Title of Invention

REAGENTS THAT BIND CCX-CKR2

Abstract Antibodies that bind to CCX-CKR2 and methods of their use are provided.
Full Text WO 2006/116319 PCT/US2006/015492
REAGENTS THAT BIND CCX-CKR2
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[01] The present patent application claims benefit of priority to U.S.
Provisional Patent Application No. 60/674,140, filed April 21, 2005, which is incorporated
by reference for all purposes.
BACKGROUND OF THE INVENTION
[02] Chemokines constitute a family of small cytoldnes 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 chernotaxis, 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 (Ca2+), granule exocytosis, integrin
upregulation, formation of bioactive lipids (e.g., leukotrienes), expression of cytoldnes, 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.
[03] 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,
EP-10, MIG, PF4, ENA-78, GCP-2, GROα, GROβ, GROγ, NAP-2, NAP-4,1-TAC) or are
adjacent residues (as in CC chemokines MIP-1α, MIP-1β, RANTES, MCP-1, MCP-2, MCP-
3,I-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-γ inducible
10 kDa protein) are particularly active in inducing chemotaxis of activated peripheral blood

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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 la and 1β (MlP-1α and
MIP-1β) have been characterized as chemoattractants and activators of monocytes or
lymphocytes, but do not appear to be chemoattractants for neutrophils.
[04] 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.
[05] 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 U S A, 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 a and
SDF-1 p, by alternative splicing. These two proteins are identical except for the four amino
acid residues that are present in the N-terminus of SDF-1β and absent from SDF-1α.
[06] 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).
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[07] 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
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
[08] 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:
SEQ ID NO: 12 and SEQ ID NO: 14; or
SEQ ID NO:16 and SEQ ID NO: 18.
[09] 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.
[10] 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.
[11] 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.
[12] 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.
[13] 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:
SEQ ID NO: 12 and SEQ ID NO:14; or
SEQ ID NO: 16 and SEQ ID NO: 18.
[14] 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.
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[15] 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.
[16] 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.
[17] 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:
SEQ ID NO:12 and SEQ ID NO: 14; or
SEQ ID NO: 16 and SEQ ID NO: 18.
[18] In some embodiments, the antibody is linked to a detectable label.
[19] 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:
SEQ ID NO: 12 and SEQ ID NO:14; or
SEQ ID NO: 16 and SEQ ID NO: 18, thereby inhibiting angiogenesis or
proliferation of a cancer cell.
[20] 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.
[21] In some embodiments, the antibody comprises the complementarity
determining regions (CDRs) of SEQ ED 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.
[22] In some embodiments, the antibody comprises the complementarity
determining regions (CDRs) of SEQ ED NO: 16 and/or SEQ ID NO: 18 or CDRs substantially
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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.
[23] 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.
[24] The present invention also provides methods for identifying a
modulator of CCX-CKR2. hi some embodiments, the method comprises:
(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.
[25] 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.
[26] 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.
[27] 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. En some embodiments, the methods comprise:
(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
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(c) comparing the effect of the test reagent on the first animal to the effect
of the antibody on the second antibody.
[28] 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.
[29] 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.
[30] 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.
[31] 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:1 l,SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID
NO: 17.
[32] The present invention also provides method of producing a chimeric
antibody. In some embodiments, the method comprises:
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.
DEFINITIONS
[33] "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
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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.
[34] A "subject" or "individual" refers to an animal, including a human,
non-human primate, mouse, rat, dog or other mammal.
[35] 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.
[36] "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.
[37] 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 immunogiobulins 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,
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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)).
[38] 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-
tenninus 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 (VL) and
variable heavy chain (VH) refer to these light and heavy chains respectively.
[39] 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)'2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide
bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'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)).
[40] 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
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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)).
[41] 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.
[42] 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).
[43] 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.
[44] 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 biologies. 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.
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[45] 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., -CH2NH-, -CH2S-, -CH2-
CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. 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.
[46] A "ligand" refers to an agent, e.g., a polypeptide or other molecule,
capable of binding to a receptor.
[47] "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
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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.
[48] 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.
[49] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[50] "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.
