U.S. patent application number 15/099848 was filed with the patent office on 2016-08-11 for engineered cxcl12 alpha locked dimer polypeptide.
The applicant listed for this patent is The Medical College of Wisconsin, Inc., The Rockefeller University. Invention is credited to Francis C. Peterson, Thomas Sakmar, Christoph H. Seibert, Christopher T. Veldkamp, Brian F. Volkman.
Application Number | 20160229900 15/099848 |
Document ID | / |
Family ID | 40766044 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160229900 |
Kind Code |
A1 |
Volkman; Brian F. ; et
al. |
August 11, 2016 |
ENGINEERED CXCL12 ALPHA LOCKED DIMER POLYPEPTIDE
Abstract
The present invention provides a novel CXCL12-.alpha..sub.2
locked dimer polypeptide, pharmaceutical compositions thereof, and
methods of using said dimer in the treatment of cancer,
inflammatory disorders, autoimmune disease, and HIV/AIDS.
Inventors: |
Volkman; Brian F.; (Muskego,
WI) ; Veldkamp; Christopher T.; (Milwaukee, WI)
; Peterson; Francis C.; (Racine, WI) ; Sakmar;
Thomas; (New York, NY) ; Seibert; Christoph H.;
(Frankfurt am Main, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Medical College of Wisconsin, Inc.
The Rockefeller University |
Milwaukee
New York |
WI
NY |
US
US |
|
|
Family ID: |
40766044 |
Appl. No.: |
15/099848 |
Filed: |
April 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13936650 |
Jul 8, 2013 |
9346871 |
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15099848 |
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12956514 |
Nov 30, 2010 |
8524670 |
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13936650 |
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12380308 |
Feb 26, 2009 |
7923016 |
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12956514 |
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61067273 |
Feb 27, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/02 20180101; A61P 37/06 20180101; C07K 14/522 20130101;
A61P 31/18 20180101; C07K 14/521 20130101; A61K 38/00 20130101 |
International
Class: |
C07K 14/52 20060101
C07K014/52 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
AI058072 awarded by the National Institutes of Health-NIAID. The
government has certain rights in the invention.
Claims
1. A CXCL12-.alpha..sub.2 locked dimer polypeptide, wherein the
dimer comprises two monomers locked together.
2. The dimer of claim 1 wherein the monomers are not identical.
3. The dimer of claim 1 wherein the monomers are identical.
4. The dimer of claim 1 wherein at least one of the monomers has
the amino acid sequence as shown in SEQ ID NO:1.
5. The dimer of claim 1 wherein the monomers are locked together at
residues L36 and A65 of SEQ ID NO: 1.
6. A composition comprising the dimer of claim 1, and a
pharmaceutically acceptable carrier or diluent.
7. An isolated CXCL12-.alpha..sub.2 locked dimer polypeptide,
wherein the dimer consists of two monomers locked together.
8. The dimer of claim 7 wherein at least one of the monomers has
the amino acid sequence as shown in SEQ ID NO:1.
9. A kit comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide
wherein the dimer comprises monomers having the amino acid sequence
as shown in SEQ ID NO:1, a pharmaceutically acceptable carrier or
diluent, and instructional material.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/936,650, filed on Jul. 8, 2013, which is a divisional of
U.S. application Ser. No. 12/956,514, filed on Nov. 30, 2010 and
issued as U.S. Pat. No. 8,524,670 on Sep. 3, 2013, which is a
continuation of U.S. application Ser. No. 12/380,308, filed on Feb.
26, 2009 and issued as U.S. Pat. No. 7,923,016 on Apr. 4, 2011,
which claims priority to U.S. Provisional Application No.
61/067,273 filed Feb. 27, 2008. Each of these applications and
patents is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to a novel
CXCL12-.alpha..sub.2 locked dimer polypeptide, pharmaceutical
compositions thereof, and methods of using said dimer in the
treatment of cancer and autoimmune, inflammatory disease and
HIV/AIDS.
BACKGROUND
[0004] Chemokines are a superfamily of chemoattractant cytokine
proteins which primarily serve to regulate a variety of biological
responses and promote the recruitment and migration of multiple
lineages of leukocytes and lymphocytes to a body organ tissue.
Chemokines are classified into four families according to the
relative position of the first two cysteine residues in the
protein. In one family, the first two cysteines are separated by
one amino acid residue (the CXC chemokines) and in another family
the first two cysteines are adjacent (the CC chemokines). In a
third family, the first two cysteines are separated by three amino
acids (CX.sub.3C chemokines). In a fourth family there is only one
cysteine in the amino terminus (C chemokines).
[0005] The molecular targets for chemokines are cell surface
receptors. One such receptor is CXC chemokine receptor 4 (CXCR4),
which is a seven transmembrane G-protein coupled receptor (GPCR).
CXCR4 is widely expressed on cells of hematopoietic origin, and is
a major co-receptor with CD4.sup.+ for certain strains of human
immunodeficiency virus 1 (HIV-1).
[0006] CXCL12, formerly known as stromal cell-derived factor-1
(SDF-1), is an alpha or CXC type 7.8 kDa CXC chemokine. CXCL12 is
the only known natural ligand for CXCR4, as high affinity CXCL12
binding requires the CXCR4 amino terminus. CXCL12 comprises two
closely related members: CXCL12-.alpha. and CXCL12-.beta., the
native amino acid sequences of which are known, as are the genomic
sequences encoding these proteins (U.S. Pat. No. 5,563,048 and U.S.
Pat. No. 5,756,084, both of which are incorporated by reference
herein for all purposes).
[0007] Originally described as a growth factor for bone marrow
developing B cells, CXCL12 was subsequently characterized as a
chemoattractant for T cells and monocytes. Genetic ablation of
CXCR4 or CXCL12 results in defects in haematopoiesis,
vascularization of the intestines, cerebellar formation and heart
development. Similar embryonic defects in either of those chemokine
receptor or chemokine gene deficient animals has revealed roles for
CXCR4-CXCL12 signaling in cardiovascular, neuronal, and
hematopoietic stem cell development as well as gastrointestinal
vascularization. Previous studies have also established a role for
CXCL12 and CXCR4 in gut vascularization, a key process in mucosal
immunity and homeostasis. In vitro, CXCL12 stimulates chemotaxis of
a wide range of cells including monocytes and bone marrow derived
progenitor cells. Particularly notable is its ability to stimulate
a high percentage of resting and activated T-lymphocytes.
