U.S. patent application number 12/691576 was filed with the patent office on 2010-11-04 for reagents that bind ccx-ckr2.
This patent application is currently assigned to ChemoCentryx, Inc.. Invention is credited to Maureen Howard, Thomas Schall.
Application Number | 20100278820 12/691576 |
Document ID | / |
Family ID | 37215373 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100278820 |
Kind Code |
A1 |
Howard; Maureen ; et
al. |
November 4, 2010 |
REAGENTS THAT BIND CCX-CKR2
Abstract
Antibodies that bind to CCX-CKR2 and methods of their use are
provided.
Inventors: |
Howard; Maureen; (Los Altos,
CA) ; Schall; Thomas; (Palo Alto, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
ChemoCentryx, Inc.
Mountain View
CA
|
Family ID: |
37215373 |
Appl. No.: |
12/691576 |
Filed: |
January 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11407729 |
Apr 19, 2006 |
7678891 |
|
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12691576 |
|
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60674140 |
Apr 21, 2005 |
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Current U.S.
Class: |
424/133.1 ;
424/145.1; 424/158.1; 435/69.6; 435/7.1; 530/387.3; 530/388.1;
530/389.2; 530/391.3; 536/23.53 |
Current CPC
Class: |
G01N 2333/715 20130101;
A61P 35/00 20180101; G01N 33/6863 20130101; C07K 16/2866 20130101;
G01N 2500/10 20130101; A61P 19/02 20180101; A61P 7/00 20180101 |
Class at
Publication: |
424/133.1 ;
530/389.2; 530/391.3; 530/388.1; 530/387.3; 424/158.1; 424/145.1;
536/23.53; 435/7.1; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/24 20060101 C07K016/24; C07K 16/46 20060101
C07K016/46; C07H 21/04 20060101 C07H021/04; A61P 35/00 20060101
A61P035/00; G01N 33/53 20060101 G01N033/53; C12P 21/04 20060101
C12P021/04 |
Claims
1. An antibody that competitively inhibits binding of a competitor
antibody to CCX-CKR2, wherein the competitor antibody comprises the
complementarity determining region (CDR) of: SEQ ID NO:12 and SEQ
ID NO:14; or SEQ ID NO:16 and SEQ ID NO:18.
2. The antibody of claim 1, wherein the antibody is linked to a
detectable label.
3. The antibody of claim 1, which is a monoclonal antibody.
4. The antibody of claim 1, which is a humanized antibody.
5. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the antibody of claim 1.
6. The pharmaceutical composition of claim 5, wherein the antibody
is a monoclonal antibody.
7. The pharmaceutical composition of claim 5, wherein the antibody
is a humanized antibody.
8. The pharmaceutical composition of claim 5, wherein the antibody
comprises the complementarity determining regions (CDRs) of SEQ ID
NO:12 and SEQ ID NO:14.
9. The pharmaceutical composition of claim 5, wherein the antibody
comprises SEQ ID NO:12 and SEQ ID NO:14.
10. The pharmaceutical composition of claim 5, wherein the antibody
comprises the complementarity determining regions (CDRs) of SEQ ID
NO:16 and SEQ ID NO:18.
11. The pharmaceutical composition of claim 5, wherein the antibody
comprises SEQ ID NO:16 and SEQ ID NO:18.
12. A method of inhibiting angiogenesis or proliferation of a
cancer cell, the method comprising the step of contacting the cell
with an antibody of claim 1.
13. The method of claim 12, wherein the cell is in an
individual.
14. The method of claim 13, wherein the individual has or is
pre-disposed to have arthritis.
15. The method of claim 13, wherein the individual is not a
human.
16. A method for identifying a modulator of CCX CKR2, comprising:
(a) combining a cell expressing a CCX CKR2 polypeptide or an
extract of the cell with a test agent; and (b) conducting an assay
to detect whether the test agent competes with a competitor
antibody for binding to the CCX CKR2 polypeptide, wherein the
competitor antibody comprises the complementarity determining
region (CDR) of: SEQ ID NO:12 and SEQ ID NO:14; or SEQ ID NO:16 and
SEQ ID NO:18, wherein competition between the competitor antibody
and the test agent for binding to the CCX-CKR2 polypeptide is an
indication that the test agent is a modulator of CCX CKR2
activity.
17. A method for testing the efficacy of a test agent that
modulates CCX-CKR2 activity, the method comprising, (a)
administering the test reagent to a first animal; (b) administering
to a second animal an antibody that competes with a competitor
antibody for binding to the CCX CKR2 polypeptide, wherein the
competitor antibody comprises the complementarity determining
region (CDR) of: SEQ ID NO:12 and SEQ ID NO:14; or SEQ ID NO:16 and
SEQ ID NO:18; and (c) comparing the effect of the test reagent on
the first animal to the effect of the antibody on the second
antibody, thereby determining the efficacy of a test agent.
18. A polynucleotide encoding SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, or SEQ ID NO:18.
19. The polynucleotide of claim 18, wherein the polynucleotide
comprises SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, or SEQ ID
NO:17.
20. A method of producing a chimeric antibody, the method
comprising operably linking a polynucleotide encoding at least one
complementarity determining region (CDR) from SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, or SEQ ID NO:18 to a heterologous
polynucleotide encoding at least the framework region of a heavy or
light chain of an antibody, to form a fusion polynucleotide
encoding a chimeric heavy or light chain of an antibody; and
expressing a chimeric heavy or light chain from the fusion
polynucleotide.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present patent application is a continuation application
of U.S. patent application Ser. No. 11/407,729, filed Apr. 19,
2006, which claims benefit of priority to U.S. Provisional Patent
Application No. 60/674,140, filed Apr. 21, 2005, which is
incorporated by reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] Chemokines constitute a family of small cytokines that are,
inter alia, produced in inflammation and regulate leukocyte
recruitment, activation and proliferation (Baggiolini, M. et al.,
Adv. Immunol. 55: 97-179 (1994); Springer, T. A., Annu. Rev.
Physiol. 57: 827-872 (1995); and Schall, T. J. and K. B. Bacon,
Curr. Opin. Immunol. 6: 865-873 (1994)). Chemokines are capable of
selectively inducing chemotaxis of the formed elements of the blood
(other than red blood cells), including leukocytes such as
neutrophils, monocytes, macrophages, eosinophils, basophils, mast
cells, and lymphocytes, including T cells and B cells. In addition
to stimulating chemotaxis, other changes can be selectively induced
by chemokines in responsive cells, including changes in cell shape,
transient rises in the concentration of intracellular free calcium
ions (Ca.sup.2+), granule exocytosis, integrin upregulation,
formation of bioactive lipids (e.g., leukotrienes), expression of
cytokines, and respiratory burst, associated with leukocyte
activation, growth and proliferation. Thus, the chemokines are
early triggers of the inflammatory response, causing inflammatory
mediator release, chemotaxis and extravasation to sites of
infection or inflammation.
[0003] Two subfamilies of chemokines, designated as CXC and CC
chemokines, are distinguished by the arrangement of the first two
of four conserved cysteine residues, which are either separated by
one amino acid (as in CXC chemokines SDF-1, IL-8, IP-10, MIG, PF4,
ENA-78, GCP-2, GRO.alpha., GRO.beta., GRO.gamma., NAP-2, NAP-4,
I-TAC) or are adjacent residues (as in CC chemokines MIP-1.alpha.,
MIP-1.beta., RANTES, MCP-1, MCP-2, MCP-3, 1-309). Most CXC
chemokines attract neutrophil leukocytes. For example, the CXC
chemokines interleukin 8 (IL-8), platelet factor 4 (PF4), and
neutrophil-activating peptide 2 (NAP-2) are potent chemoattractants
and activators of neutrophils. The CXC chemokines designated MIG
(monokine induced by gamma interferon) and IP-10
(interferon-.gamma. inducible 10 kDa protein) are particularly
active in inducing chemotaxis of activated peripheral blood
lymphocytes. CC chemokines are generally less selective and can
attract a variety of leukocyte cell types, including monocytes,
eosinophils, basophils, T lymphocytes, granulocytes and natural
killer cells. CC chemokines such as human monocyte chemotactic
proteins 1-3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on
Activation, Normal T Expressed and Secreted), and the macrophage
inflammatory proteins 1.alpha. and 1.beta. (MIP-1.alpha. and
MIP-1.beta.) have been characterized as chemoattractants and
activators of monocytes or lymphocytes, but do not appear to be
chemoattractants for neutrophils.
[0004] CC and CXC chemokines act through receptors that belong to a
superfamily of seven transmembrane spanning G protein-coupled
receptors (Murphy, P. M., Pharmacol Rev. 52:145-176 (2000)). This
family of G-protein coupled receptors comprises a large group of
integral membrane proteins, containing seven transmembrane-spanning
regions. The receptors may be coupled to G proteins, which are
heterotrimeric regulatory proteins capable of binding GTP and
mediating signal transduction from coupled receptors, for example,
by the production of intracellular mediators. Additionally
chemokine receptors may act independently of G protein coupling.
For instance the Duffy receptor expressed predominantly on red
blood cells is a promiscuous chemokine binding receptor which is
believed to act as a chemokine, removing chemokines from the
circulatory environment.
[0005] Generally speaking, chemokine and chemokine receptor
interactions tend to be promiscuous in that one chemokine can bind
many chemokine receptors and conversely a single chemokine receptor
can interact with several chemokines. There are a few exceptions to
this rule; one such exception has been the interaction between
SDF-1 and CXCR4 (Bleul et al., J Exp Med, 184(3): 1101-9 (1996);
Oberlin et al., Nature, 382(6594): 833-5 (1996)). Originally
identified as a pre-B cell growth-stimulating factor (Nagasawa et
al., Proc Natl Acad Sci USA, 91(6): 2305-9 (1994)), SDF-1 has been
the only reported human ligand for CXCR4. The SDF-1 gene encodes
two proteins, designated SDF-1.alpha. and SDF-1.beta., by
alternative splicing. These two proteins are identical except for
the four amino acid residues that are present in the N-terminus of
SDF-1.beta. and absent from SDF-1.alpha..
[0006] There are many aspects of chemokine receptor signaling and
selectivity for ligands that were not previously understood. For
example, there are a number of orphan receptors for which no
function has been previously determined. RDC1, for example, though
earlier thought to be a receptor for vasoactive intestinal peptide
(VIP), until recently has been considered to be an orphan receptor
because its endogenous ligand has not been identified. See, e.g.,
Cook et al., FEBS Letts. 300(2):149-152 (1992).
[0007] Recently, RDC1, renamed as CCX-CKR2, was determined to bind
to both chemokines SDF-1 and I-TAC. See, e.g., PCT/US04/34807 and
U.S. patent application Ser. Nos. 10/698,541, 10/912,638 and
11/050,345 each of which are incorporated by reference in their
entirety.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides antibodies that competitively
inhibit binding of a competitor antibody to CCX-CKR2, wherein the
competitor antibody comprises the complementarity determining
region (CDR) of:
[0009] SEQ ID NO:12 and SEQ ID NO:14; or
[0010] SEQ ID NO:16 and SEQ ID NO:18.
[0011] In some embodiments, the antibody is linked to a detectable
label. In some embodiments, the antibody is linked to a
radioisotope or a cytotoxic chemical.
[0012] In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is an antibody fragment. In some
embodiments, the antibody is a humanized antibody.
[0013] In some embodiments, the antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:12 and/or
SEQ ID NO:14 or CDRs substantially identical to the CDRs of SEQ ID
NO:12 and/or SEQ ID NO:14. In some embodiments, the antibody
comprises SEQ ID NO:12 and/or SEQ ID NO:14.
[0014] In some embodiments, the antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:16 and/or
SEQ ID NO:18 or CDRs substantially identical to the CDRs of SEQ ID
NO:16 and/or SEQ ID NO:18. In some embodiments, the antibody
comprises SEQ ID NO:16 and/or SEQ ID NO:18.
[0015] The present invention also provides pharmaceutical
compositions comprising a pharmaceutically acceptable excipient and
an antibody that competitively inhibits binding of a competitor
antibody to CCX-CKR2, wherein the competitor antibody comprises the
complementarity determining region (CDR) of:
[0016] SEQ ID NO:12 and SEQ ID NO:14; or
[0017] SEQ ID NO:16 and SEQ ID NO:18.
[0018] In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is an antibody fragment. In some
embodiments, the antibody is a humanized antibody.