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[51] 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 Figure 1.
[52] 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.
[53] 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 7. 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
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Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel
et al., Current Protocols in Molecular Biology (1995 supplement)).
[54] 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 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.
[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
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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.
[56] 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
[57] Figure 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
/. Antibodies of the invention
[58] 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.
[59] 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.
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[60] 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+ T cells.
[61] 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 ED NO.1S.
[62] 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 Figure 1.
[63] 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
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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.
[64] 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.
[65] 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.
[66] 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
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(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization
protocol may be selected by one skilled in the art without undue experimentation.
[67] 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.
[68] 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.
[69] 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.
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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. Patent 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).
[70] In some embodiments, the antibodies of the invention are single chain
Fvs (scFvs). The VH and the VL regions (e.g., SEQ ID NO: 12 and SEQ ED 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 VH and VL 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.
Peptide linkers and their use are well-known in the art. See, e.g., Huston et al., Proc. Nat'I
Acad. Sci. USA 8:5879 (1988); Bird et al., Science 242:4236 (1988); Glockshuber et al.,
Biochemistry 29:1362 (1990); U.S. Patent No. 4,946,778, U.S. Patent 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 VH and VL. 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.
[71] 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
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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.
[72] 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.
[73] 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.
[74] 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.
[75] 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.
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[76] 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.
//. Immunoassays
[77] 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. Patents 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 &
Terr, eds. (1991).
[78] 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.
[79] 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,
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e.g., goat anti-mouse FITC. A general overview of the applicable technology can be found in
Harlow & Lane, Antibodies: A Laboratory Manual (1988).
[80] 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
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 ceil 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.
[81] 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.
[82] 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;
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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.
///. Modulators of CCX-CKR2
A. Methods of Identifying Modulators of Chemokine Receptors
[83] 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.
1. Chemokine Receptor Binding Assays
[84] 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.
[85] 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
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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.
2. Cells and Reagents
[86] 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.
[87] 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, ieukemias, 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.
[88] 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.
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3. Signaling or adhesion activity
[89] 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).
[90] Signaling can also be measured by determining, qualitatively and
quantitatively, secondary messengers, such as cyclic AMP or inositol phosphates, as well as
phosphorylarion 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).
[91] In addition, other events downstream of CCX-CKK2 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. Patent 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).
[92] 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
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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.
{93] 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.
[94] 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 TEMP-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.
[95] 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 endotheh'al cells mimic the
lymphatic vasculature and can be stimulated with various cytokines and growth factors (e.g.,
TNFalpha and IL-lbeta). 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.
Microdrculation. 3(3):287-300 (1996)).
4. Validation
[96] 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
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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.
[97] 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.
B. Agents that interact with CCX-CKR2
[98] 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.
[99] 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.
[100] 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
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were described by Lipinski et al. as follows: having more than 5 H-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 10 H-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.
[101] 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.
[102] 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.
[103] 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. Patent 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 etal.,J. Org. Chem. 59:658 (1994)),
nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid
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libraries {see, e.g., U.S. Patent 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. Patent 5,593,853), small organic
molecule libraries {see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993);
isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent
5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S.
Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
[104] 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, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore,
Bedford, MA). 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
[105] 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.
[106] 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
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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 AEDS-associated primary effusion lymphoma.
[107] 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 No. 11/050,345.
[108] 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; Osier-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
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amyloidosis. Antibodies of the present invention maybe used prophylactically or
therapeutically for any of the disorders or diseases described herein.
[109] 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 bums, inflammatory
diseases, ischeric heart, and peripheral vascular diseases.
[110] 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.
[111] 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.
[112] Inhibitors of angiogenesis have yet other therapeutic uses. For
example, the antibodies of the present invention maybe used for the following:
(a) Adipose tissue ablation and treatment of obesity. See, e.g. Kolonin et al.,
Nature Medicine 10(6):625-632 (2004);
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(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
(c) Treatment of cardiovascular disease. See, e.g., March, et al., Am. J. Physiol.
Heart Circ. Physiol. 287:H458-H463 (2004); Rehman etal, Circulation 109: 1292-1298
(2004).
