytosis or inflammatory mediator release by leukocytes). The
term promoter refers to substances including agonists which bind
receptor (e.g., an antibody, a homolog of a natural ligand from
another species), and substances which promote receptor function
without binding thereto (e.g., by activating an associated
protein). In a preferred embodiment, the agonist is other than a
homolog of a natural ligand.
[0077] Thus, the invention also relates to a method of detecting or
identifying an agent which binds a mammalian CC-chemokine receptor
2 or ligand binding variant thereof, including ligands, inhibitors,
promoters, and other substances which bind a mammalian CCR2
receptor or functional variant. According to the method, an agent
to be tested, an antibody or antigen-binding fragment of the
present invention (e.g., 8G2, 1D9, an antibody having an epitopic
specificity which is the same as or similar to that of 8G2 or 1D9,
and antigen-binding fragments thereof) and a composition comprising
a mammalian CC-chemokine receptor 2 or a ligand binding variant
thereof can be combined. The foregoing components are combined
under conditions suitable for binding of the antibody or
antigen-binding fragment to mammalian CC-chemokine receptor 2 or a
ligand binding variant thereof, and binding of the antibody or
fragment to the mammalian CC-chemokine receptor 2 or ligand binding
variant is detected or measured, either directly or indirectly,
according to methods described herein or other suitable methods. A
decrease in the amount of complex formed relative to a suitable
control (e.g., in the absence of the agent to be tested) is
indicative that the agent binds said receptor or variant. The
composition comprising a mammalian CC-chemokine receptor 2 or a
ligand binding variant thereof can be a membrane fraction of a cell
bearing recombinant chemokine receptor 2 protein or ligand binding
variant thereof. The antibody or fragment thereof can be labeled
with a label such as a radioisotope, spin label, antigen or epitope
label, enzyme label, fluorescent group and chemiluminescent
group.
[0078] In one embodiment, the invention relates to a method of
detecting or identifying an agent which binds a mammalian
CC-chemokine receptor 2 or a ligand binding variant thereof,
comprising combining an agent to be tested, an antibody or
antigen-binding fragment of the present invention (e.g., 1D9, 8G2,
an antibody having an epitopic specificity which is the same as or
similar to that of 1D9 or 8G2, or antigen-binding fragments
thereof) and a cell bearing a mammalian CC-chemokine receptor 2 or
a ligand binding variant thereof. The foregoing components are
combined under conditions suitable for binding of the antibody or
antigen-binding fragment to the CCR2 protein or ligand binding
variant thereof, and binding of the antibody or fragment to the
mammalian CC-chemokine receptor 2 or variant is detected or
measured, either directly or indirectly, by methods described
herein and or other suitable methods. A decrease in the amount of
complex formed relative to a suitable control is indicative that
the agent binds the receptor or variant. The antibody or fragment
thereof can be labeled with a label selected from the group
consisting of a radioisotope, spin label, antigen or epitope label,
enzyme label, fluorescent group and chemiluminescent group. These
and similar assays can be used to detect agents, including ligands
(e.g., chemokines or strains of HIV which interact with CCR2) or
other substances, including inhibitors or promoters of receptor
function, which can bind CCR2 and compete with the antibodies
described herein for binding to the receptor.
[0079] The assays described above can be used, alone or in
combination with each other or other suitable methods, to identify
ligands or other substances which bind a mammalian CCR2 protein,
and inhibitors or promoters of a mammalian CCR2 protein or variant.
The in vitro methods of the present invention can be adapted for
high-throughput screening in which large numbers of samples are
processed (e.g., a 96-well format). Cells expressing mammalian CCR2
(e.g., human CCR2) at levels suitable for high-throughput screening
can be used, and thus, are particularly valuable in the
identification and/or isolation of ligands or other substances
which bind receptor, and inhibitors or promoters of mammalian CCR2
proteins. Expression of receptor can be monitored in a variety of
ways. For instance, expression can be monitored using antibodies of
the present invention which bind receptor or a portion thereof.
Also, commercially available antibodies can be used to detect
expression of an antigen- or epitope-tagged fusion protein
comprising a receptor protein or polypeptide (e.g., FLAG tagged
receptors), and cells expressing the desired level can be
selected.
[0080] Nucleic acid encoding a mammalian CCR2 protein or functional
variant thereof can be incorporated into an expression system to
produce a receptor protein or polypeptide. An isolated and/or
recombinant mammalian CCR2 protein or variant, such as a receptor
expressed in cells stably or transiently transfected with a
construct comprising a recombinant nucleic acid encoding a
mammalian CCR2 protein or variant, or in a cell fraction containing
receptor (e.g., a membrane fraction from transfected cells,
liposomes incorporating receptor), can be used in tests for
receptor function. The receptor can be further purified if desired.
Testing of receptor function can be carried out in vitro or in
vivo.
[0081] An isolated and/or recombinant mammalian CCR2 protein or
functional variant thereof, such as a human CCR2, can be used in
the present method, in which the effect of a compound is assessed
by monitoring receptor function as described herein or using other
suitable techniques. For example, stable or transient transfectants
(e.g., baculovirus infected Sf9 cells, stable tranfectants of mouse
L1/2 pre-B cells), can be used in binding assays. Stable
transfectants of Jurkat cells or of other suitable cells capable of
chemotaxis can be used (e.g., mouse L1/2 pre-B cells) in chemotaxis
assays, for example.
[0082] According to the method of the present invention, compounds
can be individually screened or one or more compounds can be tested
simultaneously according to the methods herein. Where a mixture of
compounds is tested, the compounds selected by the processes
described can be separated (as appropriate) and identified by
suitable methods (e.g., PCR, sequencing, chromatography, mass
spectroscopy). The presence of one or more compounds (e.g., a
ligand, inhibitor, promoter) in a test sample can also be
determined according to these methods.
[0083] Large combinatorial libraries of compounds (e.g., organic
compounds, recombinant or synthetic peptides, "peptoids", nucleic
acids) produced by combinatorial chemical synthesis or other
methods can be tested (see e.g., Zuckerman, R. N. et al., J. Med.