[0019] In some embodiments, the antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:12 and/or
SEQ ID NO:14 or CDRs substantially identical to the CDRs of SEQ ID
NO:12 and/or SEQ ID NO:14. In some embodiments, the antibody
comprises SEQ ID NO:12 and/or SEQ ID NO:14.
[0020] In some embodiments, the antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:16 and/or
SEQ ID NO:18 or CDRs substantially identical to the CDRs of SEQ ID
NO:16 and/or SEQ ID NO:18. In some embodiments, the antibody
comprises SEQ ID NO:16 and/or SEQ ID NO:18.
[0021] The present invention also provides methods of detecting a
cell expressing CCX-CKR2 in a biological sample. In some
embodiments, the methods comprise contacting the biological sample
with an antibody and detecting the presence of the antibody,
wherein the antibody competitively inhibits binding of a competitor
antibody to CCX-CKR2, wherein the competitor antibody comprises the
complementarity determining region (CDR) of:
[0022] SEQ ID NO:12 and SEQ ID NO:14; or
[0023] SEQ ID NO:16 and SEQ ID NO:18.
[0024] In some embodiments, the antibody is linked to a detectable
label.
[0025] The present invention also provides methods of inhibiting
angiogenesis or proliferation of a cancer cell. In some
embodiments, the method comprises the step of contacting the cell
with an antibody that competitively inhibits binding of a
competitor antibody to CCX-CKR2, wherein the competitor antibody
comprises the complementarity determining region (CDR) of:
[0026] SEQ ID NO:12 and SEQ ID NO:14; or
[0027] SEQ ID NO:16 and SEQ ID NO:18, thereby inhibiting
angiogenesis or proliferation of a cancer cell.
[0028] In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is a monoclonal antibody. In some
embodiments, the antibody is an antibody fragment. In some
embodiments, the antibody is a humanized antibody.
[0029] In some embodiments, the antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:12 and/or
SEQ ID NO:14 or CDRs substantially identical to the CDRs of SEQ ID
NO:12 and/or SEQ ID NO:14. In some embodiments, the antibody
comprises SEQ ID NO:12 and/or SEQ ID NO:14.
[0030] In some embodiments, the antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:16 and/or
SEQ ID NO:18 or CDRs substantially identical to the CDRs of SEQ ID
NO:16 and/or SEQ ID NO:18. In some embodiments, the antibody
comprises SEQ ID NO:16 and/or SEQ ID NO:18.
[0031] In some embodiments, the cell is in an individual. In some
embodiments, the individual has or is pre-disposed to have
arthritis. In some embodiments, the individual is not a human.
[0032] The present invention also provides methods for identifying
a modulator of CCX-CKR2. In some embodiments, the method
comprises:
[0033] (a) combining a cell expressing a CCX CKR2 polypeptide or an
extract of the cell with a test agent; and
[0034] (b) conducting an assay to detect whether the test agent
competes with a competitor antibody for binding to the CCX CKR2
polypeptide, wherein the competitor antibody comprises the
complementarity determining region (CDR) of:
[0035] SEQ ID NO:12 and SEQ ID NO:14; or
[0036] SEQ ID NO:16 and SEQ ID NO:18,
[0037] wherein competition between the competitor antibody and the
test agent for binding to the CCX-CKR2 polypeptide is an indication
that the test agent is a modulator of CCX CKR2 activity.
[0038] In some embodiments, the competitor antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:12 and SEQ
ID NO:14. In some embodiments, the competitor antibody comprises
SEQ ID NO:12 and SEQ ID NO:14.
[0039] In some embodiments, the competitor antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:16 and SEQ
ID NO:18. In some embodiments, the competitor antibody comprises
SEQ ID NO:16 and SEQ ID NO:18.
[0040] The present invention also provides for methods of testing
the efficacy of a test agent that modulates CCX-CKR2 activity. This
is useful, for example, when using the antibodies of the invention
as a control drug in an analysis of CCX-CKR2 small molecule
agonists or antagonists. In some embodiments, the methods
comprise:
[0041] (a) administering the test reagent to a first animal;
[0042] (b) administering to a second animal an antibody that
competes with a competitor antibody for binding to the CCX CKR2
polypeptide, wherein the competitor antibody comprises the
complementarity determining region (CDR) of:
[0043] SEQ ID NO:12 and SEQ ID NO:14; or
[0044] SEQ ID NO:16 and SEQ ID NO:18; and
[0045] (c) comparing the effect of the test reagent on the first
animal to the effect of the antibody on the second antibody.
[0046] In some embodiments, the competitor antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:12 and SEQ
ID NO:14. In some embodiments, the competitor antibody comprises
SEQ ID NO:12 and SEQ ID NO:14.
[0047] In some embodiments, the competitor antibody comprises the
complementarity determining regions (CDRs) of SEQ ID NO:16 and SEQ
ID NO:18. In some embodiments, the competitor antibody comprises
SEQ ID NO:16 and SEQ ID NO:18.
[0048] The present invention also provides polypeptides comprising
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 or at
least one CDR from SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ
ID NO:18. In some embodiments, the polypeptides are antibodies.
[0049] The present invention also provides polynucleotides encoding
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 or at
least one CDR from SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ
ID NO:18. In some embodiments, the polynucleotide comprises SEQ ID
NO:11,SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17.
[0050] The present invention also provides method of producing a
chimeric antibody. In some embodiments, the method comprises:
[0051] operably linking a polynucleotide encoding at least one
complementarity determining region (CDR) from SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, or SEQ ID NO:18 to a heterologous
polynucleotide encoding at least the framework region of a heavy or
light chain of an antibody, to form a fusion polynucleotide
encoding a chimeric heavy or light chain of an antibody; and
[0052] expressing a chimeric heavy or light chain from the fusion
polynucleotide.
Definitions
[0053] "RDC1," designated herein as "CCX-CKR2" refers to a
seven-transmembrane domain presumed G-protein coupled receptor
(GPCR). The CCX-CKR2 dog ortholog was originally identified in
1991. See, Libert et al. Science 244:569-572 (1989). The dog
sequence is described in Libert et al., Nuc. Acids Res. 18(7):1917
(1990). The mouse sequence is described in, e.g., Heesen et al.,
Immunogenetics 47:364-370 (1998). The human sequence is described
in, e.g., Sreedharan et al., Proc. Natl. Acad. Sci. USA
88:4986-4990 (1991), which mistakenly described the protein as a
receptor of vasoactive intestinal peptide. "CCX-CKR2" includes
sequences that are conservatively modified variants of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. Fragments
of CCX-CKR2 are fragments of at least 5, and sometimes at least 10,
20, 50, 100, 200, 300 or up to 300 contiguous amino acids of one of
the above-listed sequences, or a conservatively modified variant
thereof.
[0054] A "subject" or "individual" refers to an animal, including a
human, non-human primate, mouse, rat, dog or other mammal.
[0055] A "chemotherapeutic agent" refers to an agent, which when
administered to an individual is sufficient to cause inhibition,
slowing or arresting of the growth of cancerous cells, or is
sufficient to produce a cytotoxic effect in cancerous cells.
Accordingly, the phrase "chemotherapeutically effective amount"
describes an amount of a chemotherapeutic agent administered to an
individual, which is sufficient to cause inhibition, slowing or
arresting of the growth of cancerous cells, or which is sufficient
to produce (directly or indirectly) a cytotoxic effect in cancerous
cells. A "sub-therapeutic amount" refers to an amount less than is
sufficient to cause inhibition, slowing or arresting of the growth
of cancerous cells, or which is less than sufficient to produce a
cytotoxic effect in cancerous cells.
[0056] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0057] Naturally occurring immunoglobulins have a common core
structure in which two identical light chains (about 24 kD) and two
identical heavy chains (about 55 or 70 kD) form a tetramer. The
amino-terminal portion of each chain is known as the variable (V)
region and can be distinguished from the more conserved constant
(C) regions of the remainder of each chain. Within the variable
region of the light chain is a C-terminal portion known as the J
region. Within the variable region of the heavy chain, there is a D
region in addition to the J region. Most of the amino acid sequence
variation in immunoglobulins is confined to three separate
locations in the V regions known as hypervariable regions or
complementarity determining regions (CDRs) which are directly
involved in antigen binding. Proceeding from the amino-terminus,
these regions are designated CDR1, CDR2 and CDR3, respectively. The
CDRs are held in place by more conserved framework regions (FRs).
Proceeding from the amino-terminus, these regions are designated
FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR
regions and a numbering system have been defined by, e.g., Kabat et
al. (Kabat et al., Sequences of Proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, U.S.
Government Printing Office (1991)).
[0058] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms variable light chain (V.sub.L) and variable heavy chain
(V.sub.H) refer to these light and heavy chains respectively.
[0059] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990)).
[0060] For preparation of monoclonal or polyclonal antibodies, any
technique known in the art can be used (see, e.g., Kohler &
Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology
Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies
and Cancer Therapy (1985)). "Monoclonal" antibodies refer to
antibodies derived from a single clone. Techniques for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce antibodies to polypeptides of this invention.
Also, transgenic mice, or other organisms such as other mammals,
may be used to express humanized antibodies. Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)).
[0061] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0062] A "humanized" antibody is an antibody that retains the
reactivity of a non-human antibody while being less immunogenic in
humans. This can be achieved, for instance, by retaining the
non-human CDR regions and replacing the remaining parts of the
antibody with their human counterparts. See, e.g., Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi,
Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991);
Padlan, Molec. Immun., 31(3):169-217 (1994).
[0063] The term "isolated," when applied to a protein, denotes that
the protein is essentially free of other cellular components with
which it is associated in the natural state. It is preferably in a
homogeneous state although it can be in either a dry or aqueous
solution. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified. The term "purified" denotes that a protein
gives rise to essentially one band in an electrophoretic gel.
Particularly, it means that the protein is at least 85% pure, more
preferably at least 95% pure, and most preferably at least 99%
pure.
[0064] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein at least two times the background and
do not substantially bind in a significant amount to other proteins
present in the sample. Typically a specific or selective reaction
will be at least twice background signal or noise and more
typically more than 10 to 100 times background.
[0065] The terms "peptidomimetic" and "mimetic" refer to a
synthetic chemical compound that has substantially the same
structural and functional characteristics of a naturally or
non-naturally occurring polypeptide (e.g., a reagent that binds to
CCX-CKR2). Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. Adv.
Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);
and Evans et al. J. Med. Chem. 30:1229 (1987), which are
incorporated herein by reference). Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used
to produce an equivalent or enhanced therapeutic or prophylactic
effect. Generally, peptidomimetics are structurally similar to a
paradigm polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as found in a polypeptide of
interest, but have one or more peptide linkages optionally replaced
by a linkage selected from the group consisting of, e.g.,
--CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CH.sub.2SO--. The mimetic can be either entirely composed of
synthetic, non-natural analogues of amino acids, or, is a chimeric
molecule of partly natural peptide amino acids and partly
non-natural analogs of amino acids. The mimetic can also
incorporate any amount of natural amino acid conservative
substitutions as long as such substitutions also do not
substantially alter the mimetic's structure and/or activity. For
example, a mimetic composition is within the scope of the invention
if it is capable of carrying out at least one of the binding or
enzymatic activities of a polypeptide of interest.
[0066] A "ligand" refers to an agent, e.g., a polypeptide or other
molecule, capable of binding to a receptor.
[0067] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids that encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a number of nucleic acid sequences
will encode any given protein. For instance, the codons GCA, GCC,
GCG and GCU all encode the amino acid alanine. Thus, at every
position where an alanine is specified by a codon, the codon can be
altered to any of the corresponding codons described without
altering the encoded polypeptide. Such nucleic acid variations are
"silent variations," which are one species of conservatively
modified variations. Every nucleic acid sequence herein which
encodes a polypeptide also describes every possible silent
variation of the nucleic acid. One of skill will recognize that
each codon in a nucleic acid (except AUG, which is ordinarily the
only codon for methionine, and TGG, which is ordinarily the only
codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0068] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0069] The following eight groups each contain amino acids that are
conservative substitutions for one another: [0070] 1) Alanine (A),
Glycine (G); [0071] 2) Aspartic acid (D), Glutamic acid (E); [0072]
3) Asparagine (N), Glutamine (Q); [0073] 4) Arginine (R), Lysine
(K); [0074] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V); [0075] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0076] 7) Serine (S), Threonine (T); and [0077] 8) Cysteine (C),
Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0078] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0079] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%,
90%, or 95% identity over a specified region, e.g., of the entire
polypeptide sequences of the invention or the extra-cellular
domains of the polypeptides of the invention), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
Such sequences are then said to be "substantially identical." This
definition also refers to the complement of a test sequence.