V. Administration and pharmaceutical compositions
[113] 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, 17th ed. 1985)).
[114] 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.
[115] 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.
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[116] 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.
[117] 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 rumor 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.
[118] 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.
[119] 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.
[120] 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
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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
[121] 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 immunornodulatory agent, an anti-vascular agent, a signal transduction
inhibitor, an antiproliferative agent, or an apoptosis inducer.
[122] 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.
[123] Anti-angiogenesis agents, such as MMP-2 (matrix-metalloprotienase
2) inhibitors, MMP-9 (matrix-metalloprotienase 9) inhibitors, and COX-II (cyclooxygenase
fl) 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
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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™
(Genentech, Inc. of South San Francisco, Calif., USA).
[124] 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.
[125] 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 α (TNFα), is associated with multiple
inflammatory events, including arthritis, and anti-TNFα therapies are being used clinically.
VII. Kits for use in diagnostic and/or prognostic applications
[126] 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.
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[127] 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
[128] 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.
[129] 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).
[130] The antibodies were also neutralizing, as demonstrated by radioligand
competitive binding assays. Both antibodies 6E10 and 11G8 compete with both SDF-1 and
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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.
[131] 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 E17 fetal or adult mouse. CCX CKR2 staining was
also seen in cytospins of cells stably expressing the human CCX-CKR2.
[132] The heavy and light chain variable region coding sequence, and
predicted amino acid sequences were determined. 6E10'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). 1 lG8's heavy chain variable
region is contained in SEQ ID NO: 16 (encoded by SEQ ID NO: 15). 11 G8's light chain
variable region is contained in SEQ ID NO: 18 (encoded by SEQ ID NO: 17)
[133] 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.
[134] 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.