Chem., 37: 2678-2685 (1994) and references cited therein; see also,
Ohlmeyer, M. H. J. et al., Proc. Natl. Acad. Sci. USA
90:10922-10926 (1993) and DeWitt, S. H. et al., Proc. Natl. Acad.
Sci. USA 90:6909-6913 (1993), relating to tagged compounds; Rutter,
W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D. et al., U.S.
Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No. 4,833,092).
Where compounds selected from a combinatorial library by the
present method carry unique tags, identification of individual
compounds by chromatographic methods is possible.
[0084] In one embodiment, phage display methodology is used. For
example, a mammalian CCR2 protein or functional variant, an
antibody or functional portion thereof of the present invention,
and a phage (e.g., a phage or collection of phage such as a
library) displaying a polypeptide, can be combined under conditions
appropriate for binding of the antibody or portion thereof to the
mammalian CCR2 protein or variant (e.g., in a suitable binding
buffer). Phage which can compete with the antibody or portion
thereof and bind to the mammalian CCR2 protein or variant can be
detected or selected using standard techniques or other suitable
methods. Bound phage can be separated from receptor using a
suitable elution buffer. For example, a change in the ionic
strength or pH can lead to a release of phage. Alternatively, the
elution buffer can comprise a release component or components
designed to disrupt binding of compounds (e.g., one or more
compounds which can disrupt binding of the displayed peptide to the
receptor, such as a ligand, inhibitor, and/or promoter which
competitively inhibits binding). Optionally, the selection process
can be repeated or another selection step can be used to further
enrich for phage which bind receptor. The displayed polypeptide can
be characterized (e.g., by sequencing phage DNA). The polypeptides
identified can be produced and further tested for binding, and for
inhibitor or promoter function. Analogs of such peptides can be
produced which will have increased stability or other desirable
properties.
[0085] In one embodiment, phage expressing and displaying fusion
proteins comprising a coat protein with an N-terminal peptide
encoded by random sequence nucleic acids can be produced. Suitable
host cells expressing a mammalian CCR2 protein or variant and an
anti-CCR2 antibody or functional portion thereof, are combined with
the phage, bound phage are selected, recovered and characterized.
(See e.g., Doorbar, J. and G. Winter, J. Mol. Biol., 244: 361
(1994) discussing a phage display procedure used with a G
protein-coupled receptor).
[0086] Other sources of potential ligands or other substances which
bind to, or inhibitors and/or promoters of, mammalian CCR2 proteins
include, but are not limited to, variants of CCR2 ligands,
including naturally occurring, synthetic or recombinant variants of
MCP-1, MCP-2, MCP-3 and/or MCP-4, substances such as other
chemoattractants or chemokines, variants thereof, low molecular
weight organic molecules, other inhibitors and/or promoters (e.g.,
anti-CCR2 antibodies, antagonists, agonists), other G
protein-coupled receptor ligands, inhibitors and/or promoters
(e.g., antagonists or agonists), and soluble portions of a
mammalian CCR2 receptor, such as a suitable receptor peptide or
analog which can inhibit receptor function (see e.g., Murphy, R.
B., WO 94/05695).
[0087] Models of Inflammation
[0088] In vivo models of inflammation are available which can be
used to assess the effects of antibodies and fragments of the
invention in vivo as therapeutic agents. For example, leukocyte
infiltration upon intradermal injection of a chemokine and an
antibody or fragment thereof reactive with mammalian CCR2 into a
suitable animal, such as rabbit, mouse, rat, guinea pig or rhesus
macaque can be monitored (see e.g., Van Damme, J. et al., J. Exp.
Med., 176: 59-65 (1992); Zachariae, C. O. C. et al., J. Exp. Med.
171: 2177-2182 (1990); Jose, P. J. et al., J. Exp. Med. 179:
881-887 (1994)). In one embodiment, skin biopsies are assessed
histologically for infiltration of leukocytes (e.g., eosinophils,
granulocytes). In another embodiment, labeled cells (e.g., stably
transfected cells expressing a mammalian CCR2, labeled with
.sup.111In for example) capable of chemotaxis and extravasation are
administered to the animal. For example, an antibody or fragment to
be assessed can be administered, either before, simultaneously with
or after ligand or agonist is administered to the test animal. A
decrease of the extent of infiltration in the presence of antibody
as compared with the extent of infiltration in the absence of
inhibitor is indicative of inhibition.
[0089] Diagnostic and Therapeutic Applications
[0090] The antibodies and fragments of the present invention are
useful in a variety of applications, including research, diagnostic
and therapeutic applications. In one embodiment, the antibodies are
labeled with a suitable label (e.g., fluorescent label,
chemiluminescent label, isotope label, antigen or epitope label or
enzyme label). For instance, they can be used to isolate and/or
purify receptor or portions thereof, and to study receptor
structure (e.g., conformation) and function.
[0091] In addition, the various antibodies of the present invention
can be used to detect CCR2 or to measure the expression of
receptor, for example, on T cells (e.g., CD8+ cells, CD45RO+
cells), monocytes and/or on cells transfected with a receptor gene.
Thus, they also have utility in applications such as cell sorting
(e.g., flow cytometry, fluorescence activated cell sorting), for
diagnostic or research purposes.
[0092] The anti-CCR2 antibodies of the present invention have value
in diagnostic applications. An anti-CCR2 antibody or fragment
thereof can be used to monitor expression of this receptor in HIV
infected individuals, similar to the way anti-CD4 has been used as
a diagnostic indicator of disease stage.
[0093] Typically, diagnostic assays entail detecting the formation
of a complex resulting from the binding of an antibody or fragment
thereof to CCR2. For diagnostic purposes, the antibodies or
antigen-binding fragments can be labeled or unlabeled. The
antibodies or fragments can be directly labeled. A variety of
labels can be employed, including, but not limited to,
radionuclides, fluorescers, enzymes, enzyme substrates, enzyme
cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).
Numerous appropriate immunoassays are known to the skilled artisan
(see, for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654
and 4,098,876). When unlabeled, the antibodies or fragments can be
detected using suitable means, as in agglutination assays, for
example. Unlabeled antibodies or fragments can also be used in
combination with another (i.e., one or more) suitable reagent which
can be used to detect antibody, such as a labeled antibody (e.g., a
second antibody) reactive with the first antibody (e.g.,
anti-idiotype antibodies or other antibodies that are specific for
the unlabeled immunoglobulin) or other suitable reagent (e.g.,
labeled protein A).