Optionally, the identity exists over a region that is at least
about 50 nucleotides in length, or more preferably over a region
that is 100 to 500 or 1000 or more nucleotides in length. The
present invention includes polypeptides that are substantially
identical to SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and/or SEQ ID
NO:18 and/or CDR1 or CDR2 within SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16 and/or SEQ ID NO:18, as displayed in FIG. 1.
[0080] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0081] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of, e.g., a full length sequence or from
20 to 600, about 50 to about 200, or about 100 to about 150 amino
acids or nucleotides in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are well-known in the art. Optimal
alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith and Waterman (1970) Adv.
Appl. Math. 2:482c, by the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for
similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad.
Sci. USA 85:2444, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0082] An example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) or 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands.
[0083] [55] The BLAST algorithm also performs a statistical
analysis of the similarity between two sequences (see, e.g., Karlin
and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0084] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1 depicts some embodiments of some of the
complementarity determining regions (CDRs) of the antibodies of the
invention (SEQ ID NOS:19-22).
DETAILED DESCRIPTION OF THE INVENTION
I. Antibodies of the Invention
[0086] The present invention provides reagents and methods for
treatment, diagnosis and prognosis for diseases and disorders
related to CCX-CKR2 using antibodies against CCX-CKR2. Diseases and
disorders related to CCX-CKR2 are exemplified more below and
include, but are not limited to, cancer, diseases involving
excessive or abnormal angiogenesis and arthritis.
[0087] In some embodiments, the antibodies are isolated. In some
embodiments of the invention, the antibodies recognize the same
epitope as the epitope bound by the CDRs in SEQ ID NO:12 and SEQ ID
NO:14. In some embodiments of the invention, the antibodies
recognize the same epitope as the epitope bound by the CDRs in SEQ
ID NO:16 and SEQ ID NO:18. Antibodies comprising SEQ ID NO:12 and
SEQ ID NO:14, or SEQ ID NO:16 and SEQ ID NO:18, bind to CCX-CKR2
and compete with the chemokines SDF-1 and I-TAC for binding to
CCX-CKR2. Competition assays for CCX-CKR2 binding are described in,
e.g., See, e.g., PCT/US04/34807 and U.S. Patent Publication Nos.
US2004/0170634 and 2005/0074826.
[0088] In some embodiments of the invention, the antibodies bind to
CCX-CKR2 but do not bind to human peripheral blood. For example, in
some embodiments, the antibodies of the invention do not bind to at
least one of the following: basophils, monocytes, plasmacytoid
dendritic cells; B cells, or CD4.sup.+ T cells.
[0089] In some embodiments, the antibodies of the present invention
comprise SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID
NO:18. In some embodiments, the antibodies of the present invention
comprise SEQ ID NO:12 and SEQ ID NO:14, or SEQ ID NO:16 and SEQ ID
NO:18. In some embodiments, the antibodies of the present invention
comprise the CDRs of SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16
or SEQ ID NO:18. In some embodiments, the antibodies of the present
invention comprise the CDRs of SEQ ID NO:12 and SEQ ID NO:14, or
SEQ ID NO:16 and SEQ ID NO:18.
[0090] The locations of CDR and FR regions and a numbering system
have been described previously, e.g., Kabat et al. (Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, U.S. Government
Printing Office (1991)). CDRs can generally be identified using the
NCBI IgBLAST algorithm. Those of skill in the art will recognize
that different sequence algorithms can provide slightly different
descriptions of the location of the CDRs in a particular antibody
amino acid sequence. In some cases, the heavy chain CDRs occur at
amino acid positions 31-35 (CDR1), 50-65 (CDR2) and 96-102 (CDR3).
In some cases, the light chain CDRs occur at amino acid positions
24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3). In some embodiments,
the CDRs are represented as depicted in FIG. 1.
[0091] The ability of a particular antibody to recognize the same
epitope as another antibody is typically determined by the ability
of one antibody to competitively inhibit binding of the second
antibody to the antigen, e.g., to CCX-CKR2 or a fragment or fusion
thereof. Any of a number of competitive binding assays can be used
to measure competition between two antibodies to the same antigen.
An exemplary assay is a Biacore assay. Briefly in these assays,
binding sites can be mapped in structural terms by testing the
ability of interactants, e.g. different antibodies, to inhibit the
binding of another. Injecting two consecutive antibody samples in
sufficient concentration can identify pairs of competing antibodies
for the same binding epitope. The antibody samples should have the
potential to reach a significant saturation with each injection.
The net binding of the second antibody injection is indicative for
binding epitope analysis. Two response levels can be used to
describe the boundaries of perfect competition versus non-competing
binding due to distinct epitopes. The relative amount of binding
response of the second antibody injection relative to the binding
of identical and distinct binding epitopes determines the degree of
epitope overlap. Antibodies may recognize linear or conformational
epitopes, hence antibodies may be competitive while recognizing
dissimilar and distal epitopes.
[0092] Other conventional immunoassays known in the art can be used
in the present invention. For example, antibodies can be
differentiated by the epitope to which they bind using a sandwich
ELISA assay. This is carried out by using a capture antibody to
coat the surface of a well. A subsaturating concentration of
tagged-antigen is then added to the capture surface. This protein
will be bound to the antibody through a specific antibody:epitope
interaction. After washing a second antibody, which has been
covalently linked to a detectable moiety (e.g., HRP, with the
labeled antibody being defined as the detection antibody) is added
to the ELISA. If this antibody recognizes the same epitope as the
capture antibody it will be unable to bind to the target protein as
that particular epitope will no longer be available for binding. If
however this second antibody recognizes a different epitope on the
target protein it will be able to bind and this binding can be
detected by quantifying the level of activity (and hence antibody
bound) using a relevant substrate. The background is defined by
using a single antibody as both capture and detection antibody,
whereas the maximal signal can be established by capturing with an
antigen specific antibody and detecting with an antibody to the tag
on the antigen. By using the background and maximal signals as
references, antibodies can be assessed in a pair-wise manner to
determine epitope specificity.
[0093] A first antibody is considered to competitively inhibit
binding of a second antibody, if binding of the second antibody to
the antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the first antibody using any of the assays described above.
[0094] Methods of preparing polyclonal antibodies are known to the
skilled artisan. Polyclonal antibodies can be raised in a mammal,
e.g., by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include a
protein encoded by a nucleic acid or fragment thereof or a fusion
protein thereof. It may be useful to conjugate the immunizing agent
to a protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited
to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin,
and soybean trypsin inhibitor. Examples of adjuvants which may be
employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0095] The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such
as those described by Kohler & Milstein, Nature 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. The immunizing agent
will typically include a CCX-CKR2 polypeptide, or a fragment or
fusion thereof. Generally, either peripheral blood lymphocytes
("PBLs") are used if cells of human origin are desired, or spleen
cells or lymph node cells are used if non-human mammalian sources
are desired. The lymphocytes are then fused with an immortalized
cell line using a suitable fusing agent, such as polyethylene
glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:
Principles and Practice, pp. 59-103 (1986)). Immortalized cell
lines are usually transformed mammalian cells, particularly myeloma
cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell lines are employed. The hybridoma cells can be
cultured in a suitable culture medium that contains one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of HGPRT-deficient cells.
[0096] In some embodiments the antibodies of the invention are
chimeric or humanized antibodies that compete with antibodies
comprising SEQ ID NO:12 and SEQ ID NO:14, or SEQ ID NO:16 and SEQ
ID NO:18 for binding to CCX-CKR2. As noted above, humanized forms
of antibodies are chimeric immunoglobulins in which residues from a
complementary determining region (CDR) of human antibody are
replaced by residues from a CDR of a non-human species such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity. For example, the CDRs of SEQ ID NO:12 and SEQ ID NO:14,
or SEQ ID NO:16 and SEQ ID NO:18, can be inserted into the
framework of a human antibody.
[0097] Human antibodies can be produced using various techniques
known in the art, including phage display libraries (Hoogenboom
& Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol.
Biol. 222:581 (1991)). The techniques of Cole et al. and Boerner et
al. are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
p. 77 (1985) and Boerner et al., J. Immunol. 147(1):86-95 (1991)).
Similarly, human antibodies can be made by introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, e.g., in U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
and in the following scientific publications: Marks et al.,
Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0098] [70] In some embodiments, the antibodies of the invention
are single chain Fvs (scFvs). The V.sub.H and the V.sub.L regions
(e.g., SEQ ID NO:12 and SEQ ID NO:14, or SEQ ID NO:16 and SEQ ID
NO:18) of a scFv antibody comprise a single chain which is folded
to create an antigen binding site similar to that found in two
chain antibodies. Once folded, noncovalent interactions stabilize
the single chain antibody. While the V.sub.H and V.sub.L regions of
some antibody embodiments can be directly joined together, one of
skill will appreciate that the regions may be separated by a
peptide linker consisting of one or more amino acids.
[0099] Peptide linkers and their use are well-known in the art.
See, e.g., Huston et al., Proc. Nat'l Acad. Sci. USA 8:5879 (1988);
Bird et al., Science 242:4236 (1988); Glockshuber et al.,
Biochemistry 29:1362 (1990); U.S. Pat. No. 4,946,778, U.S. Pat. No.
5,132,405 and Stemmer et al., Biotechniques 14:256-265 (1993).
Generally the peptide linker will have no specific biological
activity other than to join the regions or to preserve some minimum
distance or other spatial relationship between the V.sub.H and
V.sub.L. However, the constituent amino acids of the peptide linker
may be selected to influence some property of the molecule such as
the folding, net charge, or hydrophobicity. Single chain Fv (scFv)
antibodies optionally include a peptide linker of no more than 50
amino acids, generally no more than 40 amino acids, preferably no
more than 30 amino acids, and more preferably no more than 20 amino
acids in length. In some embodiments, the peptide linker is a
concatamer of the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:23),
preferably 2, 3, 4, 5, or 6 such sequences. However, it is to be
appreciated that some amino acid substitutions within the linker
can be made. For example, a valine can be substituted for a
glycine.
[0100] Methods of making scFv antibodies have been described. See,
Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature
341:544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314
(1996). In brief, mRNA from B-cells from an immunized animal is
isolated and cDNA is prepared. The cDNA is amplified using primers
specific for the variable regions of heavy and light chains of
immunoglobulins. The PCR products are purified and the nucleic acid
sequences are joined. If a linker peptide is desired, nucleic acid
sequences that encode the peptide are inserted between the heavy
and light chain nucleic acid sequences. The nucleic acid which
encodes the scFv is inserted into a vector and expressed in the
appropriate host cell. The scFv that specifically bind to the
desired antigen are typically found by panning of a phage display
library. Panning can be performed by any of several methods.
Panning can conveniently be performed using cells expressing the
desired antigen on their surface or using a solid surface coated
with the desired antigen. Conveniently, the surface can be a
magnetic bead. The unbound phage are washed off the solid surface
and the bound phage are eluted.
[0101] Finding the antibody with the highest affinity is dictated
by the efficiency of the selection process and depends on the
number of clones that can be screened and the stringency with which
it is done. Typically, higher stringency corresponds to more
selective panning If the conditions are too stringent, however, the
phage will not bind. After one round of panning, the phage that
bind to CCX-CKR2 coated plates or to cells expressing CCX-CKR2 on
their surface are expanded in E. coli and subjected to another
round of panning In this way, an enrichment of many fold occurs in
3 rounds of panning Thus, even when enrichment in each round is
low, multiple rounds of panning will lead to the isolation of rare
phage and the genetic material contained within which encodes the
scFv with the highest affinity or one which is better expressed on
phage.
[0102] Regardless of the method of panning chosen, the physical
link between genotype and phenotype provided by phage display makes
it possible to test every member of a cDNA library for binding to
antigen, even with large libraries of clones.
[0103] In one embodiment, the antibodies are bispecific antibodies.
Bispecific antibodies are monoclonal, including, but not limited
to, human or humanized, antibodies that have binding specificities
for at least two different antigens or that have binding
specificities for two epitopes on the same antigen. In one
embodiment, one of the binding specificities is for a CCK-CKR2
protein, the other one is for another different cancer antigen.
Alternatively, tetramer-type technology may create multivalent
reagents.
[0104] In some embodiments, the antibody is conjugated to an
effector moiety. The effector moiety can be any number of
molecules, including detectable labeling moieties such as
radioactive labels or fluorescent labels, or can be a therapeutic
moiety.