[0008] Consistent with the fact that CXCR4 is a major co-receptor
for HIV, CXCL12 has also been shown to block HIV entry into CD4+
cells. CXCR4 is a co-receptor for T-tropic (X4) strains of HIV,
which target CD4.sup.+ T cells, and CXCL12 can inhibit HIV-1
infection by preventing gp120 binding to CXCR4 and the subsequent
gp41 mediated fusion. CXCR4 co-receptor usage correlates with AIDS
onset, even though CCR5 is the primary co-receptor for most HIV
infections.
[0009] Efforts have been made to identify CXCL12-derived peptides
that interfere selectively with HIV entry, and not with CXCL12
signaling. A wide range of potential CXCR4 binding fragments of
CXCL12 have been proposed for use in blocking HIV infection,
indicating that the anti-HIV activity of CXCL12, or fragments of
CXCL12, does not depend on antagonism of the CXCR4 receptor.
[0010] CXCL12 also directs homeostatic immune cell trafficking and
inflammatory responses. Chemokine activation of specific G-protein
coupled receptors (GPCR) directs cell migration toward higher
chemokine concentration.
[0011] Additionally, CXCL12 and CXCR4 mediate cancer cell migration
and metastasis. Treatment with CXCR4-neutralizing antibodies
reduced metastatic tumor formation in a mouse model for human
breast cancer. Subsequently, over twenty cancer types have been
shown to express CXCR4 and metastasize to tissues that secrete
CXCL12, such as bone marrow, lung, liver and lymph nodes.
[0012] CXCL12 and CXCR4 also serve to establish a niche environment
for hematopoetic stem cells in bone marrow such that blocking the
function of CXCL12 leads to mobilization of said stem cells so that
they exit the bone marrow and enter the blood stream.
[0013] Accordingly, there is a current need for cost-effective
pharmaceutical agents and treatment methods for treating various
conditions including autoimmune or inflammation disorders, immune
suppression conditions, infections, blood cell deficiencies,
cancers and other described conditions and to mobilize stem cells
by manipulating and controlling CXCL12 and CXCR4.
SUMMARY OF THE INVENTION
[0014] The inventors have engineered a novel CXCL12-.alpha..sub.2
locked dimer polypeptide comprising two monomers linked together.
The dimer will/might be useful in treating various conditions
including cancer, autoimmune disorders and/or inflammation
disorders. In one preferred embodiment the dimer comprises two
monomers bound together, wherein at least one monomer has the amino
acid sequence as shown in SEQ ID NO:1.
[0015] In another embodiment, the present invention provides a
composition comprising a CXCL12-.alpha..sub.2 locked dimer
polypeptide and a pharmaceutically acceptable carrier or
diluent.
[0016] In another embodiment, the present invention provides an
isolated CXCL12-.alpha..sub.2 locked dimer polypeptide, wherein the
dimer preferably consists of at least one monomer having the amino
acid sequence as shown in SEQ ID NO:1.
[0017] In another embodiment, the present invention provides a
method of treating an autoimmune disease in a subject comprising
administering to the subject a therapeutically effective amount of
a composition comprising a CXCL12-.alpha..sub.2 locked dimer.
[0018] In another embodiment, the present invention provides a
method of treating a tumor in a subject comprising administering to
the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide.
[0019] In another embodiment, the present invention provides a
method of treating HIV/AIDS in a subject comprising administering
to the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide.
[0020] In another embodiment, the present invention provides a
method of inhibiting angiogenesis in a subject by administering to
the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide.
[0021] In another embodiment, the present invention provides a
method of inhibiting blood cancers in a subject by administering to
the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide.
[0022] In another embodiment, the present invention provides a kit
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide wherein
the dimer preferably comprises at least one monomer having the
amino acid sequence as shown in SEQ ID NO:1, a pharmaceutically
acceptable carrier or diluent, and instructional material.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1. CXCL12-.alpha..sub.2 (L36C/A65C)
(CXCL12-.alpha..sub.2) is a covalently locked dimer comprising two
CXCL12 L36C/A65C sequences (SEQ ID NO: 1), each sequence comprising
one subunit of the dimer. The lines connecting the cysteines show
where the intra- and intermolecular disulfide bonds are. A)
CXCL12-.alpha..sub.2 locked dimer amino acid sequence with the
conserved intramolecular disulfide bonds (black lines) and the
engineered intermolecular disulfide bonds (red lines) illustrated.
B) SDS-PAGE of CXCL12-.alpha. and CXCL12-.alpha..sub.2 treated with
and without dithiothreitol (DTT). CXCL12-.alpha. and
CXCL12-.alpha..sub.2 migrate near the monomeric molecular weight of
8 kDa when treated with DTT. In contrast, CXCL12-.alpha..sub.2
migrates as a dimer while CXCL12-.alpha. migrates as a monomer in
the absence of DTT. C) Translational diffusion measurements of
CXCL12-.alpha..sub.2 indicate CXCL12-.alpha..sub.2 is dimeric.
Diffusion coefficients (D.sub.s) of wild-type CXCL12 (black
circles) in 20 mM sodium phosphate at pH 7.4 plotted versus
chemokine concentration. Non-linear fitting of the CXCL12-.alpha.
D.sub.s values indicates a dimer dissociation K.sub.d of 120 .mu.M
with a pure monomer D.sub.s value of 1.6 (.times.10.sup.-6
cm.sup.2s.sup.-1) and a dimer value of .about.1.0 (.times.10.sup.-6
cm.sup.2s.sup.-1). D.sub.s values for 10, 50, and 150 .mu.M
SDF1.sub.2 (red triangles) range from 1.08-1.09 (.times.10.sup.-6
cm.sup.2s.sup.-1) consistent with those expected for CXCL12-.alpha.
in the dimeric state. D) N-terminal peptides corresponding to the
first thirty-eight amino acids of CXCR4 are illustrated. The
sequence for p38 is identical to that of CXCR4 except for an
additional N-terminal gly-ser dipeptide (cloning artifact) and the
C28A substitution (green) introduced to prevent oxidative peptide
dimer formation. The sulfated peptides are identical to p38 except
for the inclusion of sulfotyrosine at position 21 for p38-sY.sub.1
and at positions 7, 12 and 21 for p38-sY.sub.3.