[0094] In one embodiment, the antibodies or fragments of the
present invention can be utilized in enzyme immunoassays, wherein
the subject antibody or fragment, or second antibodies, are
conjugated to an enzyme. When a biological sample comprising a
mammalian CCR2 protein is combined with the subject antibodies,
binding occurs between the antibodies and CCR2 protein. In one
embodiment, a sample containing cells expressing a mammalian CCR2
protein, such as human blood, is combined with the subject
antibodies, and binding occurs between the antibodies and cells
bearing a human CCR2 protein comprising an epitope recognized by
the antibody. These bound cells can be separated from unbound
reagents and the presence of the antibody-enzyme conjugate
specifically bound to the cells can be determined, for example, by
contacting the sample with a substrate of the enzyme which produces
a color or other detectable change when acted on by the enzyme. In
another embodiment, the subject antibodies can be unlabeled, and a
second, labeled antibody can be added which recognizes the subject
antibody.
[0095] Kits for use in detecting the presence of a mammalian CCR2
protein in a biological sample can also be prepared. Such kits will
include an antibody or functional fragment thereof which binds to a
mammalian CC-chemokine receptor 2 or portion of said receptor, as
well as one or more ancillary reagents suitable for detecting the
presence of a complex between the antibody or fragment and CCR2 or
portion thereof. The antibody compositions of the present invention
can be provided in lyophilized form, either alone or in combination
with additional antibodies specific for other epitopes. The
antibodies, which can be labeled or unlabeled, can be included in
the kits with adjunct ingredients (e.g., buffers, such as Tris,
phosphate and carbonate, stabilizers, excipients, biocides and/or
inert proteins, e.g., bovine serum albumin). For example, the
antibodies can be provided as a lyophilized mixture with the
adjunct ingredients, or the adjunct ingredients can be separately
provided for combination by the user. Generally these adjunct
materials will be present in less than about 5% weight based on the
amount of active antibody, and usually will be present in a total
amount of at least about 0.001% weight based on antibody
concentration. Where a second antibody capable of binding to the
monoclonal antibody is employed, such antibody can be provided in
the kit, for instance in a separate vial or container. The second
antibody, if present, is typically labeled, and can be formulated
in an analogous manner with the antibody formulations described
above.
[0096] Similarly, the present invention also relates to a method of
detecting and/or quantitating expression of a mammalian CCR2 or a
portion of the receptor by a cell, in which a composition
comprising a cell or fraction thereof (e.g., membrane fraction) is
contacted with an antibody or functional fragment thereof (e.g.,
1D9 and/or 8G2) which binds to a mammalian CCR2 or portion of the
receptor under conditions appropriate for binding of the antibody
or fragment thereto, and binding is monitored. Detection of the
antibody, indicative of the formation of a complex between antibody
and CCR2 or a portion thereof, indicates the presence of the
receptor. Binding of antibody to the cell can be determined as
described above under the heading "Binding Assays", for example.
The method can be used to detect expression of CCR2 on cells from
an individual (e.g., in a sample, such as a body fluid, such as
blood, saliva or other suitable sample). The level of expression of
CCR2 on the surface of T cells or monocytes can also be determined,
for instance, by flow cytometry, and the level of expression (e.g.,
staining intensity) can be correlated with disease susceptibility,
progression or risk.
[0097] Chemokine receptors function in the migration of leukocytes
throughout the body, particularly to inflammatory sites.
Inflammatory cell emigration from the vasculature is regulated by a
three-step process involving interactions of leukocyte and
endothelial cell adhesion proteins and cell specific
chemoattractants and activating factors (Springer, T. A., Cell,
76:301-314 (1994); Butcher, E. C., Cell, 67:1033-1036 (1991);
Butcher, E. C. and Picker, L. J., Science (Wash. D.C.), 272:60-66
(1996)). These are: (a) a low affinity interaction between
leukocyte selectins and endothelial cell carbohydrates; (b) a
high-affinity interaction between leukocyte chemoattractant
receptors and chemoattractant/activating factors; and (c) a
tight-binding between leukocyte integrins and endothelial cell
adhesion proteins of the immunoglobulin superfamily. Different
leukocyte subsets express different repertoires of selecting,
chemoattractant receptors and integrins. Additionally, inflammation
alters the expression of endothelial adhesion proteins and the
expression of chemoattractant and leukocyte activating factors. As
a consequence, there is a great deal of diversity for regulating
the selectivity of leukocyte recruitment to extravascular sites.
The second step is crucial in that the activation of the leukocyte
chemoattractant receptors is thought to cause the transition from
the selectin-mediated cell rolling to the integrin-mediated tight
binding. This results in the leukocyte being ready to transmigrate
to perivascular sites. The chemoattractant/chemoattractant receptor
interaction is also crucial for transendothelial migration and
localization within a tissue (Campbell, J. J., et al., J. Cell
Biol., 134:255-266 (1996); Carr, M. W., et al., Immunity, 4:179-187
(1996)). This migration is directed by a concentration gradient of
chemoattractant leading towards the inflammatory focus.
[0098] CCR2 has an important role in leukocyte trafficking. It is
likely that CCR2 is a key chemokine receptor for T cell or T cell
subset or monocyte migration to certain inflammatory sites, and so
anti-CCR2 mAbs can be used to inhibit (reduce or prevent) T cell or
monocyte migration, particularly that associated with T cell
dysfunction, such as autoimmune disease, or allergic reactions or
with monocyte-mediated disorders such as atherosclerosis.
Accordingly, the antibodies and fragments thereof of the present
invention can also be used to modulate receptor function in
research and therapeutic applications. For instance, the antibodies
and functional fragments described herein can act as inhibitors to
inhibit (reduce or prevent) (a) binding (e.g., of a ligand, an
inhibitor or a promoter) to the receptor, (b) a receptor signaling
function, and/or (c) a stimulatory function. Antibodies which act
as inhibitors of receptor function can block ligand or promoter
binding directly or indirectly (e.g., by causing a conformational
change). For example, antibodies can inhibit receptor function by
inhibiting binding of a ligand, or by desensitization (with or
without inhibition of binding of a ligand). Antibodies which bind
receptor can also act as agonists of receptor function, triggering
or stimulating a receptor function, such as a signaling and/or a
stimulatory function of a receptor (e.g., leukocyte trafficking)
upon binding to receptor.