[0105] In other embodiments, the therapeutic moiety is a cytotoxic
agent. In this method, targeting the cytotoxic agent to cancer
tissue or cells, results in a reduction in the number of afflicted
cells, thereby reducing symptoms associated with the cancer.
Cytotoxic agents are numerous and varied and include, but are not
limited to, cytotoxic drugs or toxins or active fragments of such
toxins. Suitable toxins and their corresponding fragments include
diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,
curcin, crotin, phenomycin, enomycin, auristatin and the like.
Cytotoxic agents also include radiochemicals made by conjugating
radioisotopes to antibodies of the invention.
II. Immunoassays
[0106] The antibodies of the invention can be used to detect
CCX-CKR2 or CCX-CKR2-expressing cells using any of a number of well
recognized immunological binding assays (see, e.g., U.S. Pat. Nos.
4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of
the general immunoassays, see also Methods in Cell Biology, Vol.
37, Asai, ed. Academic Press, Inc. New York (1993); Basic and
Clinical Immunology 7th Edition, Stites & Ten, eds. (1991).
[0107] Thus, the present invention provides methods of detecting
cells that express CCX-CKR2. In one method, a biopsy is performed
on the subject and the collected tissue is tested in vitro. The
tissue or cells from the tissue is then contacted, with an
anti-CCX-CKR2 antibody of the invention. Any immune complexes which
result indicate the presence of a CCX-CKR2 protein in the biopsied
sample. To facilitate such detection, the antibody can be
radiolabeled or coupled to an effector molecule which is a
detectable label, such as a fluorescent label. In another method,
the cells can be detected in vivo using imaging systems. Then, the
localization of the label is determined. A conventional method for
visualizing diagnostic imaging can be used. For example,
paramagnetic isotopes can be used for MRI. Internalization of the
antibody may be important to extend the life within the organism
beyond that provided by extracellular binding, which will be
susceptible to clearance by the extracellular enzymatic environment
coupled with circulatory clearance.
[0108] CCX-CKR2 proteins can also be detected using standard
immunoassay methods and the antibodies of the invention. Standard
methods include, for example, radioimmunoassay, sandwich
immunoassays (including ELISA), immunofluorescence assays, Western
blot, affinity chromatography (affinity ligand bound to a solid
phase), and in situ detection with labeled antibodies. A secondary
detection agent may also be employed, e.g., goat anti-mouse FITC. A
general overview of the applicable technology can be found in
Harlow & Lane, Antibodies: A Laboratory Manual (1988).
[0109] The present invention provides methods of detecting a cancer
cell, including methods of providing a prognosis or diagnosis of
cancer. CCX-CKR2 is expressed in nearly every cancer cell tested to
date, whereas normal (non-cancer) expression of CCX-CKR2 appears to
be limited to the kidney and some brain cells as well as in certain
developmental stages of fetal liver. See, e.g., PCT/US04/34807 and
U.S. patent application Ser. Nos. 10/698,541 and 10/912,638.
Therefore, expression of CCX-CKR2 in a cell, and in particular, in
a non-fetal cell and/or a cell other than a kidney or brain cell,
indicates the likely presence of a cancer cell. The presence of CCX
CKR2 in the vascular endothelium of a tissue may also indicate the
presence of a cancer. In some cases, samples containing
CCX-CKR2-expressing cells are confirmed for the presence of cancer
cells using other methods known in the art.
[0110] According to yet another aspect of the invention, methods
for selecting a course of treatment of a subject having or
suspected of having cancer are provided. The methods include
obtaining from the subject a biological sample, contacting the
sample with antibodies or antigen-binding fragments thereof that
bind specifically to CCX-CKR2, detecting the presence or absence of
antibody binding, and selecting a course of treatment appropriate
to the cancer of the subject. In some embodiments, the treatment is
administering CCX-CKR2 antibodies of the invention to the
subject.
[0111] The present invention provides for methods of diagnosing
human diseases including, but not limited to cancer, e.g.,
carcinomas, gliomas, mesotheliomas, melanomas, lymphomas,
leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervical
cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and
Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal
cancer, non-small cell lung cancer, small cell lung cancer, cancer
of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary
cancer, cancer of the gallbladder, cancer of the small intestine,
rectal cancer, kidney cancer, bladder cancer, prostate cancer,
penile cancer, urethral cancer, testicular cancer, cervical cancer,
vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer,
parathyroid cancer, adrenal cancer, pancreatic endocrine cancer,
carcinoid cancer, bone cancer, skin cancer, retinoblastomas,
Hodgkin's lymphoma, non-Hodgkin's lymphoma (see, CANCER:PRINCIPLES
AND PRACTICE (DeVita, V. T. et al. eds 1997) for additional
cancers); as well as brain and neuronal dysfunction, such as
Alzheimer's disease and multiple sclerosis; kidney dysfunction;
rheumatoid arthritis; cardiac allograft rejection; atherosclerosis;
asthma; glomerulonephritis; contact dermatitis; inflammatory bowel
disease; colitis; psoriasis; reperfusion injury; as well as other
disorders and diseases described herein. In some embodiments, the
subject does not have Kaposi's sarcoma, multicentric Castleman's
disease or AIDS-associated primary effusion lymphoma.
III. Modulators of CCX-CKR2
[0112] A. Methods of Identifying Modulators of Chemokine
Receptors
[0113] A number of different screening protocols can be utilized to
identify agents that modulate the level of activity or function of
CCX-CKR2 in cells, particularly in mammalian cells, and especially
in human cells. In general terms, the screening methods involve
screening a plurality of agents to identify an agent that interacts
with CCX-CKR2 (or an extracellular domain thereof), for example, by
binding to CCX-CKR2 and preventing antibodies of the invention from
binding to CCX-CKR2 or activating CCX-CKR2. In some embodiments, an
agent binds CCX-CKR2 with at least about 1.5, 2, 3, 4, 5, 10, 20,
50, 100, 300, 500, or 1000 times the affinity of the agent for
another protein.
[0114] 1. Chemokine Receptor Binding Assays
[0115] In some embodiments, CCX-CKR2 modulators are identified by
screening for molecules that compete with antibody of the invention
from binding to a CCX-CKR2 polypeptide. Those of skill in the art
will recognize that there are a number of ways to perform
competition analyses. In some embodiments, samples with CCX-CKR2
are pre-incubated with a labeled antibody of the invention (e.g.,
an antibody comprising at least the CDRs of SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16 and/or SEQ ID NO:18) and then contacted with a
potential competitor molecule. Alteration (e.g., a decrease) of the
quantity of antibody bound to CCX-CKR2 in the presence of a test
compound indicates that the test compound is a potential CCX-CKR2
modulator.
[0116] Preliminary screens can be conducted by screening for agents
capable of binding to a CCX-CKR2, as at least some of the agents so
identified are likely chemokine receptor modulators. The binding
assays usually involve contacting CCX-CKR2 with one or more test
agents and allowing sufficient time for the protein and test agents
to form a binding complex. Any binding complexes formed can be
detected using any of a number of established analytical
techniques. Protein binding assays include, but are not limited to,
immunohistochemical binding assays, flow cytometry, radioligand
binding, europium labeled ligand binding, biotin labeled ligand
binding or other assays which maintain the conformation of
CCX-CKR2. The chemokine receptor utilized in such assays can be
naturally expressed, cloned or synthesized. Binding assays may be
used to identify agonists or antagonists. For example, by
contacting CCX-CKR2 with a potential agonist and measuring for
CCX-CKR2 activity, it is possible to identify those molecules that
stimulate CCX-CKR2 activity.
[0117] 2. Cells and Reagents
[0118] The screening methods of the invention can be performed as
in vitro or cell-based assays. In vitro assays are performed for
example, using membrane fractions or whole cells comprising
CCX-CKR2. Cell based assays can be performed in any cells in which
CCX-CKR2 is expressed.
[0119] Cell-based assays involve whole cells or cell fractions
containing CCX-CKR2 to screen for agent binding or modulation of
activity of CCX-CKR2 by the agent. Exemplary cell types that can be
used according to the methods of the invention include, e.g., any
mammalian cells including leukocytes such as neutrophils,
monocytes, macrophages, eosinophils, basophils, mast cells, and
lymphocytes, such as T cells and B cells, leukemias, Burkitt's
lymphomas, tumor cells, endothelial cells, pericytes, fibroblasts,
cardiac cells, muscle cells, breast tumor cells, ovarian cancer
carcinomas, cervical carcinomas, glioblastomas, liver cells, kidney
cells, and neuronal cells, as well as fungal cells, including
yeast. Cells can be primary cells or tumor cells or other types of
immortal cell lines. Of course, CCX-CKR2 can be expressed in cells
that do not express an endogenous version of CCX-CKR2.
[0120] In some cases, fragments of CCX-CKR2, as well as protein
fusions, can be used for screening. When molecules that compete for
binding with CCX-CKR2 ligands are desired, the CCX-CKR2 fragments
used are fragments capable of binding the antibodies of the
invention. Alternatively, any fragment of CCX-CKR2 can be used as a
target to identify molecules that bind CCX-CKR2. CCX-CKR2 fragments
can include any fragment of, e.g., at least 20, 30, 40, 50 amino
acids up to a protein containing all but one amino acid of
CCX-CKR2. Typically, ligand-binding fragments will comprise
transmembrane regions and/or most or all of the extracellular
domains of CCX-CKR2.
[0121] 3. Signaling or Adhesion Activity
[0122] In some embodiments, signaling triggered by CCX-CKR2
activation is used to identify CCX-CKR2 modulators. Signaling
activity of chemokine receptors can be determined in many ways. For
example, signaling can be determined by detecting chemokine
receptor-mediated cell adhesion. Interactions between chemokines
and chemokine receptors can lead to rapid adhesion through the
modification of integrin affinity and avidity. See, e.g., Laudanna,
Immunological Reviews 186:37-46 (2002).
[0123] Signaling can also be measured by determining, qualitatively
and quantitatively, secondary messengers, such as cyclic AMP or
inositol phosphates, as well as phosphorylation or
dephosphorylation events can also be monitored. See, e.g., Premack,
et al. Nature Medicine 2: 1174-1178 (1996) and Bokoch, Blood
86:1649-1660 (1995).
[0124] In addition, other events downstream of CCX-CKR2 activation
can also be monitored to determine signaling activity. Downstream
events include those activities or manifestations that occur as a
result of stimulation of a chemokine receptor. Exemplary downstream
events include, e.g., changed state of a cell (e.g., from normal to
cancer cell or from cancer cell to non-cancerous cell). Cell
responses include adhesion of cells (e.g., to endothelial cells).
Established signaling cascades involved in angiogenesis (e.g.,
VEGF-mediated signaling) can also be monitored for effects caused
by CCX-CKR2 modulators. The ability of agents to promote
angiogenesis can be evaluated, for example, in chick
chorioallantoic membrane, as discussed by Leung et al. (1989)
Science 246:1306-1309. Another option is to conduct assays with rat
corneas, as discussed by Rastinejad et al. (1989) Cell 56:345-355.
Other assays are disclosed in U.S. Pat. No. 5,840,693. Ovarian
angiogenesis models can also be used (see, e.g., Zimmerman, R. C.,
et al. (2003) J. Clin. Invest. 112:659-669; Zimmerman, R. C., et
al. (2001) Microvasc. Res. 62:15-25; and Hixenbaugh, E. A., et al.
(1993) Anat. Rec. 235: 487-500).
[0125] Other screening methods are based on the observation that
expression of certain regulatory proteins is induced by the
presence or activation of CCX-CKR2. Detection of such proteins can
thus be used to indirectly determine the activity of CCX-CKR2. A
series of ELISA investigations were conducted to compare the
relative concentration of various secreted proteins in the cell
culture media for cells transfected with CCX-CKR2 and untransfected
cells. Through these studies it was determined that CCX-CKR2
induces the production of a number of diverse regulatory proteins,
including growth factors, chemokines, metalloproteinases and
inhibitors of metalloproteinases. Thus, some of the screening
methods that are provided involve determining whether a test agent
modulates the production of certain growth factors, chemokines,
metalloproteinases and inhibitors of metalloproteinases by
CCX-CKR2. In some instances, the assays are conducted with cells
(or extracts thereof) that have been grown under limiting serum
conditions as this was found to increase the production of the
CCX-CKR2-induced proteins.