[0024] FIG. 2. CXCR4 N-terminus binds CXCL12-.alpha..sub.2
(L36C/A65C) (CXCL12-.alpha..sub.2). A) Ensemble of 20
CXCL12-.alpha..sub.2 NMR solution structures (gray and tan)
superimposed on the crystal structure of dimeric wild-type
CXCL12-.alpha. (blue, PDB ID 2J7Z) with an .alpha.-carbon RMSD of
1.2 .ANG. for residues 9-66. Intermolecular C36-C65 disulfide bonds
are shown in yellow. Flexible N-terminal residues of CXCL12-.alpha.
(residues 1-8) are omitted for clarity. Refinement statistics for
the CXCL12-.alpha..sub.2 structure ensemble are given in table S1.
B) .sup.15N-.sup.1H HSQC spectra of 25 .mu.M
[U-.sup.15N]-CXCL12-.alpha..sub.2 alone (black contours) and after
addition of 100 .mu.M p38 peptide (green contours). C) Combined
.sup.15N-.sup.1H chemical shift perturbations plotted versus
CXCL12-.alpha..sub.2 residue number. Secondary structure elements
are indicated and regions involved in the dimer interface are
highlighted in orange. Missing values correspond to prolines
(sequence positions 2, 10, 32 and 53) or amino acids not observed
in the .sup.15N-.sup.1H HSQC spectra.
[0025] FIG. 3. CXCL12-.alpha..sub.2 CXCR4 N-terminal domain
structures. (A) Representative intermolecular NOEs for the
CXCL12-.alpha..sub.2:p38-sY.sub.1 complex. Strips from 3D
F1-.sup.13C-filtered/F3-.sup.13C-edited NOESY-HSQC spectra acquired
on a complex containing [U-.sup.15N, .sup.13C]-CXCL12-.alpha..sub.2
and unlabeled p38-sY.sub.1 (left) and a complex containing
[U-.sup.15N, .sup.13C]-p38-sY.sub.1 and unlabeled
CXCL12-.alpha..sub.2 (right) contain equivalent NOEs between the
V18 methyl of SDF1.sub.2 and sY21 .sup.1H.sup..delta. of
p38-sY.sub.1. Ensembles of the twenty lowest energy conformers for
the (B) CXCL12-.alpha..sub.2:p38, (C)
CXCL12-.alpha..sub.2:p38-sY.sub.1, and (D)
CXCL12-.alpha..sub.2:p38-sY.sub.3 complexes. CXCL12-.alpha..sub.2
is shown in gray and the CXCR4 N-termini are orange. Sulfotyrosine
residues in CXCR4 N-terminus are shown in red.
[0026] FIG. 4. Recognition of sulfotyrosine by
CXCL12-.alpha..sub.2. A) NMR structure of CXCL12-.alpha..sub.2
bound to p38-sY.sub.3. Individual monomers of the symmetric
CXCL12-.alpha..sub.2 dimer are shown in tan and white with
symmetry-related p38-sY.sub.3 peptides in blue and orange. Chemical
shift perturbations greater than 0.25 ppm (FIG. 1C) are highlighted
in green on the surface of the CXCL12-.alpha..sub.2 surface.
Flexible regions of CXCL12-.alpha..sub.2 (residues 1-8) and
p38-sY.sub.3 (residues 29-38) are omitted for clarity.
Sulfotyrosine side chains are shown in ball-and-stick
representation. In panels B-D, basic residues in
CXCL12-.alpha..sub.2 that pair with CXCR4 sulfotyrosines are shown
in blue and CXCL12-.alpha..sub.2 residues with NOEs to the
sulfotyrosines are shown in green. B) CXCR4 sY7 binds
CXCL12-.alpha..sub.2 near R20 and makes NOE contacts with V23. C)
CXCR4 sY12 occupies a cleft bounded by CXCL12-.alpha..sub.2
residues K27, P10 and L29. D) CXCR4 sY21 pairs with
CXCL12-.alpha..sub.2 R47 and makes NOE contacts with V18 and
V49.
[0027] FIG. 5. Dimeric CXCL12-.alpha..sub.2 induces CXCR4-mediated
Ca.sup.2+-flux but inhibits chemotaxis toward monomeric wild-type
CXCL12-.alpha.. A) Ca.sup.2+-flux in THP-1 cells loaded with Fluo-3
(Invitrogen) indicates robust dose-dependant CXCR4 activation by
wild-type CXCL12-.alpha. ( , EC.sub.50=3.6 nM) and
CXCL12-.alpha..sub.2 (, EC.sub.50=12.9 nM). B) Wild-type
CXCL12-.alpha. chemoattracts THP-1 cells in a biphasic,
concentration-dependent manner with maximal migratory response at
.about.30 nM. In contrast, CXCL12-.alpha..sub.2 does not
chemoattract THP-1 cells at all protein concentrations from 1-1,000
nM. C) Schematic illustration of the bell-shaped profile for
CXCL12-.alpha.-mediated chemotaxis arising from changes in relative
concentrations of chemokine monomer and dimer. At low chemokine
concentration monomeric CXCL12-.alpha. promotes chemotaxis (green
curve), while increasing CXCL12-.alpha..sub.2 dimerization at high
chemokine concentrations halts chemotaxis (red curve). D) Wild-type
CXCL12-.alpha. and the dimerization-impaired H25R variant
chemoattract THP-1 cells equally well at low concentrations (0.1-10
nM). CXCL12-.alpha. (H25R) remains monomeric at higher
concentrations relative to wild-type CXCL12-.alpha. and induces
chemotaxis over a broader range. E) Chemoattraction of THP-1 cells
induced by 10 nM wild-type CXCL12-.alpha. is inhibited by
CXCL12-.alpha..sub.2 (IC.sub.50.about.4 nM).