[0099] Thus, the present invention provides a method of inhibiting
leukocyte trafficking in a mammal (e.g., a human patient),
comprising administering to the mammal an effective amount of an
antibody or functional fragment of the present invention.
Administration of an antibody or fragment of the present invention
can result in amelioration or elimination of the disease state.
[0100] The antibody of the present invention, or a functional
fragment thereof, can also be used to treat disorders in which
activation of the CCR2 receptor by binding of chemokines is
implicated. For example, the antibodies or functional fragments
thereof (e.g., 1D9 and/or 8G2 or functional fragments thereof) can
be used to treat allergy, atherogenesis, anaphylaxis, malignancy,
chronic and acute inflammation, histamine and IgE-mediated allergic
reactions, shock, and rheumatoid arthritis, atherosclerosis,
multiple sclerosis, allograft rejection, fibrotic disease, asthma,
and inflammatory glomerulopathies.
[0101] Diseases or conditions of humans or other species which can
be treated with inhibitors of CCR2 receptor function (including
antibodies or suitable fragments thereof), include, but are not
limited to:
[0102] inflammatory or allergic diseases and conditions, including
respiratory allergic diseases such as asthma, allergic rhinitis,
hypersensitivity lung diseases, hypersensitivity pneumonitis,
interstitial lung diseases (ILD) (e.g., idiopathic pulmonary
fibrosis, or ILD associated with rheumatoid arthritis, systemic
lupus erythematosus, ankylosing spondylitis, systemic sclerosis,
Sjogren's syndrome, polymyositis or dermatomyositis); anaphylaxis
or hypersensitivity responses, drug allergies (e.g., to penicillin,
cephalosporins), insect sting allergies; inflammatory bowel
diseases, such as Crohn's disease and ulcerative colitis;
spondyloarthropathies; scleroderma; psoriasis and inflammatory
dermatoses such as dermatitis, eczema, atopic dermatitis, allergic
contact dermatitis, urticaria; vasculitis (e.g., necrotizing,
cutaneous, and hypersensitivity vasculitis);
[0103] autoimmune diseases, such as arthritis (e.g., rheumatoid
arthritis, psoriatic arthritis), multiple sclerosis, systemic lupus
erythematosus, myasthenia gravis, juvenile onset diabetes,
nephritides such as glomerulonephritis, autoimmune thyroiditis,
Behcet's disease;
[0104] graft rejection (e.g., in transplantation), including
allograft rejection or graft-versus-host disease;
[0105] atherosclerosis;
[0106] cancers with leukocyte infiltration of the skin or
organs;
[0107] other diseases or conditions (including CCR2-mediated
diseases or conditions), in which undesirable inflammatory
responses are to be inhibited can be treated, including, but not
limited to, reperfusion injury, certain hematologic malignancies,
cytokine-induced toxicity (e.g., septic shock, endotoxic shock),
polymyositis, dermatomyositis, and granulomatous diseases including
sarcoidosis.
[0108] Diseases or conditions of humans or other species which can
be treated with promoters of CCR2 receptor function (including
antibodies or fragments thereof), include, but are not limited
to:
[0109] immunosuppression, such as that in individuals with
immunodeficiency syndromes such as AIDS, individuals undergoing
radiation therapy, chemotherapy, therapy for autoimmune disease or
other drug therapy (e.g., corticosteroid therapy), which causes
immunosuppression; and immunosuppression due congenital deficiency
in receptor function or other causes.
[0110] Anti-CCR2 antibodies of the present invention can block the
binding of one or more chemokines, thereby blocking the downstream
cascade of one or more events leading to the above disorders.
[0111] Antibodies and functional fragments thereof which are
antagonists of CCR2 can be used as therapeutics for AIDS, as well
as certain inflammatory diseases. HIV-1 and HIV-2 are the etiologic
agents of acquired immunodeficiency syndrome (AIDS) in humans. AIDS
results in part from the depletion of CD4+ T lymphocytes in HIV
infected individuals. HIV-1 infects primarily T lymphocytes,
monocytes/macrophages, dendritic cells and, in the central nervous
system, microglia. All of these cells express the CD4 glycoprotein,
which serves as a receptor for HIV-1 and HIV-2. Efficient entry of
HIV into target cells is dependent upon binding of the viral
exterior envelope glycoprotein, gp120, to the amino-terminal CD4
domain. After virus binding, the HIV-1 envelope glycoproteins
mediate the fusion of viral and host cell membranes to complete the
entry process. Membrane fusion directed by HIV-1 envelope
glycoproteins expressed on the infected cell surface leads to
cell-cell fusion, resulting in syncytia.
[0112] Recently, host cell factors in addition to CD4 have been
suggested to determine the efficiency of HIV-1 envelope
glycoprotein-mediated membrane fusion. The 7 transmembrane receptor
(7TMR) termed HUMSTSR, LESTR, or "fusin" has been shown to allow a
range of CD4-expressing cells to support infection and cell fusion
mediated by laboratory-adapted HIV-1 envelope glycoproteins (Feng,
Y., et al., Science (Wash. D.C.), 272:872-877 (1996)). Antibodies
to HUMSTSR blocked cell fusion and infection by laboratory-adapted
HIV-1 isolates but not by macrophage-tropic primary viruses in
vitro (Feng, Y., et al., Science (Wash. D.C.), 272:872-877
(1996)).
[0113] The ability of chemokine receptors and related molecules to
facilitate the infection of primary clinical HIV-1 isolates has
been reported recently by several groups (see e.g., Bates, P.,
Cell, 86:1-3 (1996); Choe, H., et al., Cell, 85:1135-1148 (1996);
Doranz et al., Cell 85:1149-1158 (1996)). These studies indicated
that involvement of various members of the chemokine receptor
family in the early stages of HIV-1 infection helps to explain
viral tropism and .beta.-chemokine inhibition of primary HIV-1
isolates.