[0126] The following proteins are examples of the various classes
of proteins that were detected, as well as specific proteins within
each class: (1) growth factors (e.g., GM-CSF); (2) chemokines
(e.g., RANTES, MCP-1); (3) cytokines (eg IL-6) (4)
metalloproteinase (e.g., MMP3); and (5) inhibitor of
metalloproteinase (e.g., TIMP-1). It is expected that other
proteins in these various classes can also be detected.
[0127] These particular proteins can be detected using standard
immunological detection methods that are known in the art. One
approach that is suitable for use in a high-throughput format, for
example, are ELISAs that are conducted in multi-well plates. An
ELISA kit for detecting TIMP-1 is available from DakoCytomation
(Product Code No. EL513). ELISA kits for IL-6 and MMP3 can be
obtained from R and D Systems. Further examples of suppliers of
antibodies that specifically bind the proteins listed above are
provided in the examples below. Proteins such as the
metalloproteinases that are enzymes can also be detected by known
enzymatic assays.
[0128] In other embodiments, potential modulators of CCX-CK2 are
tested for their ability to modulate cell adhesion. Tumor cell
adhesion to endothelial cell monolayers has been studied as a model
of metastatic invasion (see, e.g., Blood and Zetter, Biovhem.
Biophys. Acta, 1032, 89-119 (1990). These monolayers of endothelial
cells mimic the lymphatic vasculature and can be stimulated with
various cytokines and growth factors (e.g., TNFalpha and IL-1beta).
Cells expressing CCX-CKR2 can be evaluated for the ability to
adhere to this monolayer in both static adhesion assays as well as
assays where cells are under flow conditions to mimic the force of
the vasculature in vivo. Additionally, assays to evaluate adhesion
can also be performed in vivo (see, e.g., von Andrian, U. H.
Microcirculation. 3(3):287-300 (1996)).
[0129] 4. Validation
[0130] Agents that are initially identified by any of the foregoing
screening methods can be further tested to validate the apparent
activity. Preferably such studies are conducted with suitable
animal models. The basic format of such methods involves
administering a lead compound identified during an initial screen
to an animal that serves as a disease model for humans and then
determining if the disease (e.g., cancer, myocardial infarction,
wound healing, or other diseases related to angiogenesis) is in
fact modulated and/or the disease or condition is ameliorated. The
animal models utilized in validation studies generally are mammals
of any kind Specific examples of suitable animals include, but are
not limited to, primates, mice, rats and zebrafish.
[0131] In some embodiments, arthritis animal models are used to
screen and/or validate therapeutic uses for agents that modulate
CCX-CKR2. Exemplary arthritis animal models include, e.g., the
collagen-induced arthritis (CIA) animal model.
[0132] B. Agents that Interact with CCX-CKR2
[0133] Modulators of CCX-CKR2 (e.g., antagonists or agonists) can
include, e.g., antibodies (including monoclonal, humanized or other
types of binding proteins that are known in the art), small organic
molecules, siRNAs, CCX-CKR2 polypeptides or variants thereof,
chemokines (including but not limited to SDF-1 and/or I-TAC),
chemokine mimetics, chemokine polypeptides, etc.
[0134] The agents tested as modulators of CCX-CKR2 can be any small
chemical compound, or a biological entity, such as a polypeptide,
sugar, nucleic acid or lipid. Alternatively, modulators can be
genetically altered versions, or peptidomimetic versions, of a
chemokine or other ligand. Typically, test compounds will be small
chemical molecules and peptides. Essentially any chemical compound
can be used as a potential modulator or ligand in the assays of the
invention, although most often compounds that can be dissolved in
aqueous or organic (especially DMSO-based) solutions are used. The
assays are designed to screen large chemical libraries by
automating the assay steps and providing compounds from any
convenient source to assays, which are typically run in parallel
(e.g., in microtiter formats on microtiter plates in robotic
assays). It will be appreciated that there are many suppliers of
chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.
Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka
Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.
[0135] In some embodiments, the agents have a molecular weight of
less than 1,500 daltons, and in some cases less than 1,000, 800,
600, 500, or 400 daltons. The relatively small size of the agents
can be desirable because smaller molecules have a higher likelihood
of having physiochemical properties compatible with good
pharmacokinetic characteristics, including oral absorption than
agents with higher molecular weight. For example, agents less
likely to be successful as drugs based on permeability and
solubility were described by Lipinski et al. as follows: having
more than 5H-bond donors (expressed as the sum of OHs and NHs);
having a molecular weight over 500; having a LogP over 5 (or MLogP
over 4.15); and/or having more than 10H-bond acceptors (expressed
as the sum of Ns and Os). See, e.g., Lipinski et al. Adv Drug
Delivery Res 23:3-25 (1997). Compound classes that are substrates
for biological transporters are typically exceptions to the
rule.
[0136] In one embodiment, high throughput screening methods involve
providing a combinatorial chemical or peptide library containing a
large number of potential therapeutic compounds (potential
modulator or ligand compounds). Such "combinatorial chemical
libraries" or "ligand libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0137] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks." For example, a linear combinatorial chemical library such
as a polypeptide library is formed by combining a set of chemical
building blocks (amino acids) in every possible way for a given
compound length (i.e., the number of amino acids in a polypeptide
compound). Millions of chemical compounds can be synthesized
through such combinatorial mixing of chemical building blocks.
[0138] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091),
benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., J.
Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses
of small compound libraries (Chen et al., J. Amer. Chem. Soc.
116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303
(1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger
and Sambrook, all supra), peptide nucleic acid libraries (see,
e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g.,
Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
U.S. Pat. No. 5,288,514, and the like).
[0139] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa.,
Martek Biosciences, Columbia, Md., etc.).
IV. Cancer, Angiogenesis and other Biological Aspects of
CCX-CKR2
[0140] The antibodies of the invention can be contacted to a cell
expressing CCX-CKR2 in vitro, in vivo, or ex vivo (i.e., removed
from a body, treated and returned to the body). The antibodies of
the invention can be administered directly to the mammalian subject
for modulation of chemokine receptor activity in vivo. In some
embodiments, the antibodies compete with SDF1 and/or I-TAC for
binding to CCX-CKR2. In some embodiments of the invention, the
antibodies recognize the same epitope as the epitope bound by the
CDRs in SEQ ID NO:12 and SEQ ID NO:14, or SEQ ID NO:16 and SEQ ID
NO:18. In some embodiments, the antibodies comprise SEQ ID NO:12
and/or SEQ ID NO:14, or SEQ ID NO:16 and/or SEQ ID NO:18.
[0141] In some embodiments, the CCX-CKR2 antibodies are
administered to a subject having cancer. In some cases, CCX-CKR2
modulators are administered to treat cancer, e.g., carcinomas,
gliomas, mesotheliomas, melanomas, lymphomas, leukemias,
adenocarcinomas, breast cancer, ovarian cancer, cervical cancer,
glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's
lymphoma, head and neck cancer, colon cancer, colorectal cancer,
non-small cell lung cancer, small cell lung cancer, cancer of the
esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer,
cancer of the gallbladder, cancer of the small intestine, rectal
cancer, kidney cancer, bladder cancer, prostate cancer, penile
cancer, urethral cancer, testicular cancer, cervical cancer,
vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer,
parathyroid cancer, adrenal cancer, pancreatic endocrine cancer,
carcinoid cancer, bone cancer, skin cancer, retinoblastomas,
Hodgkin's lymphoma, non-Hodgkin's lymphoma (see, CANCER:PRINCIPLES
AND PRACTICE (DeVita, V. T. et al. eds 1997) for additional
cancers); as well as brain and neuronal dysfunction, such as
Alzheimer's disease and multiple sclerosis; kidney dysfunction;
rheumatoid arthritis; cardiac allograft rejection; atherosclerosis;
asthma; glomerulonephritis; contact dermatitis; inflammatory bowel
disease; colitis; psoriasis; reperfusion injury; as well as other
disorders and diseases described herein. In some embodiments, the
subject does not have Kaposi's sarcoma, multicentric Castleman's
disease or AIDS-associated primary effusion lymphoma.
[0142] The present invention also encompasses decreasing
angiogenesis in any subject in need thereof by administering
antibodies of the invention. For example, decreasing CCX-CKR2
activity by contacting CCX-CKR2 with an antibody of the invention,
thereby decreasing angiogenesis, is useful to inhibit formation,
growth and/or metastasis of tumors, especially solid tumors.
Description of embodiments relating to modulated CCX-CKR2 and
angiogenesis are described in, e.g., U.S. patent application Ser.
No. 11/050,345.
[0143] Other disorders involving unwanted or problematic
angiogenesis include rheumatoid arthritis; psoriasis; ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, comeal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis;
Osler-Webber Syndrome; myocardial angiogenesis; plaque
neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; disease of excessive or abnormal stimulation of
endothelial cells, including intestinal adhesions, Crohn's disease,
skin diseases such as psoriasis, excema, and scleroderma, diabetes,
diabetic retinopathy, retinopathy of prematurity, age-related
macular degeneration, atherosclerosis, scleroderma, wound
granulation and hypertrophic scars, i.e., keloids, and diseases
that have angiogenesis as a pathologic consequence such as cat
scratch disease and ulcers (Helicobacter pylori), can also be
treated with antibodies of the invention. Angiogenic inhibitors can
be used to prevent or inhibit adhesions, especially
intra-peritoneal or pelvic adhesions such as those resulting after
open or laproscopic surgery, and bum contractions. Other conditions
which should be beneficially treated using the angiogenesis
inhibitors include prevention of scarring following
transplantation, cirrhosis of the liver, pulmonary fibrosis
following acute respiratory distress syndrome or other pulmonary
fibrosis of the newborn, implantation of temporary prosthetics, and
adhesions after surgery between the brain and the dura.
Endometriosis, polyposis, cardiac hypertrophyy, as well as obesity,
may also be treated by inhibition of angiogenesis. These disorders
may involve increases in size or growth of other types of normal
tissue, such as uterine fibroids, prostatic hypertrophy, and
amyloidosis. Antibodies of the present invention may be used
prophylactically or therapeutically for any of the disorders or
diseases described herein.
[0144] Decreasing CCX-CKR2 activity with the antibodies of the
present invention can also be used in the prevention of
neovascularization to effectively treat a host of disorders. Thus,
for example, the decreasing angiogenesis can be used as part of a
treatment for disorders of blood vessels (e.g., hemangiomas and
capillary proliferation within atherosclerotic plaques), muscle
diseases (e.g., myocardial angiogenesis, myocardial infarction or
angiogenesis within smooth muscles), joints (e.g., arthritis,
hemophiliac joints, etc.), and other disorders associated with
angiogenesis. Promotion of angiogenesis can also aid in
accelerating various physiological processes and treatment of
diseases requiring increased vascularization such as the healing of
wounds, fractures, and burns, inflammatory diseases, ischeric
heart, and peripheral vascular diseases.
[0145] The antibodies of the present invention may also be used to
enhance wound healing. Without intending to limit the invention to
a particular mechanism of action, it may be that antagonism of
CCX-CKR2 allows for endogenous ligands to instead bind to lower
affinity receptors, thereby triggering enhanced wound healing. For
example, SDF-1 binds to both CCX-CKR2 and CXCR4, but binds to CXCR4
with a lower affinity. Similarly, I-TAC binds to CXCR3 with a lower
affinity than I-TAC binds to CCX-CKR2. By preventing binding of
these ligands to CCX-CKR2, CCX-CKR2 antagonists may allow the
ligands to bind to the other receptors, thereby enhancing wound
healing. Thus, the antagonism of CCX-CKR2 to enhance wound healing
may be mediated by a different mechanism than enhancing wound
healing by stimulating CCX-CKR2 activity with an agonist.
[0146] Aside from treating disorders and symptoms associated with
neovascularization, the inhibition of angiogenesis can be used to
modulate or prevent the occurrence of normal physiological
conditions associated with neovascularization. Thus, for example
the inventive method can be used as a birth control. In accordance
with the present invention, decreasing CCX-CKR2 activity within the
ovaries or endometrium can attenuate neovascularization associated
with ovulation, implantation of an embryo, placenta formation,
etc.
[0147] Inhibitors of angiogenesis have yet other therapeutic uses.
For example, the antibodies of the present invention may be used
for the following:
[0148] (a) Adipose tissue ablation and treatment of obesity. See,
e.g, Kolonin et al., Nature Medicine 10(6):625-632 (2004);
[0149] (b) Treatment of preclampsia. See, e.g., Levine et al., N.
Engl. J. Med. 350(7): 672-683 (2004); Maynard, et al., J. Clin.