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SEQUENCE LISTING
SEQ ID NO:1 CCX-CKR2 coding sequence
ATGGATCTGCATCTCTTCGACTACTCAGAGCCAGGGAACTTCTCGGACATCAGCT
GGCCATGCAACAGCAGCGACTGCATCGTGGTGGACACGGTGATGTGTCCCAACA
TGCCCAACAAAAGCGTCCTGCTCTACACGCTCTCCTTCATTTACATTTTCATCTTC
GTCATCGGCATGATTGCCAACTCCGTGGTGGTCTGGGTGAATATCCAGGCCAAGA
CCACAGGCTATGACACGCACTGCTACATCTTGAACCTGGCCATTGCCGACCTGTG
GGTTGTCCTCACCATCCCAGTCTGGGTGGTCAGTCTCGTGCAGCACAACCAGTGG
CCCATGGGCGAGCTCACGTGCAAAGTCACACACCTCATCTTCTCCATCAACCTCT
TCGGCAGCATTTTCTTCCTCACGTGCATGAGCGTGGACCGCTACCTCTCCATCACC
TACTTCACCAACACCCCCAGCAGCAGGAAGAAGATGGTACGCCGTGTCGTCTGC
ATCCTGGTGTGGCTGCTGGCCTTCTGCGTGTCTCTGCCTGACACCTACTACCTGAA
GACCGTCACGTCTGCGTCCAACAATGAGACCTACTGCCGGTCCTTCTACCCCGAG
CACAGCATCAAGGAGTGGCTGATCGGCATGGAGCTGGTCTCCGTTGTCTTGGGCT
TTGCCGTTCCCTTCTCCATTATCGCTGTCTTCTACTTCCTGCTGGCCAGAGCCATC
TCGGCGTCCAGTGACCAGGAGAAGCACAGCAGCCGGAAGATCATCTTCTCCTAC
GTGGTGGTCTTCCTTGTCTGCTGGCTGCCCTACCACGTGGCGGTGCTGCTGGACA
TCTTCTCCATCCTGCACTACATCCCTTTCACCTGCCGGCTGGAGCACGCCCTCTTC
ACGGCCCTGCATGTCACACAGTGCCTGTCGCTGGTGCACTGCTGCGTCAACCCTG
TCCTCTACAGCTTCATCAATCGCAACTACAGGTACGAGCTGATGAAGGCCTTCAT
CTTCAAGTACTCGGCCAAAACAGGGCTCACCAAGCTCATCGATGCCTCCAGAGTC
TCAGAGACGGAGTACTCTGCCTTGGAGCAGAGCACCAAATGA
SEQ ID NO:2 CCX-CKR2 amino acid sequence
MDLHLFDYSEPGNFSDISWPCNSSDCIWDTVMCPNMPNKSVLLYTLSFIYIFIFVIGM
IANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPMGEL
TCKVTHLIFSINLFGSIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRWCILVWLLAFC
VSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYF
LLARAISASSDQEKHSSRKIIFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHAL
FTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSE
TEYSALEQSTK
SEQ ID NO:3 CCX-CKR2.2 coding sequence
ATGGATCTGCACCTCTTCGACTACGCCGAGCCAGGCAACTTCTCGGACATCAGCT
GGCCATGCAACAGCAGCGACTGCATCGTGGTGGACACGGTGATGTGTCCCAACA
TGCCCAACAAAAGCGTCCTGCTCTACACGCTCTCCTTCATTTACATTTTCATCTTC
GTCATCGGCATGATTGCCAACTCCGTGGTGGTCTGGGTGAATATCCAGGCCAAGA
CCACAGGCTATGACACGCACTGCTACATCTTGAACCTGGCCATTGCCGACCTGTG
GGTTGTCCTCACCATCCCAGTCTGGGTGGTCAGTCTCGTGCAGCACAACCAGTGG
CCCATGGGCGAGCTCACGTGCAAAGTCACACACCTCATCTTCTCCATCAACCTCT
TCAGCGGCATTTTCTTCCTCACGTGCATGAGCGTGGACCGCTACCTCTCCATCACC
TACTTCACCAACACCCCCAGCAGCAGGAAGAAGATGGTACGCCGTGTCGTCTGC
ATCCTGGTGTGGCTGCTGGCCTTCTGCGTGTCTCTGCCTGACACCTACTACCTGAA
GACCGTCACGTCTGCGTCCAACAATGAGACCTACTGCCGGTCCTTCTACCCCGAG
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CACAGCATCAAGGAGTGGCTGATCGGCATGGAGCTGGTCTCCGTTGTCTTGGGCT
TTGCCGTTCCCTTCTCCATTATCGCTGTCTTCTACTTCCTGCTGGCCAGAGCCATC
TCGGCGTCCAGTGACCAGGAGAAGCACAGCAGCCGGAAGATCATCTTCTCCTAC
GTGGTGGTCTTCCTTGTCTGCTGGCTGCCCTACCACGTGGCGGTGCTGCTGGACA
TCTTCTCCATCCTGCACTACATCCCTTTCACCTGCCGGCTGGAGCACGCCCTCTTC
ACGGCCCTGCATGTCACACAGTGCCTGTCGCTGGTGCACTGCTGCGTCAACCCTG
TCCTCTACAGCTTCATCAATCGCAACTACAGGTACGAGCTGATGAAGGCCTTCAT
CTTCAAGTACTCGGCCAAAACAGGGCTCACCAAGCTCATCGATGCCTCCAGAGTG
TCGGAGACGGAGTACTCCGCCTTGGAGCAAAACGCCAAGTGA
SEQ ID NO:4 CCX-CKR2.2 amino acid sequence