[0114] The present invention also provides a method of inhibiting
HIV infection of a cell (e.g., new infection and/or syncytium
formation) which expresses a mammalian CCR2 or portion thereof,
comprising contacting the cell with a composition comprising an
effective amount of an antibody or functional fragment thereof
which binds to a mammalian CCR2 or portion of said receptor. The
composition can also comprise one or more additional agents
effective against HIV, including, but not limited to, anti-CCR3
antibodies, anti-CCR5 antibodies, and anti-fusin antibodies.
[0115] Various methods can be used to assess binding of HIV to a
cell and/or infection of a cell by HIV in the presence of the
antibodies of the present invention. For example, assays which
assess binding of gp120 or a portion thereof to the receptor, HIV
infection and syncytium formation can be used (see, for example,
Choe, H., et al., Cell, 85:1135-1148 (1996)). The ability of the
antibody of the present invention to inhibit these processes can be
assessed using these or other suitable methods.
[0116] In addition, the present invention provides a method of
treating HIV in a patient, comprising administering to the patient
a composition comprising an effective amount of an antibody or
functional fragment thereof which binds to a mammalian CCR2 or
portion of said receptor. Again, the composition can also comprise
one or more additional agents effective against HIV, including, but
not limited to, anti-CCR3 antibodies, anti-CCR5 antibodies, and
anti-fusin antibodies. Therapeutic use of antibody to treat HIV
includes prophylactic use (e.g., for treatment of a patient who may
be or who may have been exposed to HIV). For example, health care
providers who may be exposed or who have been exposed to HIV (e.g.,
by needle-stick) can be treated according to the method. Another
example is the treatment of a patient exposed to virus after
unprotected sexual contact or failure of protection.
[0117] In AIDS, multiple drug treatment appears the most promising.
An anti-chemokine receptor antagonist that inhibits HIV infection
can be added to the drug treatment regimen, in particular by
blocking virus infection of new cells. Thus, administration of an
antibody or fragment of the present invention in combination with
one or more other therapeutic agents such as nucleoside analogues
(e.g., AZT, 3TC, ddI) and/or protease inhibitors is envisioned, and
provides an important addition to an HIV treatment regimen. In one
embodiment, a humanized anti-CCR2 mAb is used in combination with a
(i.e., one or more) therapeutic agent to reduce viral load from
patients, by preventing fusion and/or infection of new cells. Such
an antibody can also be useful in preventing perinatal
infection.
[0118] Another aspect of the invention relates to a method of
preventing HIV infection in an individual, comprising administering
to the individual an effective amount of an antibody or functional
fragment thereof which binds to CCR2. According to the method,
preventing HIV infection includes treatment in order to prevent
(reduce or eliminate) infection of new cells in an infected
individual or in order to prevent infection in an individual who
may be, may have been, or has been, exposed to HIV. For example,
individuals such as an HIV infected individual, a fetus of an HIV
infected female, or a health care worker may be treated according
to the method of the present invention.
[0119] Modes of Administration
[0120] One or more antibodies or fragments of the present invention
can be administered to an individual by an appropriate route,
either alone or in combination with (before, simultaneous with, or
after) another drug or agent. For example, the antibodies of the
present invention can also be used in combination with other
monoclonal or polyclonal antibodies (e.g., in combination with
antibodies which bind other chemokine receptors, including, but not
limited to, CCR3 and CCR5) or with existing blood plasma products,
such as commercially available gamma globulin and immune globulin
products used in prophylactic or therapeutic treatments. The
antibodies or fragments of the present invention can be used as
separately administered compositions given in conjunction with
antibiotics and/or antimicrobial agents.
[0121] An effective amount of an antibody or fragment (i.e., one or
more antibodies or fragments) is administered. An effective amount
is an amount sufficient to achieve the desired therapeutic
(including prophylactic) effect, under the conditions of
administration, such as an amount sufficient for inhibition of a
CCR2 function, and thereby, inhibition of an inflammatory response
or HIV infection, or an amount sufficient for promotion of a CCR2
function, as indicated.
[0122] A variety of routes of administration are possible
including, but not necessarily limited to, oral, dietary, topical,
parenteral (e.g., intravenous, intraarterial, intramuscular,
subcutaneous injection), inhalation (e.g., intrabronchial,
intraocular, intranasal or oral inhalation, intranasal drops),
depending on the disease or condition to be treated. Other suitable
methods of administration can also include rechargeable or
biodegradable devices and slow release polymeric devices. The
pharmaceutical compositions of this invention can also be
administered as part of a combinatorial therapy with other
agents.
[0123] Formulation of an antibody or fragment to be administered
will vary according to the route of administration and formulation
(e.g., solution, emulsion, capsule) selected. An appropriate
pharmaceutical composition comprising an antibody or functional
fragment thereof to be administered can be prepared in a
physiologically acceptable vehicle or carrier. A mixture of
antibodies and/or fragments can also be used. For solutions or
emulsions, suitable carriers include, for example, aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles can include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. A variety of appropriate aqueous
carriers are known to the skilled artisan, including water,
buffered water, buffered saline, polyols (e.g., glycerol, propylene
glycol, liquid polyethylene glycol), dextrose solution and glycine.
Intravenous vehicles can include various additives, preservatives,
or fluid, nutrient or electrolyte replenishers (See, generally,
Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980).
The compositions can optionally contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions such as pH adjusting and buffering agents and toxicity
adjusting agents, for example, sodium acetate, sodium chloride,
potassium chloride, calcium chloride and sodium lactate. The
antibodies and fragments of this invention can be lyophilized for
storage and reconstituted in a suitable carrier prior to use
according to art-known lyophilization and reconstitution
techniques. The optimum concentration of the active ingredient(s)
in the chosen medium can be determined empirically, according to
procedures well known to the skilled artisan, and will depend on
the ultimate pharmaceutical formulation desired. For inhalation,
the antibody or fragment can be solubilized and loaded into a
suitable dispenser for administration (e.g., an atomizer, nebulizer
or pressurized aerosol dispenser).
[0124] The present invention will now be illustrated by the
following Examples, which are not intended to be limiting in any
way. The teachings of all references cited herein are incorporated
herein by reference.