Invest. 111(5): 649-658 (2003); and
[0150] (c) Treatment of cardiovascular disease. See, e.g., March,
et al., Am. J. Physiol. Heart Circ. Physiol. 287:H458-H463 (2004);
Rehman et al., Circulation 109: 1292-1298 (2004).
V. Administration and Pharmaceutical Compositions
[0151] The pharmaceutical compositions of the invention may
comprise, e.g., an antibody of the present invention and a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are determined in part by the particular composition being
administered, as well as by the particular method used to
administer the composition. Accordingly, there is a wide variety of
suitable formulations of pharmaceutical compositions of the present
invention (see, e.g., Remington's Pharmaceutical Sciences,
17.sup.th ed. 1985)).
[0152] Formulations suitable for administration include aqueous and
non-aqueous solutions, isotonic sterile solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, orally, nasally, topically, intravenously,
intraperitoneally, subcutaneously, or intrathecally. The
formulations of compounds can be presented in unit-dose or
multi-dose sealed containers, such as ampoules and vials. Solutions
and suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0153] The composition can be administered by means of an infusion
pump, for example, of the type used for delivering insulin or
chemotherapy to specific organs or tumors. Compositions of the
inventions can be injected using a syringe or catheter directly
into a tumor or at the site of a primary tumor prior to or after
excision; or systemically following excision of the primary tumor.
The compositions of the invention can be administered topically or
locally as needed. For prolonged local administration, the
antibodies may be administered in a controlled release implant
injected at the site of a tumor. Alternatively an individual's
cells can be transfected ex vivo with plasmids so as to express the
antibody of the invention and subsequently injected at the site of
the tumor. For topical treatment of a skin condition, the enzyme
antibodies may be administered to the skin in an ointment or
gel.
[0154] In some embodiments, CCX-CKR2 antibodies of the present
invention can be administered in combination with other appropriate
therapeutic agents, including, e.g., chemotherapeutic agents,
radiation, etc. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders such as, e.g.,
cancer, wounds, kidney dysfunction, brain dysfunction or neuronal
dysfunction. Using this approach, one may be able to achieve
therapeutic efficacy with lower dosages of each agent, thus
reducing the potential for adverse side effects.
[0155] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
response in the subject over time (e.g., to reduce tumor size or
tumor load). The optimal dose level for any patient will depend on
a variety of factors including the efficacy of the specific
modulator employed, the age, body weight, physical activity, and
diet of the patient, on a possible combination with other drugs,
and on the severity of a particular disease. The size of the dose
also will be determined by the existence, nature, and extent of any
adverse side-effects that accompany the administration of a
particular compound or vector in a particular subject.
[0156] In determining the effective amount of the antibody to be
administered a physician may evaluate circulating plasma levels of
the antibody, antibody toxicity, and the production of
anti-antibody antibodies. In general, the dose equivalent of an
antibody is from about 1 ng/kg to 10 mg/kg for a typical
subject.
[0157] For administration, the antibodies of the present invention
can be administered at a rate determined by the LD-50 of the
antibody, and the side-effects of the antibody at various
concentrations, as applied to the mass and overall health of the
subject. Clearance of the antibody by the recipient's immune system
may also affect the suitable dosage to be administered.
Administration can be accomplished via single or divided doses.
[0158] The compositions containing antibodies of the invention can
be administered for therapeutic or prophylactic treatments. In
therapeutic applications, compositions are administered to a
patient suffering from a disease (e.g., a cancer, arthritis or
other CCX-CKR2-related disease or disorder) in an amount sufficient
to cure or at least partially arrest the disease and its
complications, e.g., decreased size of tumor, etc. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the agents of this invention to
effectively treat the patient. An amount of modulator that is
capable of preventing or slowing the development of cancer in a
mammal is referred to as a "prophylactically effective dose." The
particular dose required for a prophylactic treatment will depend
upon the medical condition and history of the mammal, the
particular cancer being prevented, as well as other factors such as
age, weight, gender, administration route, efficiency, etc. Such
prophylactic treatments may be used, e.g., in a mammal who has
previously had cancer to prevent a recurrence of the cancer, or in
a mammal who is suspected of having a significant likelihood of
developing cancer.
VI. Combination Therapies
[0159] Antibodies of the invention can be supplied alone or in
conjunction with one or more other drugs. Possible combination
partners can include, e.g., additional anti-angiogenic factors
and/or chemotherapeutic agents (e.g., cytotoxic agents) or
radiation, a cancer vaccine, an immunomodulatory agent, an
anti-vascular agent, a signal transduction inhibitor, an
antiproliferative agent, or an apoptosis inducer.
[0160] Antibodies of the invention can be used in conjunction with
antibodies and peptides that block integrin engagement, proteins
and small molecules that inhibit metalloproteinases (e.g.,
marmistat), agents that block phosphorylation cascades within
endothelial cells (e.g., herbamycin), dominant negative receptors
for known inducers of angiogenesis, antibodies against inducers of
angiogenesis or other compounds that block their activity (e.g.,
suramin), or other compounds (e.g., retinoids, IL-4, interferons,
etc.) acting by other means. Indeed, as such factors may modulate
angiogenesis by different mechanisms, employing antibodies of the
invention in combination with other antiangiogenic agents can
potentiate a more potent (and potentially synergistic) inhibition
of angiogenesis within the desired tissue.
[0161] Anti-angiogenesis agents, such as MMP-2
(matrix-metalloprotienase 2) inhibitors, MMP-9
(matrix-metalloprotienase 9) inhibitors, and COX-II (cyclooxygenase
II) inhibitors, can be used in conjunction with antibodies of the
invention and pharmaceutical compositions described herein.
Anti-CCX-CKR2 antibodies of the invention can also be used with
signal transduction inhibitors, such as agents that can inhibit
EGFR (epidermal growth factor receptor) responses, such as EGFR
antibodies, EGF antibodies, and molecules that are EGFR inhibitors;
VEGF (vascular endothelial growth factor) inhibitors, such as VEGF
receptors and molecules that can inhibit VEGF; and erbB2 receptor
inhibitors, such as organic molecules or antibodies that bind to
the erbB2 receptor, for example, HERCEPTIN.TM. (Genentech, Inc. of
South San Francisco, Calif., USA).
[0162] Anti-CCX-CKR2 antibodies of the invention can also be
combined with other drugs including drugs that promote angiogenesis
and/or wound healing. Those of skill in the art will appreciate
that one can incorporate one or more medico-surgically useful
substances or therapeutic agents, e.g., those which can further
intensify the angiogenic response, and/or accelerate and/or
beneficially modify the healing process when the composition is
applied to the desired site requiring angiogenesis. For example, to
further promote angiogenesis, repair and/or tissue growth, at least
one of several hormones, growth factors or mitogenic proteins can
be included in the composition, e.g., fibroblast growth factor,
platelet derived growth factor, macrophage derived growth factor,
etc. In addition, antimicrobial agents can be included in the
compositions, e.g., antibiotics such as gentamicin sulfate, or
erythromycin. Other medico-surgically useful agents can include
anti-inflammatories, analgesics, anesthetics, rubifacients,
enzymes, antihistamines and dyes.
[0163] Anti-CCX-CKR2 antibodies of the invention can also be
combined with other drugs including drugs for treating arthritis.
Examples of such agents include anti-inflammatory therapeutic
agents. For example, glucocorticosteroids, such as prednisolone and
methylprednisolone, are often-used anti-inflammatory drugs.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are also used to
suppress inflammation. NSAIDs inhibit the cyclooxygenase (COX)
enzymes, COX-1 and COX-2, which are central to the production of
prostaglandins produced in excess at sites of inflammation. In
addition, the inflammation-promoting cytokine, tumor necrosis
factor .alpha. (TNF.alpha.), is associated with multiple
inflammatory events, including arthritis, and anti-TNF.alpha.
therapies are being used clinically.
VII. Kits for Use in Diagnostic and/or Prognostic Applications
[0164] For use in diagnostic, research, and therapeutic
applications suggested above, kits are also provided by the
invention. In the diagnostic and research applications such kits
may include any or all of the following: assay reagents, buffers,
and the anti-CCX-CKR2 antibodies of the invention. A therapeutic
product may include sterile saline or another pharmaceutically
acceptable emulsion and suspension base.
[0165] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
methods of this invention. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
Examples
[0166] Production of antibodies to G-protein coupled receptors
(GPCRs) has been notoriously difficult. We used the method of
Genovac AG, DE outlined in Canadian Patent application CA 2 350
078. Antibodies that bind CCX-CKR2 were created by inoculation of
mice with cDNA expressing CCX-CKR2 (SEQ ID NO:1). Briefly, CCX-CKR2
was cloned into an expression vector and mice were inoculated with
the vector by the gene gun method. At an appropriate time point, B
cells were isolated, fused with myeloma cells by standard
techniques, and fused hybridoma cells selected in in vitro culture.
Supernatants from clonal cultures were analyzed for binding to
cells stably transfected with CCX-CKR2 by flow cytometry. Positive
clones were amplified and subjected to further rounds of flow
cytometric screening.
[0167] It was determined that monoclonal antibodies 6E10 and 11G8
bind to CCX-CKR2. Antibodies 6E10 and 11G8 detected CCX-CKR2 on
transfectant cell lines that do not endogenously produce CCX-CKR2,
as well as on cells that endogenously express CCX-CKR2, such as
HeLa and MCF-7 (ATCC, VA). Additionally the antibodies were able to
recognize the mouse homolog of CCX CKR2. For example, antibodies
6E10 and 11G8 detected CCX-CR2 on the mouse mammary tumor cell line
4T1 and Lewis lung carcinoma cells (ATCC, Va). Antibodies 6E10 and
11G8, but not isotype controls were detected on an HEK 293 cell
line transfected with CCX-CKR2, but did not bind to HEK 293 cells
transfected with an empty vector or those expressing other
chemokine receptors (e.g., CXCR2).
[0168] The antibodies were also neutralizing, as demonstrated by
radioligand competitive binding assays. Both antibodies 6E10 and
11G8 compete with both SDF-1 and I-TAC for binding to both mouse
and human CCX-CKR2. Antibody 11G8 typically exhibited a greater
percentage inhibition of chemokine binding than did antibody
6E10.
[0169] Antibodies 6E10 and 11G8 also recognize CCX-CKR2 in
immunohistochemical (IHC) assays on fixed paraffin embedded tissue
sections. In experiments on various tissue types, IHC staining with
antibodies 6E10 and 11G8 matched the expression patterns determined
with binding assays incorporating radiolabeled SDF or I-TAC on the
respective tissues. For instance CCX-CKR2 staining was found in
sections of E13 fetal mouse, but not in sections of E11 fetal or
adult mouse. CCX CKR2 staining was also seen in cytospins of cells
stably expressing the human CCX-CKR2.
[0170] The heavy and light chain variable region coding sequence,
and predicted amino acid sequences were determined. 6E 10's heavy
chain variable region is contained in SEQ ID NO:12 (encoded by SEQ
ID NO:11). 6E10's light chain variable region is contained in SEQ
ID NO:14 (encoded by SEQ ID NO:13). 11G8's heavy chain variable
region is contained in SEQ ID NO:16 (encoded by SEQ ID NO:15).