DETAILED DESCRIPTION OF THE INVENTION
I. In General
[0028] Before the present materials and methods are described, it
is understood that this invention is not limited to the particular
methodology, protocols, materials, and reagents described, as these
may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to limit the scope of the present invention,
which will be limited only by any later-filed nonprovisional
applications.
[0029] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
references unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprising", "including", and "having" can be used
interchangeably.
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described. All
publications and patents specifically mentioned herein are
incorporated by reference for all purposes including describing and
disclosing the materials, instruments, statistical analysis and
methodologies which are reported in the publications which might be
used in connection with the invention. All references cited in this
specification are to be taken as indicative of the level of skill
in the art. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
II. The Invention
[0031] CXCL12-.alpha..sub.2 Locked Dimer Polypeptide. In one
embodiment, the invention provides a CXCL12-.alpha..sub.2 locked
dimer polypeptide comprising at least two monomers. The monomers
may be identical or may be non-identical. In one embodiment, at
least one of the monomers has the amino acid sequence according to
SEQ ID NO: 1. In alternate embodiments, both monomers have the
amino acid sequence according to SEQ ID NO:1.
[0032] By "locked" we mean the monomer components of the
polypeptide are linked to each other via at least one covalent
bond. The monomer and dimer forms do not interconvert. In a
preferred embodiment, at least one of residues L36 and A65 are
replaced with cysteine residues to create at least one
intermolecular disulfide bond between cysteine residues at position
36 of one subunit and/or position 65 of the other monomer subunit.
As shown in FIG. 1A, either or both cysteine residues at positions
L36 and A65 can be replaced with cysteines to form the locked dimer
with at least one, but preferably two, disulfide bonds.
[0033] Other residue(s) besides L36C and A65C in CXCL12 could be
mutated to cysteines in order to form the locked dimer similar to
the one of the present invention. For instance, a locked dimer can
be created by mutating amino acid(s) in the CXCL12 dimer interface
to cysteines that are positioned opposite one another yielding a
disulfide bond that covalently links two CXCL12 monomers. For
example, residue K27 is directly across the CXCL12 dimer interface
from residue K27 of the opposing subunit and K27C mutation would
likely make a locked dimer. Residues L26 and I28 are also on the
CXCL12 dimer interface, and a L26C/I28C variant should form a
locked dimer with L26C of one monomer subunit forming a disulfide
bond with I28C of the opposing subunit and I28C of one monomer
subunit forming a disulfide bond with L26C of the opposing
subunit.
[0034] In a preferred embodiment, the CXCL12.alpha..sub.2 locked
dimer of the present invention has substitutions at both L36C/A65C
residues. Residue L36 is on beta strand 2 and A65 is near the end
of the alpha helix of the dimer. Thus, disulfide bonds form between
beta strand 2 and the end of the helix generate the locked dimer. A
similar locked dimer could be created using disulfide bonds
introduced between beta strand 1 and the middle of the alpha helix.
For example, CXCL12 with I28C/Y61C or I28C/L62C would form a locked
dimer with beta strand one of one monomer having a disulfide bond
to the middle of the alpha helix of the second monomer thus making
a locked dimer. Additionally, a locked dimer may be created by
generating a construct that produces two CXCL12 monomers where the
C-terminus of one is linked to the N-terminus of the other through
an amino acid linker.
[0035] Additional methods for making locked dimers of CXCL12 could
also include other types of covalent linkages besides disulfide
bonds including, but not limited to, chemical cross-linking
reagents.
[0036] In a preferred embodiment, the locked dimer of the present
invention comprises a substantially pure preparation. By
"substantially pure" we mean a preparation in which more than 90%,
e.g., 95%, 98% or 99% of the preparation is that of the locked
dimer.
[0037] In a preferred embodiment, at least one of the monomers
comprising the locked dimer of the present invention has the amino
acid sequence as shown in SEQ ID NO:1 or a homologue or fragment
thereof. In a further preferred embodiment, the dimer comprises two
monomers having the amino acid sequence as shown in SEQ ID NO:1 or
a homologue or variant thereof. By "homologue" we mean an amino
acid sequence generally being at least 80%, preferably at least 90%
and more preferably at least 95% homologous to the polypeptide of
SEQ ID NO:1 over a region of at least twenty contiguous amino
acids. By "fragment," we mean peptides, oligopeptides,
polypeptides, proteins and enzymes that comprise a stretch of
contiguous amino acid residues, and exhibit substantially a
similar, but not necessarily identical, functional activity as the
complete sequence. Fragments of SEQ ID NO:1, or their homologues,
will generally be at least ten, preferably at least fifteen, amino
acids in length, and are also encompassed by the term "a CXCL12
monomer" as used herein.
[0038] Mutations known to prevent degradation of CXCL12 or increase
the in vivo half life may also be incorporated into the
CXCL12.alpha..sub.2 sequence. For instance, adding a serine to the
N-terminus along with a S4V substitution prevent CXCL12 degradation
by proteases. Therefore, adding a serine to the N-terminus would
likely similarly prevent protease degradation of the
CXCL12.alpha..sub.2 locked dimer of the present invention.
[0039] Further, in addition to binding CXCR4, CXCL12 also binds to
heparin found in the extracellular matrix on cell surfaces. The
inventors have shown that the CXCL12.alpha..sub.2 locked dimer of
the present invention also binds heparin. Amino acid substitutions
in CXCL12, including K24S, K27S, or K24S/K27S can prevent heparin
binding and increase the half-life of CXCL12 in vivo; therefore,
similar mutations in CXCL12.alpha..sub.2 would likely prevent
heparin binding and increase the in vivo half-life of the
dimer.