EXAMPLES
[0125] Materials:
[0126] The following materials were obtained from the indicated
sources:
[0127] PE-conjugated anti-CD16, PE-conjugated streptavidin, and
biotinylated anti-human IgE were from Pharmingen (San Diego,
Calif.). FITC-conjugated goat anti-mouse IgG was from Jackson
Immunoresearch Laboratories (West Grove, Pa.). FACS Lysing Buffer
was from Becton Dickenson (Mountain View, Calif.) and
[.sup.125I]-MCP-1 was from NEN (Boston, Mass.).
[0128] Cells, Cell Lines, and Tissue Culture
[0129] The murine pre-B lymphoma cell line L1/2 was maintained in
RPMI-1640 supplemented with 10% Fetal Clone I (Gibco BRL,
Gaithersburg, Md.) 50 Units/mL penicillin (Gibco BRL), 50 .mu.g/mL
streptomycin (Gibco BRL), 2 mM L-Glutamine (Gibco BRL), and 55
.mu.M .beta.-mercaptoethanol (Gibco BRL). Other cell lines included
transfectants of L1/2 cells expressing either CCR1 (Campbell, J. et
al. (1996) J. Cell Bio., 134:255-266), CCR5 (Wu et al., Nature
384:179-183 (1996)) grown in the above culture medium supplemented
with 800 .mu.g/ml active G418. THP-1 cells (ATCC No. TIB202) were
grown in accordance with ATCC instructions. PBMC were purified from
heparinized blood as described in Ponath et al., J. Clin. Invest.,
97:604-612 (1996).
[0130] Preparation of CCR2b Expression Construct and Stable
Transfectants
[0131] The coding region for the human CCR2b (Charo et al. (1994)
Proc. Natl. Acad. Sci. USA, 91:2752) was obtained by RT-PCR
amplification as described (Qin, S. et al. (1996) Eur. J. Immunol.,
26:640-647). cDNA was made using oligo(dT)-priming, and
amplification of the CCR2b coding region was achieved by nested PCR
with the following sets of primers which correspond to the
positions of the CCR2b sequence (GenBank Accession No. U03905;
Charo et al., Proc. Natl. Acad. Sci. USA 91:2752-2756 (1994)) as
indicated:
[0132] 1) 5' primer: 5'-TGAGACAAGCCACAAGCTGAAC-3' (nucleotides 11
to 32; SEQ ID NO: 1);
[0133] 3' Primer: 5'-TCTGTATTAGTACACACAGCCC-3' (nucleotides 1301 to
1280; SEQ ID NO: 2);
[0134] 2) 5' Primer: 5' -ATGCTGTCCACATCTCGTTCTCGG-3' (nucleotides
81 to 104; SEQ ID NO: 3);
[0135] 3' Primer: 5'-TTATAAACCAGCCGAGACTTCCTGCTC-3' (nucleotides
1164 to 1137; SEQ ID NO: 4).
[0136] The CCR2B cDNA coding region was modified to contain the CD5
signal peptide leader sequence (Aruffo et al., Cell 61:1303-1313
(1990)). The predicted amino acid sequence of this peptide is:
NH.sub.2-Met-Pro-Met-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Thr-Leu-Tyr-Leu-Leu-Gly-M-
et-Leu-Val-Ala-Ser-Val-Leu-Ala . . . (SEQ ID NO: 5)
[0137] Using PCR with the CCR2b cDNA as template and two
overlapping 5' primers that contain a BamHI restriction site,
encode the CD5 signal peptide sequence and the amino terminal
sequence of CCR2b, and a 3' primer located internally in the CCR2b
coding region.
[0138] 5' CD5 Seq1 primer
[0139] 5'-GGGGATCCAGAAACCATGCCCATGGGGTCTCTGCAACCGCTG
GCCACCTTGTACCTGCTG-3' (SEQ ID NO: 6)
[0140] 5' CD5 Seq2 primer
[0141] 5'-GCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTGCTAG
CGATGCTGTCCACATCTCGTTC-3' (SEQ ID NO: 7)
[0142] 3' CCR2AB2 primer-5'-GACGACCAGCATGTTGCC-3' (SEQ ID NO: 8;
U03905 nucleotides 272 to 255)
[0143] The 278 base pair amplified fragment was digested with BamHI
and Apal and the resulting 209 base pair fragment was inserted at
the Apal site at position 206 of the CCR2b cDNA (GenBank Accession
No. U03905) to replace the endogenous 5' base pair fragment of
CCR2. The resulting sequence that encodes a CCR2b with the CD5
signal peptide leader sequence immediately preceding the receptor
initiator methionine was inserted into the BamHI and Xhol sites of
pcDNA3 (Invitrogen, San Diego, Calif.) to create the mammalian
expression plasmid pCD5MCPRB. The CD5-CCR2b fragment was subcloned
into the BamH I-Not I site of pCDEF3 (Goldman et al., (1996)
Biotechniques 21:1013-1015), and this construct was designated
CCR2bDEF3. In this expression vector, the expression of the
inserted gene is driven by the EF-1.alpha. promoter.
[0144] Fifty milliliters of L1/2 cells were seeded at
4.times.10.sup.5 cells/mL the day before the electroporation. On
the day of the electroporation, the cells, which had grown up to a
density of 1.times.10.sup.6/mL, were centrifuged out of their
medium and resuspended in 800 .mu.l room temperature
electroporation buffer (Zajac et al., DNA 7:509-513). 120 mM
L-Glutamic Acid (Sigma), 7 mM Mg Acetate (EM Science), 4.3 mM
Glucose (Sigma), 17 mM K Pipes, pH 6.9 (Sigma), 1 mM EGTA (Sigma),
5 mM ATP, pH 7.0 (Sigma). Twenty-five micrograms Sca I linearized,
phenol/chloroform/isoamyl alcohol extracted and isopropanol
precipitated CCR2bDEF3 plasmid DNA was placed in an 0.4 cm gap
electroporation curvette. The resuspended cells were added to the
curvette, and a single pulse applied at 450 volts, 960 .mu.Fd. The
cells were then transferred from the curvette to a T-75 flask
containing 15 mL L1/2 growth medium (described above, and grown for
three days, at which time the cells were centrifuged out of their
medium and resuspended in L1/2 growth medium additionally
supplemented with 1 mM sodium pyruvate (Gibco BRL) and 0.8 mg/mL
active G418 (Gibco BRL).