11G8's light chain variable region is contained in SEQ ID NO:18
(encoded by SEQ ID NO:17)
[0171] Although the invention has been described in some detail by
way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to one of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
[0172] All publications, databases, Genbank sequences, patents, and
patent applications cited in this specification are herein
incorporated by reference as if each was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
2311089DNAHomo sapiensG-protein coupled receptor (GPCR) CCX-CKR2
(RDC1) cDNA 1atggatctgc atctcttcga ctactcagag ccagggaact tctcggacat
cagctggcca 60tgcaacagca gcgactgcat cgtggtggac acggtgatgt gtcccaacat
gcccaacaaa 120agcgtcctgc tctacacgct ctccttcatt tacattttca
tcttcgtcat cggcatgatt 180gccaactccg tggtggtctg ggtgaatatc
caggccaaga ccacaggcta tgacacgcac 240tgctacatct tgaacctggc
cattgccgac ctgtgggttg tcctcaccat cccagtctgg 300gtggtcagtc
tcgtgcagca caaccagtgg cccatgggcg agctcacgtg caaagtcaca
360cacctcatct tctccatcaa cctcttcggc agcattttct tcctcacgtg
catgagcgtg 420gaccgctacc tctccatcac ctacttcacc aacaccccca
gcagcaggaa gaagatggta 480cgccgtgtcg tctgcatcct ggtgtggctg
ctggccttct gcgtgtctct gcctgacacc 540tactacctga agaccgtcac
gtctgcgtcc aacaatgaga cctactgccg gtccttctac 600cccgagcaca
gcatcaagga gtggctgatc ggcatggagc tggtctccgt tgtcttgggc
660tttgccgttc ccttctccat tatcgctgtc ttctacttcc tgctggccag
agccatctcg 720gcgtccagtg accaggagaa gcacagcagc cggaagatca
tcttctccta cgtggtggtc 780ttccttgtct gctggctgcc ctaccacgtg
gcggtgctgc tggacatctt ctccatcctg 840cactacatcc ctttcacctg
ccggctggag cacgccctct tcacggccct gcatgtcaca 900cagtgcctgt
cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt catcaatcgc
960aactacaggt acgagctgat gaaggccttc atcttcaagt actcggccaa
aacagggctc 1020accaagctca tcgatgcctc cagagtctca gagacggagt
actctgcctt ggagcagagc 1080accaaatga 10892362PRTHomo
sapiensG-protein coupled receptor (GPCR) CCX-CKR2 2Met Asp Leu His
Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp 1 5 10 15Ile Ser
Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val 20 25 30Met
Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser 35 40
45Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val
50 55 60Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr
His 65 70 75 80Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val
Val Leu Thr 85 90 95Ile Pro Val Trp Val Val Ser Leu Val Gln His Asn
Gln Trp Pro Met 100 105 110Gly Glu Leu Thr Cys Lys Val Thr His Leu
Ile Phe Ser Ile Asn Leu 115 120 125Phe Gly Ser Ile Phe Phe Leu Thr
Cys Met Ser Val Asp Arg Tyr Leu 130 135 140Ser Ile Thr Tyr Phe Thr
Asn Thr Pro Ser Ser Arg Lys Lys Met Val145 150 155 160Arg Arg Val
Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser 165 170 175Leu
Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn 180 185
190Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp
195 200 205Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly Phe Ala
Val Pro 210 215 220Phe Ser Ile Ile Ala Val Phe Tyr Phe Leu Leu Ala
Arg Ala Ile Ser225 230 235 240Ala Ser Ser Asp Gln Glu Lys His Ser
Ser Arg Lys Ile Ile Phe Ser 245 250 255Tyr Val Val Val Phe Leu Val
Cys Trp Leu Pro Tyr His Val Ala Val 260 265 270Leu Leu Asp Ile Phe
Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg 275 280 285Leu Glu His
Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser 290 295 300Leu
Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn Arg305 310
315 320Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser
Ala 325 330 335Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val
Ser Glu Thr 340 345 350Glu Tyr Ser Ala Leu Glu Gln Ser Thr Lys 355
36031089DNAHomo sapiensG-protein coupled receptor (GPCR) CCX-CKR2.2
coding sequence 3atggatctgc acctcttcga ctacgccgag ccaggcaact
tctcggacat cagctggcca 60tgcaacagca gcgactgcat cgtggtggac acggtgatgt
gtcccaacat gcccaacaaa 120agcgtcctgc tctacacgct ctccttcatt
tacattttca tcttcgtcat cggcatgatt 180gccaactccg tggtggtctg
ggtgaatatc caggccaaga ccacaggcta tgacacgcac 240tgctacatct
tgaacctggc cattgccgac ctgtgggttg tcctcaccat cccagtctgg
300gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg agctcacgtg
caaagtcaca 360cacctcatct tctccatcaa cctcttcagc ggcattttct
tcctcacgtg catgagcgtg 420gaccgctacc tctccatcac ctacttcacc
aacaccccca gcagcaggaa gaagatggta 480cgccgtgtcg tctgcatcct
ggtgtggctg ctggccttct gcgtgtctct gcctgacacc 540tactacctga
agaccgtcac gtctgcgtcc aacaatgaga cctactgccg gtccttctac
600cccgagcaca gcatcaagga gtggctgatc ggcatggagc tggtctccgt
tgtcttgggc 660tttgccgttc ccttctccat tatcgctgtc ttctacttcc
tgctggccag agccatctcg 720gcgtccagtg accaggagaa gcacagcagc
cggaagatca tcttctccta cgtggtggtc 780ttccttgtct gctggctgcc
ctaccacgtg gcggtgctgc tggacatctt ctccatcctg 840cactacatcc
ctttcacctg ccggctggag cacgccctct tcacggccct gcatgtcaca
900cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt
catcaatcgc 960aactacaggt acgagctgat gaaggccttc atcttcaagt
actcggccaa aacagggctc 1020accaagctca tcgatgcctc cagagtgtcg
gagacggagt actccgcctt ggagcaaaac 1080gccaagtga 10894362PRTHomo
sapiensG-protein coupled receptor (GPCR) CCX-CKR2.2 4Met Asp Leu
His Leu Phe Asp Tyr Ala Glu Pro Gly Asn Phe Ser Asp 1 5 10 15Ile
Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val 20 25
30Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser
35 40 45Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser
Val 50 55 60Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp
Thr His 65 70 75 80Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp
Val Val Leu Thr 85 90 95Ile Pro Val Trp Val Val Ser Leu Val Gln His
Asn Gln Trp Pro Met 100 105 110Gly Glu Leu Thr Cys Lys Val Thr His
Leu Ile Phe Ser Ile Asn Leu 115 120 125Phe Ser Gly Ile Phe Phe Leu
Thr Cys Met Ser Val Asp Arg Tyr Leu 130 135 140Ser Ile Thr Tyr Phe
Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val145 150 155 160Arg Arg
Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser 165 170
175Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn
180 185 190Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys
Glu Trp 195 200 205Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly
Phe Ala Val Pro 210 215 220Phe Ser Ile Ile Ala Val Phe Tyr Phe Leu
Leu Ala Arg Ala Ile Ser225 230 235 240Ala Ser Ser Asp Gln Glu Lys
His Ser Ser Arg Lys Ile Ile Phe Ser 245 250 255Tyr Val Val Val Phe
Leu Val Cys Trp Leu Pro Tyr His Val Ala Val 260 265 270Leu Leu Asp
Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg 275 280 285Leu
Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser 290 295
300Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn
Arg305 310 315 320Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe
Lys Tyr Ser Ala 325 330 335Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala
Ser Arg Val Ser Glu Thr 340 345 350Glu Tyr Ser Ala Leu Glu Gln Asn
Ala Lys 355 36051089DNAHomo sapiensG-protein coupled receptor
(GPCR) CCX-CKR2.3 coding sequence 5atggatctgc atctcttcga ctactcagag
ccagggaact tctcggacat cagctggcca 60tgcaacagca gcgactgcat cgtggtggac
acggtgatgt gtcccaacat gcccaacaaa 120agcgtcctgc tctacacgct
ctccttcatt tacattttca tcttcgtcat cggcatgatt 180gccaactccg
tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac
240tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat
cccagtctgg 300gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg
agctcacgtg caaagtcaca 360cacctcatct tctccatcaa cctcttcggc
agcattttct tcctcacgtg catgagcgtg 420gaccgctacc tctccatcac
ctacttcacc aacaccccca gcagcaggaa gaagatggta 480cgccgtgtcg
tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc
540tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg
gtccttctac 600cccgagcaca gcatcaagga gtggctgatc ggcatggagc
tggtctccgt tgtcttgggc 660tttgccgttc ccttctccat tgtcgctgtc
ttctacttcc tgctggccag agccatctcg 720gcgtccagtg accaggagaa
gcacagcagc cggaagatca tcttctccta cgtggtggtc 780ttccttgtct
gctggttgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg
840cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct
gcatgtcaca 900cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc
tctacagctt catcaatcgc 960aactacaggt acgagctgat gaaggccttc
atcttcaagt actcggccaa aacagggctc 1020accaagctca tcgatgcctc
cagagtctca gagacggagt actctgcctt ggagcagagc 1080accaaatga
10896362PRTHomo sapiensG-protein coupled receptor (GPCR) CCX-CKR2.3
6Met Asp Leu His Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp 1
5 10 15Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr
Val 20 25 30Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr
Leu Ser 35 40 45Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala
Asn Ser Val 50 55 60Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly
Tyr Asp Thr His 65 70 75 80Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp
Leu Trp Val Val Leu Thr 85 90 95Ile Pro Val Trp Val Val Ser Leu Val
Gln His Asn Gln Trp Pro Met 100 105 110Gly Glu Leu Thr Cys Lys Val
Thr His Leu Ile Phe Ser Ile Asn Leu 115 120 125Phe Gly Ser Ile Phe
Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu 130 135 140Ser Ile Thr
Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val145 150 155
160Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser
165 170 175Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser
Asn Asn 180 185 190Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser
Ile Lys Glu Trp 195 200 205Leu Ile Gly Met Glu Leu Val Ser Val Val
Leu Gly Phe Ala Val Pro 210 215 220Phe Ser Ile Val Ala Val Phe Tyr
Phe Leu Leu Ala Arg Ala Ile Ser225 230 235 240Ala Ser Ser Asp Gln
Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser 245 250 255Tyr Val Val
Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val 260 265 270Leu
Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg 275 280
285Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser
290 295 300Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile
Asn Arg305 310 315 320Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile
Phe Lys Tyr Ser Ala 325 330 335Lys Thr Gly Leu Thr Lys Leu Ile Asp
Ala Ser Arg Val Ser Glu Thr 340 345 350Glu Tyr Ser Ala Leu Glu Gln
Ser Thr Lys 355 36071089DNAHomo sapiensG-protein coupled receptor
(GPCR) CCX-CKR2.4 coding sequence 7atggatctgc atctcttcga ctactcagag
ccagggaact tctcggacat cagctggcca 60tgcaacagca gcgactgcat cgtggtggac
acggtgatgt gtcccaacat gcccaacaaa 120agcgtcctgc tctacacgct
ctccttcatt tacattttca tcttcgtcat cggcatgatt 180gccaactccg
tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac
240tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat
cccagtctgg 300gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg
agctcacgtg caaagtcaca 360cacctcatct tctccatcaa cctcttcggc
agcattttct tcctcacgtg catgagcgtg 420gaccgctacc tctccatcac
ctacttcacc aacaccccca gcagcaggaa gaagatggta 480cgccgtgtcg
tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc
540tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg
gtccttctac 600cccgagcaca gcatcaagga gtggctgatc ggcatggagc
tggtctccgt tgtcttgggc 660tttgccgttc ccttctccat tatcgctgtc
ttctacttcc tgctggccag agccatctcg 720gcgtccagtg accaggagaa
gcacagcagc cggaagatca tcttctccta cgtggtggtc 780ttccttgtct
gctggctgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg
840cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct
gcatgtcaca 900cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc
tctacagctt catcaatcgc 960aactacaggt acgagctgat gaaggccttc
atcttcaagt actcggccaa aacagggctc 1020accaagctca tcgatgcctc
cagagtctca gagacggagt actctgcctt ggagcagagc 1080accaaatga
10898362PRTHomo sapiensG-protein coupled receptor (GPCR) CCX-CKR2.4
8Met Asp Leu His Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp 1
5 10 15Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr
Val 20 25 30Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr
Leu Ser 35 40 45Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala
Asn Ser Val 50 55 60Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly
Tyr Asp Thr His 65 70 75 80Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp
Leu Trp Val Val Leu Thr 85 90 95Ile Pro Val Trp Val Val Ser Leu Val