[0040] CXCL12.alpha..sub.2 variants have been generated that have a
gly-met dipeptide on the N-terminus. N-terminal extensions to
CXCL12 prevent CXCR4 activation and thus inclusion in
CXCL12.alpha..sub.2 may increase its effectiveness. Additionally,
it may be useful to create CXCL12.alpha..sub.2 variants where both
subunits are not identical. For example, only one monomer of the
CXCL12.alpha..sub.2 dimer may need to include the addition of an
N-terminal serine and a S4V substitution or the lysine
substitutions for the prevention of heparin binding. Alternatively,
a CXCL12.alpha..sub.2 variant where the N-terminus of one monomer
has the native sequence but the other has been extended may have
different or enhanced pharmacological properties compared to
CXCL12.alpha..sub.2.
[0041] The locked CXCL12 dimer could also be incorporated into a
larger protein or attached to a fusion protein that may function to
increase the half life of the dimer in vivo or be used as a
mechanism for time released and/or local delivery (U.S. Patent
Appn. No. 20060088510).
[0042] In another embodiment, the invention provides an isolated
CXCL12-.alpha..sub.2 locked dimer polypeptide as described above.
By "isolated" we mean a nucleic acid sequence that is identified
and separated from at least one component or contaminant with which
it is ordinarily associated. An isolated nucleic acid is present in
a form or setting that is different from that in which it is found
in nature. In contrast, non-isolated nucleic acids such as DNA and
RNA are found in the state they exist in nature. For example, a
given DNA sequence (e.g., a gene) is found on the host cell
chromosome in proximity to neighboring genes; RNA sequences, such
as a specific mRNA sequence encoding a specific protein, are found
in the cell as a mixture with numerous other mRNAs that encode a
multitude of proteins. However, an isolated nucleic acid encoding a
given protein includes, by way of example, such nucleic acid in
cells ordinarily expressing the given protein where the nucleic
acid is in a chromosomal location different from that of natural
cells, or is otherwise flanked by a different nucleic acid sequence
than that found in nature. The isolated nucleic acid,
oligonucleotide, or polynucleotide can be present in
single-stranded or double-stranded form. When an isolated nucleic
acid, oligonucleotide or polynucleotide is to be utilized to
express a protein, the oligonucleotide or polynucleotide will
contain at a minimum the sense or coding strand (i.e., the
oligonucleotide or polynucleotide can be single-stranded), but can
contain both the sense and anti-sense strands (i.e., the
oligonucleotide or polynucleotide can be double-stranded).
[0043] CXCL12-.alpha..sub.2 locked dimer polypeptides of the
present invention can be prepared by standard techniques known in
the art. The peptide component of CXCL12-.alpha..sub.2 is composed,
at least in part, of a peptide, which can be synthesized using
standard techniques such as those described in Bodansky, M.
Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and
Grant, G. A. (ed.). Synthetic Peptides: A User's Guide, W. H.
Freeman and Company, New York (1992). Automated peptide
synthesizers are commercially available (e.g., Advanced ChemTech
Model 396; Milligen/Biosearch 9600). Additionally, one or more
modulating groups can be attached to the CXCL12-.alpha..sub.2
derived peptidic component by standard methods, such as by using
methods for reaction through an amino group (e.g., the alpha-amino
group at the amino-terminus of a peptide), a carboxyl group (e.g.,
at the carboxy terminus of a peptide), a hydroxyl group (e.g., on a
tyrosine, serine or threonine residue) or other suitable reactive
group on an amino acid side chain (see e.g., Greene, T. W. and
Wuts, P. G. M. Protective Groups in Organic Synthesis, John Wiley
and Sons, Inc., New York (1991)). Exemplary syntheses of preferred
CXCL12-.alpha..sub.2 locked dimer polypeptides according to the
present invention are described further in the Examples below.
[0044] Peptides of the invention may be chemically synthesized
using standard techniques such as those described in Bodansky, M.
Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and
Grant, G. A. (ed.). Synthetic Peptides: A User's Guide, W.H.
Freeman and Company, New York, (1992) (all of which are
incorporated herein by reference).
[0045] In another aspect of the invention, peptides may be prepared
according to standard recombinant DNA techniques using a nucleic
acid molecule encoding the peptide. A nucleotide sequence encoding
the peptide can be determined using the genetic code and an
oligonucleotide molecule having this nucleotide sequence can be
synthesized by standard DNA synthesis methods (e.g., using an
automated DNA synthesizer). Alternatively, a DNA molecule encoding
a peptide compound can be derived from the natural precursor
protein gene or cDNA (e.g., using the polymerase chain reaction
(PCR) and/or restriction enzyme digestion) according to standard
molecular biology techniques.
[0046] CXCL12-.alpha..sub.2 Locked Dimer Polypeptide Pharmaceutical
Compositions. In another embodiment, the invention provides a
composition comprising a substantially pure CXCL12-.alpha..sub.2
locked dimer polypeptide of the present invention, and a
pharmaceutically acceptable carrier. By "pharmaceutically
acceptable carrier" we mean any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. In one embodiment, the carrier may be suitable for
parenteral administration. Alternatively, the carrier can be
suitable for intravenous, intraperitoneal, intramuscular,
sublingual or oral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0047] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, membrane nanoparticle or other ordered structure suitable
to high drug concentration. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. In many
cases, it will be preferable to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, or sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, such as, monostearate salts and
gelatin.
[0048] Moreover, the CXCL12-.alpha..sub.2 locked dimer polypeptide
of the present invention can be administered in a time-release
formulation, such as in a composition which includes a slow release
polymer. The active compounds can be prepared with carriers that
will protect the compound against rapid release, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are patented or
generally known to those skilled in the art.
[0049] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g. CXCR4 antagonist) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
CXCL12-.alpha..sub.2 locked dimer polypeptide of the present
invention also may be formulated with one or more additional
compounds that enhance the solubility of the CXCL12-.alpha..sub.2
locked dimer polypeptide.
[0050] Administration. The CXCL12-.alpha..sub.2 locked dimer
polypeptide of the present invention, optionally comprising other
pharmaceutically active compounds, can be administered to a patient
orally, rectally, parenterally, (e.g., intravenously,
intramuscularly, or subcutaneously) intracisternally,
intravaginally, intraperitoneally, intravesically, locally (for
example, powders, ointments or drops), or as a buccal or nasal
spray. Other contemplated formulations include projected
nanoparticles, liposomal preparations, resealed erythrocytes
containing the active ingredient, and immunologically-based
formulations.