MDLHLFDYAEPGNFSDISWPCNSSDCIVVDTVMCPNMPNKSVLLYTLSFIYIFIFVIGM
IANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPMGEL
TCKVTHLIFSINLFSGIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAPC
VSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYF
LLARAISASSDQEKHSSRKIIFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHAL
FTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSE
TEYSALEQNAK
SEQ ID NO:5 CCX-CKR2.3 coding sequence
ATGGATCTGCATCTCTTCGACTACTCAGAGCCAGGGAACTTCTCGGACATCAGCT
GGCCATGCAACAGCAGCGACTGCATCGTGGTGGACACGGTGATGTGTCCCAACA
TGCCCAACAAAAGCGTCCTGCTCTACACGCTCTCCTTCATTTACATTTTCATCTTC
GTCATCGGCATGATTGCCAACTCCGTGGTGGTCTGGGTGAATATCCAGGCCAAGA
CCACAGGCTATGACACGCACTGCTACATCTTGAACCTGGCCATTGCCGACCTGTG
GGTTGTCCTCACCATCCCAGTCTGGGTGGTCAGTCTCGTGCAGCACAACCAGTGG
CCCATGGGCGAGCTCACGTGCAAAGTCACACACCTCATCTTCTCCATCAACCTCT
TCGGCAGCATTTTCTTCCTCACGTGCATGAGCGTGGACCGCTACCTCTCCATCACC
TACTTCACCAACACCCCCAGCAGCAGGAAGAAGATGGTACGCCGTGTCGTCTGC
ATCCTGGTGTGGCTGCTGGCCTTCTGCGTGTCTCTGCCTGACACCTACTACCTGAA
GACCGTCACGTCTGCGTCCAACAATGAGACCTACTGCCGGTCCTTCTACCCCGAG
CACAGCATCAAGGAGTGGCTGATCGGCATGGAGCTGGTCTCCGTTGTCTTGGGCT
TTGCCGTTCCCTTCTCCATTGTCGCTGTCTTCTACTTCCTGCTGGCCAGAGCCATC
TCGGCGTCCAGTGACCAGGAGAAGCACAGCAGCCGGAAGATCATCTTCTCCTAC
GTGGTGGTCTTCCTTGTCTGCTGGTTGCCCTACCACGTGGCGGTGCTGCTGGACAT
CTTCTCCATCCTGCACTACATCCCTTTCACCTGCCGGCTGGAGCACGCCCTCTTCA
CGGCCCTGCATGTCACACAGTGCCTGTCGCTGGTGCACTGCTGCGTCAACCCTGT
CCTCTACAGCTTCATCAATCGCAACTACAGGTACGAGCTGATGAAGGCCTTCATC
TTCAAGTACTCGGCCAAAACAGGGCTCACCAAGCTCATCGATGCCTCCAGAGTCT
CAGAGACGGAGTACTCTGCCTTGGAGCAGAGCACCAAATGA
SEQ ID NO:6 CCX-CKR2.3 amino acid sequence
MDLHLFDYSEPGNFSDISWPCNSSDCIVVDTVMCPNMPNKSVLLYTLSFIYIFIFVIGM
IANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPMGEL
TCKVTHLIFSINLFGSIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAFC
VSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIVAVFY
FLLARAISASSDQEKHSSRKHFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHA
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LFTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLEDASRVS
ETEYSALEQSTK
SEQ ID NO:7 CCX-CKR2.4 coding sequence
ATGGATCTGCATCTCTTCGACTACTCAGAGCCAGGGAACTTCTCGGACATCAGCT
GGCCATGCAACAGCAGCGACTGCATCGTGGTGGACACGGTGATGTGTCCCAACA
TGCCCAACAAAAGCGTCCTGCTCTACACGCTCTCCTTCATTTACATTTTCATCTTC
GTCATCGGCATGATTGCCAACTCCGTGGTGGTCTGGGTGAATATCCAGGCCAAGA
CCACAGGCTATGACACGCACTGCTACATCTTGAACCTGGCCATTGCCGACCTGTG
GGTTGTCCTCACCATCCCAGTCTGGGTGGTCAGTCTCGTGCAGCACAACCAGTGG
CCCATGGGCGAGCTCACGTGCAAAGTCACACACCTCATCTTCTCCATCAACCTCT
TCGGCAGCATTTTCTTCCTCACGTGCATGAGCGTGGACCGCTACCTCTCCATCACC
TACTTCACCAACACCCCCAGCAGCAGGAAGAAGATGGTACGCCGTGTCGTCTGC
ATCCTGGTGTGGCTGCTGGCCTTCTGCGTGTCTCTGCCTGACACCTACTACCTGAA
GACCGTCACGTCTGCGTCCAACAATGAGACCTACTGCCGGTCCTTCTACCCCGAG
CACAGCATCAAGGAGTGGCTGATCGGCATGGAGCTGGTCTCCGTTGTCTTGGGCT
TTGCCGTTCCCTTCTCCATTATCGCTGTCTTCTACTTCCTGCTGGCCAGAGCCATC
TCGGCGTCCAGTGACCAGGAGAAGCACAGCAGCCGGAAGATCATCTTCTCCTAC
GTGGTGGTCTTCCTTGTCTGCTGGCTGCCCTACCACGTGGCGGTGCTGCTGGACA