[0145] Selection of Cells Expressing CCR2b by Chemotaxis
[0146] The transfected cells were allowed to grow for eleven days,
at which point they were split 1:20 into fresh growth medium. On
the sixteenth day, the cells were selected by chemotaxis. 600 .mu.L
1 nM MCP-1 in RPMI 1640 supplemented with 0.5% BSA (RPMI/BSA) was
placed in the lower chamber and 1.times.10.sup.6 CCR2bDEF3 cells in
100 .mu.l of RPMI/BSA were placed in the upper chamber of a 3.0
micron pore 24-well chemotaxis plate (Becton Dickinson). The cells
were allowed to chemotax for four hours and twenty minutes in a
37.degree. C., 5% CO.sub.2, humidified incubator, at which time the
upper chamber was removed. This incubation time was chosen at the
time of the experiment because it was sufficiently long for cells
responding to the MCP-1 to chemotax, but short enough to keep the
background low.
[0147] Secondary Selection of CCR2b Expressing-Cells by FACS
Sorting
[0148] The cells which had chemotaxed through the membrane and into
the lower chamber were grown up, and further purified by sterile
FACS sorting. Ten million CCR2bDEF3 cells were centrifuged out of
their medium, resuspended in 2.5 mL PBS(+Ca, Mg) supplemented with
1% heat-inactivated Fetal Calf Serum ("HI FCS") (Gibco BRL) and 2.5
mL sterile filtered anti-CCR2b amino-terminal peptide antibody
supernatant 5A11. The cells and the antibody were mixed and allowed
to incubate on ice for thirty minutes. The cells were then washed
twice with PBS (+) (Gibco BRL), and resuspended in 5 mL of a
sterile filtered, 1:250 dilution of FITC-conjugated,
affinity-purified F(ab.sup.1).sub.2 goat anti-mouse lgG (Jackson
ImmunoResearch Laboratories) in PBS (+) supplemented with 1% HI
FCS. The cells were incubated for thirty minutes on ice in the
dark, and then washed twice with PBS(+) (GIBCO BRL). The cells were
sorted on the FACSCalibur.RTM. and the brightest 4% of cells were
collected. (FL1.gtoreq.3.times.10.sup.2).
[0149] The sorted cells were allowed to grow up, and they were
resorted using the same protocol as above. The brightest 1% of
cells were collected. (FL1.gtoreq.3.times.10.sup.3).
[0150] Monoclonal Antibody Production
[0151] To produce mAbs to CCR2b, transfectants were continually
monitored to ensure that levels of expression did not drift
downward. FACS staining was performed periodically to ascertain
receptor expression on the transfectants using the anti CCR2b
antibody supernatant 5A11 with goat anti-mouse IgG FITC as the
secondary antibody.
[0152] Twenty million CCR2bDEF3.L1/2 cells were washed in RPMI 1640
(Gibco BRL) and incubated in RPMI 1640 plus 0.2 mg/mL Mitomycin C
for 30 minutes at 37.degree. C. The cells were then washed twice
with PBS (+) and 2.times.10.sup.7 cells in 0.5 mL PBS (+) were
injected intraperitoneally into a C57 BL/6 female mouse. This was
repeated two more times at two week intervals. The fourth time,
2.times.10.sup.7 cells were resuspended in 0.25 mL and injected
intravenously. Three days after the intravenous injection, the
mouse was sacrificed and the spleen removed and the cells fused
with the SP2/0 cell line as described (Current Protocols in
Immunology, John Wiley and Sons, New York, 1992).
[0153] This set of mice had previously been immunized many times
with 2 different cell lines as well as a synthetic peptide, but no
antibodies that stained CCR2 positive cells were generated from
several fusions. The above four immunizations with the
CCR2bDEF3.L1/2 cell line expressing high levels of CCR2b were
critical to obtain the described antibody.
[0154] Selecting Single Cell Clone of CCR2 Transfectants by
Limiting Dilution
[0155] After the mouse received the last injection, the twice
sorted cells were allowed to grow up again, and then they were
further purified by limiting dilution. The cells were plated at 1
and 0.5 cell per well in 96 well plates. Subcloned cells from the
0.5 cell per well dilution were grown up and tested for CCR2b
expression by indirect immunofluorescent FACS analysis using the
anti-CCR2b antibody supernatant 5A11 with goat anti-mouse IgG FITC
as the secondary antibody. The procedure was the same as described
above, except that the staining volume was 100 .mu.l. Four
positives were selected and frozen down.
[0156] Identification of Positive Monoclonal Antibodies
[0157] Immunofluorescent staining analysis using a FACScan.RTM.
(Becton Dickinson & Co., Mountain View, Calif.) was used to
identify the monoclonal antibodies which were reactive with the
CCR2b receptor. Hybridoma culture supernatants were assayed in a
96-well format using goat anti-mouse IgG FITC as the secondary
antibody. CCR2bDEF3.L1/2 cells were used to identify monoclonal
antibodies reactive with CCR2b, and untransfected L1/2 cells were
used to eliminate monoclonal antibodies reactive with other cell
surface proteins.
[0158] FACS Staining--Cultured Cells
[0159] For the staining of cultured transfectant cell lines
0.5.times.10.sup.6 cells in 50 .mu.l were resuspended in PBS+1% FCS
in a 96 well polystyrene V-bottom plate. 50 .mu.l of primary
antibody supernatants or HT medium (negative control) were added,
and the samples were incubated at 4.degree. C. for 30 min. 100
.mu.l of PBS were added and the cells were pelleted by
centrifugation and washed once with PBS. The pellet was resuspended
in 100 .mu.l PBS+1% FCS containing FITC-conjugated goat anti-mouse
IgG antibody (a 1:250 dilution) and incubated for thirty minutes at
4.degree. C. in the dark. The cells were washed twice with PBS,
resuspended in PBS, and analyzed by flow cytrometry with a FacScan
cytometer using the CellQuest software (Becton-Dickenson) Cells
were fixed with PBS/1% formaldehyde if they were not to be analyzed
the same day. Monoclonal antibodies 1D9 and 8G2 stain CCR2
transfectants but not CCR1 or CCR5 transfectants (FIGS. 1A-1O).