[0051] Parenteral administration of a pharmaceutical composition
includes any route of administration characterized by physical
breaching of a tissue of a human and administration of the
pharmaceutical composition through the breach in the tissue.
Parenteral administration thus includes administration of a
pharmaceutical composition by injection of the composition, by
application of the composition through a surgical incision, by
application of the composition through a tissue-penetrating
non-surgical wound, and the like. In particular, parenteral
administration includes subcutaneous, intraperitoneal, intravenous,
intraarterial, intramuscular, or intrasternal injection and
intravenous, intraarterial, or kidney dialytic infusion
techniques.
[0052] Compositions suitable for parenteral injection comprise the
CXCL12-.alpha..sub.2 locked dimer of the invention combined with a
pharmaceutically acceptable carrier such as physiologically
acceptable sterile aqueous or nonaqueous solutions, dispersions,
suspensions, or emulsions, or may comprise sterile powders for
reconstitution into sterile injectable solutions or dispersions.
Examples of suitable aqueous and nonaqueous carriers, diluents,
solvents, or vehicles include water, isotonic saline, ethanol,
polyols (e.g., propylene glycol, polyethylene glycol, glycerol, and
the like), suitable mixtures thereof, triglycerides, including
vegetable oils such as olive oil, or injectable organic esters such
as ethyl oleate. Proper fluidity can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersions, and/or by the
use of surfactants. Such formulations can be prepared, packaged, or
sold in a form suitable for bolus administration or for continuous
administration. Injectable formulations can be prepared, packaged,
or sold in unit dosage form, such as in ampules, in multi-dose
containers containing a preservative, or in single-use devices for
auto-injection or injection by a medical practitioner.
[0053] Formulations for parenteral administration include
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations can further comprise one or more
additional ingredients including suspending, stabilizing, or
dispersing agents. In one embodiment of a formulation for
parenteral administration, the CXCL12-.alpha..sub.2 locked dimer
polypeptide is provided in dry (i.e., powder or granular) form for
reconstitution with a suitable vehicle (e.g., sterile pyrogen-free
water) prior to parenteral administration of the reconstituted
composition.
[0054] The pharmaceutical compositions can be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution can be
formulated according to the known art. Such sterile injectable
formulations can be prepared using a non-toxic
parenterally-acceptable diluent or solvent, such as water or
1,3-butanediol, for example. Other acceptable diluents and solvents
include Ringer's solution, isotonic sodium chloride solution, and
fixed oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation can comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0055] The CXCL12-.alpha..sub.2 locked dimer polypeptide of the
present invention may also contain adjuvants such as suspending,
preserving, wetting, emulsifying, and/or dispersing agents,
including, for example, parabens, chlorobutanol, phenol, sorbic
acid, and the like. It may also be desirable to include isotonic
agents, for example, sugars, sodium chloride, and the like.
Prolonged absorption of injectable pharmaceutical compositions can
be brought about by the use of agents capable of delaying
absorption, such as aluminum monostearate and/or gelatin.
[0056] Dosage forms can include solid or injectable implants or
depots. In preferred embodiments, the implant comprises an
effective amount of the .alpha..sub.2 locked dimer polypeptide and
a biodegradable polymer. In preferred embodiments, a suitable
biodegradable polymer can be selected from the group consisting of
a polyaspartate, polyglutamate, poly(L-lactide), a
poly(D,L-lactide), a poly(lactide-co-glycolide), a
poly(c-caprolactone), a polyanhydride, a poly(beta-hydroxy
butyrate), a poly(ortho ester) and a polyphosphazene. In other
embodiments, the implant comprises an effective amount of
CXCL12-.alpha..sub.2 locked dimer polypeptide and a silastic
polymer. The implant provides the release of an effective amount of
CXCL12-.alpha..sub.2 locked dimer polypeptide for an extended
period ranging from about one week to several years.
[0057] Solid dosage forms for oral administration include capsules,
tablets, powders, and granules. In such solid dosage forms, the
CXCL12-.alpha..sub.2 locked dimer polypeptide is admixed with at
least one inert customary excipient (or carrier) such as sodium
citrate or dicalcium phosphate or (a) fillers or extenders, as for
example, starches, lactose, sucrose, mannitol, or silicic acid; (b)
binders, as for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidone, sucrose, or acacia; (c) humectants,
as for example, glycerol; (d) disintegrating agents, as for
example, agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, certain complex silicates, or sodium carbonate; (e)
solution retarders, as for example, paraffin; (f) absorption
accelerators, as for example, quaternary ammonium compounds; (g)
wetting agents, as for example, cetyl alcohol or glycerol
monostearate; (h) adsorbents, as for example, kaolin or bentonite;
and/or (i) lubricants, as for example, talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, or mixtures thereof. In the case of capsules and tablets,
the dosage forms may also comprise buffering agents.
[0058] A tablet comprising the active ingredient can, for example,
be made by compressing or molding the active ingredient, optionally
with one or more additional ingredients. Compressed tablets can be
prepared by compressing, in a suitable device, the active
ingredient in a free-flowing form such as a powder or granular
preparation, optionally mixed with one or more of a binder, a
lubricant, an excipient, a surface active agent, and a dispersing
agent. Molded tablets can be made by molding, in a suitable device,
a mixture of the active ingredient, a pharmaceutically acceptable
carrier, and at least sufficient liquid to moisten the mixture.
[0059] Tablets may be manufactured with pharmaceutically acceptable
excipients such as inert diluents, granulating and disintegrating
agents, binding agents, and lubricating agents. Known dispersing
agents include potato starch and sodium starch glycolate. Known
surface active agents include sodium lauryl sulfate. Known diluents
include calcium carbonate, sodium carbonate, lactose,
microcrystalline cellulose, calcium phosphate, calcium hydrogen
phosphate, and sodium phosphate. Known granulating and
disintegrating agents include corn starch and alginic acid. Known
binding agents include gelatin, acacia, pre-gelatinized maize
starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.
Known lubricating agents include magnesium stearate, stearic acid,
silica, and talc.