TCTTCTCCATCCTGCACTACATCCCTTTCACCTGCCGGCTGGAGCACGCCCTCTTC
ACGGCCCTGCATGTCACACAGTGCCTGTCGCTGGTGCACTGCTGCGTCAACCCTG
TCCTCTACAGCTTCATCAATCGCAACTACAGGTACGAGCTGATGAAGGCCTTCAT
CTTCAAGTACTCGGCCAAAACAGGGCTCACCAAGCTCATCGATGCCTCCAGAGTC
TCAGAGACGGAGTACTCTGCCTTGGAGCAGAGCACCAAATGA
SEQ ED NO:8 CCX-CKR2.4 amino acid sequence
MDLHLFDYSEPGNFSDISWPCNSSDCIVVDTVMCPNMPNKSVLLYTLSFIYrFrFVIGM
IANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPMGEL
TCKVTHLIFSE^LFGSIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAFC
VSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYF
LLARAISASSDQEKHSSRKIIFSYVWFLVCWLPYHVAVLLDIFSILHY1PFTCRLEHAL
FTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSE
TEYSALEQSTK
SEQ ID NO:9 CCX-CKR2.5 coding sequence
ATGGATCTGCATCTCTTCGACTACTCAGAGCCAGGGAACTTCTCGGACATCAGCT
GGCCGTGCAACAGCAGCGACTGCATCGTGGTGGACACGGTGATGTGTCCCAACA
TGCCCAACAAAAGCGTCCTGCTCTACACGCTCTCCTTCATTTACATTTTCATCTTC
GTCATCGGCATGATTGCCAACTCCGTGGTGGTCTGGGTGAATATCCAGGCCAAGA
CCACAGGCTATGACACGCACTGCTACATCTTGAACCTGGCCATTGCCGACCTGTG
GGTTGTCCTCACCATCCCAGTCTGGGTGGTCAGTCTCGTGCAGCACAACCAGTGG
CCCATGGGCGAGCTCACGTGCAAAGTCACACACCTCATCTTCTCCATCAACCTCT
TCAGCAGCATTTTCTTCCTCACGTGCATGAGCGTGGACCGCTACCTCTCCATCACC
TACTTCACCAACACCCCCAGCAGCAGGAAGAAGATGGTACGCCGTGTCGTCTGC
ATCCTGGTGTGGCTGCTGGCCTTCTGCGTGTCTCTGCCTGACACCTACTACCTGAA
GACCGTCACGTCTGCGTCCAACAATGAGACCTACTGCCGGTCCTTCTACCCCGAG
CACAGCATCAAGGAGTGGCTGATCGGCATGGAGCTGGTCTCCGTTGTCTTGGGCT
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TTGCCGTTCCCTTCTCCATTATCGCTGTCTTCTACTTCCTGCTGGCCAGAGCCATC
TCGGCGTCCAGTGACCAGGAGAAGCACAGCAGCCGGAAGATCATCTTCTCCTAC
GTGGTGGTCTTCCTTGTCTGCTGGTTGCCCTACCACGTGGCGGTGCTGCTGGACAT
CTTCTCCATCCTGCACTACATCCCTTTCACCTGCCGGCTGGAGCACGCCCTCTTCA
CGGCCCTGCATGTCACACAGTGCCTGTCGCTGGTGCACTGCTGCGTCAACCCTGT
CCTCTACAGCTTCATCAATCGCAACTACAGGTACGAGCTGATGAAGGCCTTCATC
TTCAAGTACTCGGCCAAAACAGGGCTCACCAAGCTCATCGATGCCTCCAGAGTCT
CAGAGACGGAGTACTCCGCCTTGGAGCAGAGCACCAAATGA
SEQ ID NO: 10 CCX-CKR2.5 ammo acid sequence
MDLHLFDYSEPGNFSDISWPCNSSDCIVVDTVMCPNMPNKSVLLYTLSFIYIFIFVIGM
IANSVVVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQVVPMGEL
TCKVTHLIFSINLFSSIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAFC
VSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYF
LLARAISASSDQEKHSSRKIIFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHAL
FTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSE
TEYSALEQSTK
SEQ ID NO: 11 DNA sequence for antibody 6E10 heavy chain variable region
ATGTACTTGGGACTGAGCTGTGTATTCATTGTTTTTCTCTTAAAAGGTGTCCAGTG
TGAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCAT
GAAACTCTCCTGTGTTGCCTCTGGATTCACTTTTAGTGACTACTGGATGAACTGG
GTCCGCCAGTCTCCAGAAAAAGGACTGGAGTGGGTAGGACAAATTAGAAACAAA
CCTTATAATTATGAAACATATTATTCAGATTCTGTGAAAGGCAGATTCACCATCT
CAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAACTG
AAGACACGGGTATCTACTACTGTACATCCTTACGTTACTGGGGCCAAGGAACTCT
GGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTGTATCCTGTGGCCCCT
GGAAGCTTGGG
SEQ ID NO: 12 amino acid sequence for antibody 6E10 heavy chain variable region