Gln His Asn Gln Trp Pro Met 100 105 110Gly Glu Leu Thr Cys Lys Val
Thr His Leu Ile Phe Ser Ile Asn Leu 115 120 125Phe Gly Ser Ile Phe
Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu 130 135 140Ser Ile Thr
Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val145 150 155
160Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser
165 170 175Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser
Asn Asn 180 185 190Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser
Ile Lys Glu Trp 195 200 205Leu Ile Gly Met Glu Leu Val Ser Val Val
Leu Gly Phe Ala Val Pro 210 215 220Phe Ser Ile Ile Ala Val Phe Tyr
Phe Leu Leu Ala Arg Ala Ile Ser225 230 235 240Ala Ser Ser Asp Gln
Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser 245 250 255Tyr Val Val
Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val 260 265 270Leu
Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg 275 280
285Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser
290 295 300Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile
Asn Arg305 310 315 320Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile
Phe Lys Tyr Ser Ala 325 330 335Lys Thr Gly Leu Thr Lys Leu Ile Asp
Ala Ser Arg Val Ser Glu Thr 340 345 350Glu Tyr Ser Ala Leu Glu Gln
Ser Thr Lys 355 36091089DNAHomo sapiensG-protein coupled receptor
(GPCR) CCX-CKR2.5 coding sequence 9atggatctgc atctcttcga ctactcagag
ccagggaact tctcggacat cagctggccg 60tgcaacagca gcgactgcat cgtggtggac
acggtgatgt gtcccaacat gcccaacaaa 120agcgtcctgc tctacacgct
ctccttcatt tacattttca tcttcgtcat cggcatgatt 180gccaactccg
tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac
240tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat
cccagtctgg 300gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg
agctcacgtg caaagtcaca 360cacctcatct tctccatcaa cctcttcagc
agcattttct tcctcacgtg catgagcgtg 420gaccgctacc tctccatcac
ctacttcacc aacaccccca gcagcaggaa gaagatggta 480cgccgtgtcg
tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc
540tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg
gtccttctac 600cccgagcaca gcatcaagga gtggctgatc ggcatggagc
tggtctccgt tgtcttgggc 660tttgccgttc ccttctccat tatcgctgtc
ttctacttcc tgctggccag agccatctcg 720gcgtccagtg accaggagaa
gcacagcagc cggaagatca tcttctccta cgtggtggtc 780ttccttgtct
gctggttgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg
840cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct
gcatgtcaca 900cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc
tctacagctt catcaatcgc 960aactacaggt acgagctgat gaaggccttc
atcttcaagt actcggccaa aacagggctc 1020accaagctca tcgatgcctc
cagagtctca gagacggagt actccgcctt
ggagcagagc 1080accaaatga 108910362PRTHomo sapiensG-protein coupled
receptor (GPCR) CCX-CKR2.5 10Met Asp Leu His Leu Phe Asp Tyr Ser
Glu Pro Gly Asn Phe Ser Asp 1 5 10 15Ile Ser Trp Pro Cys Asn Ser
Ser Asp Cys Ile Val Val Asp Thr Val 20 25 30Met Cys Pro Asn Met Pro
Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser 35 40 45Phe Ile Tyr Ile Phe
Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val 50 55 60Val Val Trp Val
Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr His 65 70 75 80Cys Tyr
Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val Val Leu Thr 85 90 95Ile
Pro Val Trp Val Val Ser Leu Val Gln His Asn Gln Trp Pro Met 100 105
110Gly Glu Leu Thr Cys Lys Val Thr His Leu Ile Phe Ser Ile Asn Leu
115 120 125Phe Ser Ser Ile Phe Phe Leu Thr Cys Met Ser Val Asp Arg
Tyr Leu 130 135 140Ser Ile Thr Tyr Phe Thr Asn Thr Pro Ser Ser Arg
Lys Lys Met Val145 150 155 160Arg Arg Val Val Cys Ile Leu Val Trp
Leu Leu Ala Phe Cys Val Ser 165 170 175Leu Pro Asp Thr Tyr Tyr Leu
Lys Thr Val Thr Ser Ala Ser Asn Asn 180 185 190Glu Thr Tyr Cys Arg
Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp 195 200 205Leu Ile Gly
Met Glu Leu Val Ser Val Val Leu Gly Phe Ala Val Pro 210 215 220Phe
Ser Ile Ile Ala Val Phe Tyr Phe Leu Leu Ala Arg Ala Ile Ser225 230
235 240Ala Ser Ser Asp Gln Glu Lys His Ser Ser Arg Lys Ile Ile Phe
Ser 245 250 255Tyr Val Val Val Phe Leu Val Cys Trp Leu Pro Tyr His
Val Ala Val 260 265 270Leu Leu Asp Ile Phe Ser Ile Leu His Tyr Ile
Pro Phe Thr Cys Arg 275 280 285Leu Glu His Ala Leu Phe Thr Ala Leu
His Val Thr Gln Cys Leu Ser 290 295 300Leu Val His Cys Cys Val Asn
Pro Val Leu Tyr Ser Phe Ile Asn Arg305 310 315 320Asn Tyr Arg Tyr
Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser Ala 325 330 335Lys Thr
Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val Ser Glu Thr 340 345
350Glu Tyr Ser Ala Leu Glu Gln Ser Thr Lys 355 36011449DNAMus
sp.mouse monoclonal antibody 6E10 heavy chain variable region
11atgtacttgg gactgagctg tgtattcatt gtttttctct taaaaggtgt ccagtgtgag
60gtgaagctgg atgagactgg aggaggcttg gtgcaacctg ggaggcccat gaaactctcc
120tgtgttgcct ctggattcac ttttagtgac tactggatga actgggtccg
ccagtctcca 180gaaaaaggac tggagtgggt aggacaaatt agaaacaaac
cttataatta tgaaacatat 240tattcagatt ctgtgaaagg cagattcacc
atctcaagag atgattccaa aagtagtgtc 300tacctgcaaa tgaacaactt
aagaactgaa gacacgggta tctactactg tacatcctta 360cgttactggg
gccaaggaac tctggtcact gtctctgcag ccaaaacgac acccccatcc
420gtgtatcctg tggcccctgg aagcttggg 44912149PRTMus sp.mouse
monoclonal antibody 6E10 heavy chain variable region 12Met Tyr Leu
Gly Leu Ser Cys Val Phe Ile Val Phe Leu Leu Lys Gly 1 5 10 15Val
Gln Cys Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln 20 25
30Pro Gly Arg Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe
35 40 45Ser Asp Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly
Leu 50 55 60Glu Trp Val Gly Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu
Thr Tyr 65 70 75 80Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser 85 90 95Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu
Arg Thr Glu Asp Thr 100 105 110Gly Ile Tyr Tyr Cys Thr Ser Leu Arg
Tyr Trp Gly Gln Gly Thr Leu 115 120 125Val Thr Val Ser Ala Ala Lys
Thr Thr Pro Pro Ser Val Tyr Pro Val 130 135 140Ala Pro Gly Ser
Leu14513439DNAMus sp.mouse monoclonal antibody 6E10 light chain
variable region 13atggtcctca tgtccttgct gttctgggta tctggtacct
gtggggacat tgtgatgaca 60cagtctccat cctccctgac tgtgacagca ggagagaagg
tcactatgag ctgcaagtcc 120agtcacagtc tgttaaacag tggaattcaa
aagaacttct tgacctggta tcaacagaaa 180ccagggcagc ctcctaaagt
attgatctac tgggcattca ctagggaatc tggggtccct 240gaacgcttca
caggcagtgg atctggaaca gatttcactc tcaccatcag tagtgtgcag
300gctgaagacc tggcagttta ttactgtcag agtgattata cttatccatt
cacgttcggc 360tcggggacaa agttggaaat aaaacgggct gatgctgcac
caactgtatc catcttccca 420ccatccagta agcttgggg 43914146PRTMus
sp.mouse monoclonal antibody 6E10 light chain variable region 14Met
Val Leu Met Ser Leu Leu Phe Trp Val Ser Gly Thr Cys Gly Asp 1 5 10
15Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu
20 25 30Lys Val Thr Met Ser Cys Lys Ser Ser His Ser Leu Leu Asn Ser
Gly 35 40 45Ile Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Gln Pro 50 55 60Pro Lys Val Leu Ile Tyr Trp Ala Phe Thr Arg Glu Ser
Gly Val Pro 65 70 75 80Glu Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile 85 90 95Ser Ser Val Gln Ala Glu Asp Leu Ala Val
Tyr Tyr Cys Gln Ser Asp 100 105 110Tyr Thr Tyr Pro Phe Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125Arg Ala Asp Ala Ala Pro
Thr Val Ser Ile Phe Pro Pro Ser Ser Lys 130 135 140Leu
Gly14515463DNAMus sp.mouse monoclonal antibody 11G8 heavy chain
variable region 15atggagttgg ggttaaactg ggttttcctt gtccttgttt
taaaaggtgt ccagtgtgaa 60gtgaagctgg tggagtctgg gggagacttg gtccagcctg
gagggtccct gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac
tattacatgt tttgggttcg ccagactcca 180gagaagaggc tggagtgggt
cgcatacatt actaatgggg gtgatagaag ttattattca 240gacactgtaa
cgggccgatt catcatctcc agagacaatg ccaagaacac cctgtatctg
300caaatgagcc gtctgaagtc tgaggacaca gccatgtatt actgtgcaag
acaagggaac 360tgggccgcct ggtttgttta ttggggccaa gggactctgg
tcactgtttc tgcagccaaa 420acgacacccc catccgttta tcccttggcc
cctggaagct tgg 46316154PRTMus sp.mouse monoclonal antibody 11G8
heavy chain variable region 16Met Glu Leu Gly Leu Asn Trp Val Phe
Leu Val Leu Val Leu Lys Gly 1 5 10 15Val Gln Cys Glu Val Lys Leu
Val Glu Ser Gly Gly Asp Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Lys
Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45Ser Asp Tyr Tyr Met
Phe Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60Glu Trp Val Ala
Tyr Ile Thr Asn Gly Gly Asp Arg Ser Tyr Tyr Ser 65 70 75 80Asp Thr
Val Thr Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Thr
Leu Tyr Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105
110Tyr Tyr Cys Ala Arg Gln Gly Asn Trp Ala Ala Trp Phe Val Tyr Trp
115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr
Pro Pro 130 135 140Ser Val Tyr Pro Leu Ala Pro Gly Ser Leu145
15017447DNAMus sp.mouse monoclonal antibody 11G8 light chain
variable region 17atgaagttgc ctgttaggct gttggtgctg atgttctgga
ttcctgcttc caccagtgat 60gttttgatga cccaaactcc actctccctg cctgtcagtc
ttggagatca agcctccatc 120tcttgcagat ctagtcacta tattgtacat
agtgacggaa acacctattt agagtggtac 180ctgcagaaac caggccagtc
tccaaagctc ctgatctaca aagtttccaa ccgattttct 240ggggtcccag
acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc
300agagtggagg ctgaggatct gggaatttat tactgctttc aaggttcaca
tgttccgctc 360acgttcggtg ctgggaccaa gctggagctg aaacgggctg
atgctgcacc aactgtatcc 420atcttcccac catccagtaa gcttggg
44718149PRTMus sp.mouse monoclonal antibody 11G8 light chain
variable region 18Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe
Trp Ile Pro Ala 1 5 10 15Ser Thr Ser Asp Val Leu Met Thr Gln Thr
Pro Leu Ser Leu Pro Val 20 25 30Ser Leu Gly Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser His Tyr Ile 35 40 45Val His Ser Asp Gly Asn Thr Tyr
Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60Gly Gln Ser Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys 100 105 110Phe Gln
Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 115 120
125Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro
130 135 140Ser Ser Lys Leu Gly1451999PRTMus sp.mouse monoclonal
antibody 6E10 heavy chain variable region complementarity
determining region (CDR) 19Glu Val Lys Leu Asp Glu Thr Gly Gly Gly
Leu Val Gln Pro Gly Arg 1 5 10 15Pro Met Lys Leu Ser Cys Val Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Trp Met Asn Trp Val Arg Gln
Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Asn Lys
Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80Val Tyr Leu
Gln Met Asn Asn Leu Arg Thr Glu Asp Thr Gly Ile Tyr 85 90 95Tyr Cys
Thr20101PRTMus sp.mouse monoclonal antibody 6E10 light chain
variable region complementarity determining region (CDR) 20Asp Ile
Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10
15Glu Lys Val Thr Met Ser Cys Lys Ser Ser His Ser Leu Leu Asn Ser
20 25 30Gly Ile Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Gln 35 40 45Pro Pro Lys Val Leu Ile Tyr Trp Ala Phe Thr Arg Glu Ser
Gly Val 50 55 60Pro Glu Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr 65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val
Tyr Tyr Cys Gln Ser 85 90 95Asp Tyr Thr Tyr Pro 1002198PRTMus
sp.mouse monoclonal antibody 11G8 heavy chain variable region
complementarity determining region (CDR) 21Glu Val Lys Leu Val Glu
Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15Ser Leu Lys Leu
Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Phe
Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Tyr
Ile Thr Asn Gly Gly Asp Arg Ser Tyr Tyr Ser Asp Thr Val 50 55 60Thr
Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Arg22100PRTMus sp.mouse monoclonal antibody 11G8
light chain variable region complementarity determining region
(CDR) 22Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu
Gly 1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser His Tyr Ile
Val His Ser 20 25 30Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu
Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro
100235PRTArtificial SequenceDescription of Artificial
Sequencepeptide linker 23Gly Gly Gly Gly Ser 1 5
* * * * *
References