[0060] Tablets can be non-coated or coated using known methods to
achieve delayed disintegration in the gastrointestinal tract of a
human, thereby providing sustained release and absorption of the
active ingredient. By way of example, a material such as glyceryl
monostearate or glyceryl distearate can be used to coat tablets.
Further by way of example, tablets can be coated using methods
described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to
form osmotically-controlled release tablets. Tablets can further
comprise a sweetening agent, a flavoring agent, a coloring agent, a
preservative, or some combination of these in order to provide
pharmaceutically elegant and palatable preparation.
[0061] Solid dosage forms such as tablets, dragees, capsules, and
granules can be prepared with coatings or shells, such as enteric
coatings and others well known in the art. They may also contain
opacifying agents, and can also be of such composition that they
release the active compound or compounds in a delayed manner.
Examples of embedding compositions that can be used are polymeric
substances and waxes. The active compounds can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-mentioned excipients.
[0062] Solid compositions of a similar type may also be used as
fillers in soft or hard filled gelatin capsules using such
excipients as lactose or milk sugar, as well as high molecular
weight polyethylene glycols, and the like. Hard capsules comprising
the active ingredient can be made using a physiologically
degradable composition, such as gelatin. Such hard capsules
comprise the active ingredient, and can further comprise additional
ingredients including, for example, an inert solid diluent such as
calcium carbonate, calcium phosphate, or kaolin. Soft gelatin
capsules comprising the active ingredient can be made using a
physiologically degradable composition, such as gelatin. Such soft
capsules comprise the active ingredient, which can be mixed with
water or an oil medium such as peanut oil, liquid paraffin, or
olive oil.
[0063] Dose Requirements. In particular embodiments, a preferred
range for therapeutically or prophylactically effective amounts of
CXCL12-.alpha..sub.2 locked dimer polypeptide may be 0.1 nM-0.1M,
particularly 0.1 nM-0.05M, more particularly 0.05 nM-15 .mu.M and
most particularly 0.01 nM-10 .mu.M. It is to be noted that dosage
values may vary with the severity of the condition to be
alleviated, especially with multiple sclerosis. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0064] The amount of CXCL12-.alpha..sub.2 locked dimer polypeptide
in the composition may vary according to factors such as the
disease state, age, sex, and weight of the individual. Dosage
regimens may be adjusted to provide the optimum therapeutic
response. For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such as active
compound for the treatment of sensitivity in individuals.
[0065] Methods of Use. The invention also provides corresponding
methods of use, including methods of medical treatment, in which a
therapeutically effective dose of a CXCL12-.alpha..sub.2 locked
dimer polypeptide, preferably wherein the dimer comprises at least
one monomer having the amino acid sequence according to SEQ ID
NO:1, is administered in a pharmacologically acceptable
formulation. Accordingly, the invention also provides therapeutic
compositions comprising a CXCL12-.alpha..sub.2 locked dimer
polypeptide and a pharmacologically acceptable excipient or
carrier, as described above. The therapeutic composition may
advantageously be soluble in an aqueous solution at a
physiologically acceptable pH.
[0066] In one embodiment, the invention provides a method of
treating autoimmune disease in a subject comprising administering
to the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide. By
"autoimmune disease" we mean illnesses generally understood to be
caused by the over-production of cytokines, lymphotoxins and
antibodies by white blood cells, including in particular T-cells.
Such autoimmune diseases include but are not limited to Multiple
Sclerosis (MS), Guillain-Barre Syndrome, Amyotrophic Lateral
Sclerosis, Parkinson's disease, Alzheimer's disease, Diabetes Type
I, gout, lupus, and any other human illness that T-cells play a
major role in, such as tissue graft rejection. In addition,
diseases involving the degradation of extra-cellular matrix
include, but are not limited to, psoriatic arthritis, juvenile
arthritis, early arthritis, reactive arthritis, osteoarthritis,
ankylosing spondylitis. osteoporosis, muscular skeletal diseases
like tendonitis and periodontal disease, cancer metastasis, airway
diseases (COPD, asthma or other reactive airways disease), renal
and liver fibrosis, cardio-vascular diseases like atherosclerosis
and heart failure, and neurological diseases like neuroinflammation
and multiple sclerosis. Diseases involving primarily joint
degeneration include, but are not limited to, rheumatoid arthritis,
psoriatic arthritis, juvenile arthritis, early arthritis, reactive
arthritis, osteoarthritis, ankylosing spondylitis. Diseases
involving the eye include, but are not limited to autoimmune
uveitis and uveoconjunctivitis and dry eye syndrome. Diseases
involving post-infections complications of viral or bacterial
diseases such as glomerulonephritis, vasculitis,
meningoencephalitis. Diseases involving the gastrointestinal system
include but are not limited to inflammatory bowel diseases.
[0067] By "subject" we mean mammals and non-mammals. "Mammals"
means any member of the class Mammalia including, but not limited
to, humans, non-human primates such as chimpanzees and other apes
and monkey species; farm animals such as cattle, horses, sheep,
goats, and swine; domestic animals such as rabbits, dogs, and cats;
laboratory animals including rodents, such as rats, mice, and
guinea pigs; and the like. Examples of non-mammals include, but are
not limited to, birds, fish and the like. The term "subject" does
not denote a particular age or sex.
[0068] By "treating" we mean the management and care of a subject
for the purpose of combating the disease, condition, or disorder.
The terms embrace both preventative, i.e., prophylactic, and
palliative treatments. Treating includes the administration of a
compound of the present invention to prevent, ameliorate and/or
improve the onset of the symptoms or complications, alleviating the
symptoms or complications, or eliminating the disease, condition,
or disorder.