[0160] FACS Staining--Whole Blood
[0161] 100 .mu.l whole blood was mixed with 100 .mu.l of 1D9
antibody hybridoma supernatants or HT medium (negative control) and
incubated at 4.degree. C. for 30 min. After one wash with PBS,
100.mu.L FITC-conjugated goat anti-mouse IgG antibody (a 1:250
dilution) was added to each sample and incubated for 30 min. at
4.degree. C. in the dark. Samples were then washed once with PBS if
a second color staining is to be done, otherwise washed twice more
in PBS. For two color staining 5 .mu.l of mouse serum was added to
the cell pellets after the single wash, mixed, and incubated for
five minutes at 4.degree. C. in the dark. Second primary antibodies
(or PBS as a negative control) were added (10 .mu.l anti-CD16, 100
.mu.l 1:200 dilution of anti-IgE) and incubated for thirty minutes
at 4.degree. C. in the dark. Samples were then washed one time with
PBS and resuspended in 100 .mu.L streptavidin PE (1:200 PBS+1% BSA)
and incubated for fifteen minutes at 4.degree. C. in the dark.
Eyrythrocytes were lysed by adding 2 ml of FACS Lysing Buffer to
each sample and incubating at room temperature in the dark for
fifteen minutes or until samples were clear. The cells were
pelleted by centrifugation and all but 200 .mu.l of the supernatant
was aspirated. The samples were analyzed by flow cytometry on a
FacScan cytometer using the CellQuest software (Becton-Dickenson).
CCR2b is expressed on most monocytes, a subpopulation of
lymphocytes and a subset of granulocytes (FIGS. 2A-2L). CCR2b is
expressed on an IgE-positive population in peripheral blood
(basophils)(FIGS. 3A-3I).
[0162] MCP-1 Binding Assays
[0163] MCP-1 binding was performed in a final volume of 0.1 ml of
50 mM Hepes pH 7.4, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 0.02% sodium
azide, 0.5% BSA (HBB), containing either 2.5 .mu.g THP-1 membrane
protein or 500,000 PBMC and 0.1 nM of [.sup.125I]-MCP-1.
Competition binding experiments were performed by including
variable concentrations of unlabeled MCP-1, 1D9 antibody, or a
negative control IgG2a. Nonspecific binding was determined
following the addition of a 2500-fold excess of unlabeled MCP-1.
Samples were incubated for 60 minutes at room temperature, and
bound and free tracer were separated by filtration through 96-well
GF/B filterplates presoaked in 0.3% polyethyleneimine. The filters
were washed in HBB further supplemented with 0.5 M NaCl, dried, and
the amount of bound radioactivity determined by liquid
scintillation counting. mAb 1D9 inhibits [.sup.125I]MCP-1 binding
to THP-1 cell membranes with an IC.sub.50 of about 0.004 .mu.g/ml
(approximately 0.02 nM; FIG. 4) and to fresh PBMC with an IC.sub.50
of 0.04 .mu.g/ml (approximately 0.2 nM; FIG. 5).
[0164] Chemotaxis of PBMC
[0165] Chemotaxis was assayed using a 3 .mu.m pore size 96-well
chemotaxis plate (Neuroprobe, Cabin John, MD). PBMC isolated by
standard methods using Ficoll-Hypaque density gradient
centrifugation were washed with PBS/0.5% BSA and then resuspended
in chemotaxis assay media (HBSS/10 mM HEPES/0.5% Fatty acid free
BSA) to a final concentration of 10.times.10.sup.6 cells/ml. Cells
were princubated in chemotaxis assay media at room temperature for
20 min. with various concentrations of the anti-CCR2 antibody, 1D9,
or nonspecific murine IgG2a. The same dilutions of antibody were
mixed with chemokine and 30 .mu.l of the mixture was added to each
of the bottom wells of the chemotaxis plate. The bottom wells are
covered with the membrane, and 25 .mu.l of the cell and antibody
mixture are added to the top of the filter. The plates are
incubated at 37.degree. C. in 5% CO.sub.2 incubator for
approximately 80 min. At the completion of the migration, the
membrane is removed and the plate with the bottom wells is
incubated -80C. for 30 minutes to freeze the contents. The plates
are thawed at 37.degree. C. for 10 minutes. 6 .mu.l of a 1:400
dilution of CyQuant reagent (Molecular Probes, Eugene, Oreg.) in a
lysis buffer provided by the supplier is added to each well, and
the cell migration is quantified as indicated by fluorescence
intensity determined using a CytoFlour fluorescence plate reader at
485ex/535em. mAb 1D9 inhibits MCP-1-induced chemotaxis, but not
RANTES-induced chemotaxis, of fresh PBMC (FIGS. 6A and 6B).
Inhibition of MCP-1-induced chemotaxis of fresh PBMC has been
demonstrated with 10 .mu.g/ml (.apprxeq.40 nM).
[0166] 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
8 1 22 DNA Artificial Sequence Primer 1 tgagacaagc cacaagctga ac 22
2 22 DNA Artificial Sequence Primer 2 tctgtattag tacacacagc cc 22 3
24 DNA Artificial Sequence Primer 3 atgctgtcca catctcgttc tcgg 24 4
27 DNA Artificial Sequence Primer 4 ttataaacca gccgagactt cctgctc
27 5 24 PRT Homo sapiens 5 Met Pro Met Gly Ser Leu Gln Pro Leu Ala
Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Val Leu Ala
20 6 60 DNA Artificial Sequence Primer 6 ggggatccag aaaccatgcc
catggggtct ctgcaaccgc tggccacctt gtacctgctg 60 7 65 DNA Artificial
Sequence Primer 7 gccaccttgt acctgctggg gatgctggtc gcttccgtgc
tagcgatgct gtccacatct 60 cgttc 65 8 18 DNA Artificial Sequence
Primer 8 gacgaccagc atgttgcc 18
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