MYLGLSCVFIVFLLKGVQCEVKLDETGGGLVQPGRPMKJ.SCVASGFTFSDYWMNW
VRQSPEKGLEWVGQIRNKPYNYETYYSDSVKGRFnSRDDSKSSVYLQMNNLRTEDT
GIYYCTSLRYWGQGTLVTVSAAKTTPPSVYPVAPGSL
SEQ ID NO: 13 DNA sequence for antibody 6E10 light chain variable region
ATGGTCCTCATGTCCTTGCTGTTCTGGGTATCTGGTACCTGTGGGGACATTGTGAT
GACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAG
CTGCAAGTCCAGTCACAGTCTGTTAAACAGTGGAATTCAAAAGAACTTCTTGACC
TGGTATCAACAGAAACCAGGGCAGCCTCCTAAAGTATTGATCTACTGGGCATTCA
CTAGGGAATCTGGGGTCCCTGAACGCTTCACAGGCAGTGGATCTGGAACAGATTT
CACTCTCACCATCAGTAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAG
AGTGATTATACTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC
GGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGCTTGGGG
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SEQ ED NO: 14 amino acid sequence for antibody 6E10 light chain variable region
MVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSHSLLNSGIQKNFLTW
YQQKPGQPPKVLIYWAFTRESGVPERFTGSGSGTDFTLTISSVQAEDLAVYYCQSDY
TYPFTFGSGTKLEIKRADAAPTVSIFPPSSKLG
SEQ ID NO: 15 DNA sequence for antibody 11G8 heavy chain variable region
ATGGAGTTGGGGTTAAACTGGGTTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTG
TGAAGTGAAGCTGGTGGAGTCTGGGGGAGACTTGGTCCAGCCTGGAGGGTCCCT
GAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTTTTGGG
TTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTACTAATGGGG
GTGATAGAAGTTATTATTCAGACACTGTAACGGGCCGATTCATCATCTCCAGAGA
CAATGCCAAGAACACCCTGTATCTGCAAATGAGCCGTCTGAAGTCTGAGGACAC
AGCCATGTATTACTGTGCAAGACAAGGGAACTGGGCCGCCTGGTTTGTTTATTGG
GGCCAAGGGACTCTGGTCACTGTTTCTGCAGCCAAAACGACACCCCCATCCGTTT
ATCCCTTGGCCCCTGGAAGCTTGG
SEQ ID NO: 16 amino acid sequence for antibody 11G8 heavy chain variable region
MELGLNWVFLVLVLKGVQCEVKLVESGGDLVQPGGSLKLSCATSGFTFSDYYMFW
VRQTPEKRLEWVAYITNGGDRSYYSDTVTGRFIISRDNAKNTLYLQMSRLKSEDTAM
YYCARQGNWAAWFVYWGQGTLVTVSAAKTTPPSVYPLAPGSL
SEQ ID NO: 17 DNA sequence for antibody 11G8 light chain variable region
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCACCAG
TGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAA
GCCTCCATCTCTTGCAGATCTAGTCACTATATTGTACATAGTGACGGAAACACCT
ATTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAA
AGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGG
ACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTAT
TACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGG
AGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAA
GCTTGGG
SEQ ID NO: 18 amino acid sequence for antibody 11G8 light chain variable region
MKLPVRLLVLMFWIPASTSDVLMTQTPLSLPVSLGDQASISCRSSHYIVHSDGNTYLE
WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGS
HVPLTFGAGTKLELKRADAAPTVSIFPPSSKLG
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WHAT IS CLAIMED IS:
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 ED 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. The antibody of claim 1, which comprises the complementarity
determining regions (CDRs) of SEQ ID NO.12 and SEQ ID NO:14.
6. The antibody of claim 1, which comprises SEQ ID NO: 12 and SEQ ID
NO: 14.
7. The antibody of claim 1, which comprises the complementarity
determining regions (CDRs) of SEQ ID NO:16 and SEQ ID NO.18.
8. The antibody of claim 1, which comprises SEQ ID NO:16 and SEQ ID
NO: 18.
9. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the antibody of claim 1.
10. The pharmaceutical composition of claim 9, wherein the antibody is a
monoclonal antibody.
11. The pharmaceutical composition of claim 9, wherein the antibody is a
humanized antibody.
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WO 2006/116319 PCT/US2006/015492
12. The pharmaceutical composition of claim 9, wherein the antibody
comprises the complementarity determining regions (CDRs) of SEQ ID NO: 12 and SEQ ID
NO:14.
13. The pharmaceutical composition of claim 9, wherein the antibody
comprises SEQ ID NO: 12 and SEQ ID NO: 14.
14. The pharmaceutical composition of claim 9, wherein the antibody
comprises the complementarity determining regions (CDRs) of SEQ ID NO: 16 and SEQ ID
NO:18.
15. The pharmaceutical composition of claim 9, wherein the antibody
comprises SEQ ID NO:16 and SEQ ID NO:18.
16. A method of detecting a cell expressing CCX-CKR2 in a biological
sample, the method comprising contacting the biological sample with an antibody of claim 1
and detecting the presence of the antibody.
17. 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.
18. The method of claim 17, wherein the cell is in an individual.
19. The method of claim 18, wherein the individual has or is pre-disposed
to have arthritis.
20. The method of claim 18, wherein the individual is not a human.
21. 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
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WO 2006/116319 PCT/US2006/015492
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.
22. 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.
23. A polypeptide comprising SEQ ID NO:12, SEQ ID NO: 14, SEQ ID
NO:16, or SEQ ID NO:18.
24. A polynucleotide encoding SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO:16, or SEQ ID NO:18.
25. The polynucleotide of claim 24, wherein the polynucleotide comprises
SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17.
26. 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.
44

Antibodies that bind to CCX-CKR2 and methods of their use are provided.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=A0U4MTES+6gqioxVmorCEw==&loc=wDBSZCsAt7zoiVrqcFJsRw==


Patent Number 270286
Indian Patent Application Number 3897/KOLNP/2007
PG Journal Number 50/2015
Publication Date 11-Dec-2015
Grant Date 09-Dec-2015
Date of Filing 11-Oct-2007
Name of Patentee CHEMOCENTRYX, INC.
Applicant Address 850 MAUDE AVENUE MOUNTAIN VIEW, CALIFORNIA
Inventors:
# Inventor's Name Inventor's Address
1 SCHALL THOMAS 563 HOMER AVENUE, PALO ALTO, CALIFORNIA 94301
2 HOWARD MAUREEN 12700 VISCAINO ROAD, LOS ALTOS, CALIFORNIA 94022
PCT International Classification Number C07K 16/00
PCT International Application Number PCT/US2006/015492
PCT International Filing date 2006-04-19
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/674140 2005-04-21 U.S.A.