[0069] By "ameliorate", "amelioration", "improvement" or the like
we mean a detectable improvement or a detectable change consistent
with improvement occurs in a subject or in at least a minority of
subjects, e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%,
40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a
range about between any two of these values. Such improvement or
change may be observed in treated subjects as compared to subjects
not treated with the locked dimer of the present invention, where
the untreated subjects have, or are subject to developing, the same
or similar disease, condition, symptom or the like. Amelioration of
a disease, condition, symptom or assay parameter may be determined
subjectively or objectively, e.g., self assessment by a subject(s),
by a clinician's assessment or by conducting an appropriate assay
or measurement, including, e.g., a quality of life assessment, a
slowed progression of a disease(s) or condition(s), a reduced
severity of a disease(s) or condition(s), or a suitable assay(s)
for the level or activity(ies) of a biomolecule(s), cell(s) or by
detection of cell migration within a subject. Amelioration may be
transient, prolonged or permanent or it may be variable at relevant
times during or after the locked dimer of the present invention is
administered to a subject or is used in an assay or other method
described herein or a cited reference, e.g., within about 1 hour of
the administration or use of the locked dimer of the present
invention to about 3, 6, 9 months or more after a subject(s) has
received the locked dimer of the present invention.
[0070] By "modulation" of, e.g., a symptom, level or biological
activity of a molecule, replication of a pathogen, cellular
response, cellular activity or the like means that the cell level
or activity is detectably increased or decreased. Such increase or
decrease may be observed in treated subjects as compared to
subjects not treated with the locked dimer of the present
invention, where the untreated subjects have, or are subject to
developing, the same or similar disease, condition, symptom or the
like. Such increases or decreases may be at least about 2%, 5%,
10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more
or about within any range about between any two of these values.
Modulation may be determined subjectively or objectively, e.g., by
the subject's self assessment, by a clinician's assessment or by
conducting an appropriate assay or measurement, including, e.g.,
quality of life assessments or suitable assays for the level or
activity of molecules, cells or cell migration within a subject.
Modulation may be transient, prolonged or permanent or it may be
variable at relevant times during or after the locked dimer of the
present invention is administered to a subject or is used in an
assay or other method described herein or a cited reference, e.g.,
within about 1 hour of the administration or use of the locked
dimer of the present invention to about 3, 6, 9 months or more
after a subject(s) has received the locked dimer of the present
invention.
[0071] By "administering" we mean any means for introducing the
CXCL12-.alpha..sub.2 locked dimer polypeptide of the present
invention into the body, preferably into the systemic circulation.
Examples include but are not limited to oral, buccal, sublingual,
pulmonary, transdermal, transmucosal, as well as subcutaneous,
intraperitoneal, intravenous, and intramuscular injection.
[0072] By "therapeutically effective amount" we mean an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result, such as reduction or reversal of
angiogenesis in the case of cancers, or reduction or inhibition of
T-cells in autoimmune diseases. A therapeutically effective amount
of the CXCL12-.alpha..sub.2 locked dimer polypeptide may vary
according to factors such as the disease state, age, sex, and
weight of the subject, and the ability of the CXCL12-.alpha..sub.2
locked dimer polypeptide to elicit a desired response in the
subject. Dosage regimens may be adjusted to provide the optimum
therapeutic response. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the
CXCL12-.alpha..sub.2 locked dimer polypeptide are outweighed by the
therapeutically beneficial effects.
[0073] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result, such as preventing or inhibiting
the rate of metastasis of a tumor or the onset of bouts or episodes
of multiple sclerosis. A prophylactically effective amount can be
determined as described above for the therapeutically effective
amount. Typically, since a prophylactic dose is used in subjects
prior to or at an earlier stage of disease, the prophylactically
effective amount will be less than the therapeutically effective
amount.
[0074] In another embodiment, the invention provides a method of
treating a tumor in a subject comprising administering to the
subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide. By
"tumor" we mean any abnormal proliferation of tissues, including
solid and non-solid tumors. For instance, the composition and
methods of the present invention can be utilized to treat cancers
that manifest solid tumors such as breast cancer, colon cancer,
lung cancer, thyroid cancer, ovarian cancer and the like. The
composition and methods of the present invention can also be
utilized to treat non-solid tumor cancers such as non-Hodgkin's
lymphoma, leukemia and the like.
[0075] In another embodiment, the present invention provides a
method of inhibiting angiogenesis in a subject by administering to
the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide. By
"angiogenesis" we mean the process whereby new blood vessels
penetrate tissue thus supplying oxygen and nutrients while removing
waste in various pathological conditions including but not limited
to diabetic retinopathy, macular degeneration, rheumatoid
arthritis, inflammatory bowel disease, cancer, psoriasis,
osteoarthritis, ulcerative colitis, Crohn's disease and coronary
thrombosis.
[0076] In another embodiment, the present invention provides a
method of treating inflammation in a subject by administering to
the subject a therapeutically effective amount of a composition
comprising a CXCL12-.alpha..sub.2 locked dimer polypeptide. By
"inflammation" we mean the complex biological response of vascular
tissues to harmful stimuli, such as pathogens, damaged cells, or
irritants. It is a protective attempt by the organism to remove the
injurious stimuli as well as initiate the healing process for the
tissue. For instance, the composition and methods of the present
invention can be utilized to treat inflammation associated with: an
allergic disease such as asthma, hives, urticaria, pollen allergy,
dust mite allergy, venom allergy, cosmetics allergy, latex allergy,
chemical allergy, drug allergy, insect bite allergy, animal dander
allergy, stinging plant allergy, poison ivy allergy and food
allergy; a neurodegenerative disease; a cardiovascular disease; a
gastrointestinal disease; a tumor such as a malignant tumor, a
benign tumor, a solid tumor, a metastatic tumor and a non-solid
tumor; septic shock; anaphylactic shock; toxic shock syndrome;
cachexia; necrosis; gangrene; a prosthetic implant such as a breast
implant, a silicone implant, a dental implant, a penile implant, a
cardiac implant, an artificial joint, a bone fracture repair
device, a bone replacement implant, a drug delivery implant, a
catheter, a pacemaker and a respirator tube; menstruation; an ulcer
such as a skin ulcer, a bed sore, a gastric ulcer, a peptic ulcer,
a buccal ulcer, a nasopharyngeal ulcer, an esophageal ulcer, a
duodenal ulcer and a gastrointestinal ulcer; an injury such as an
abrasion, a bruise, a cut, a puncture wound, a laceration, an
impact wound, a concussion, a contusion, a thermal burn, frostbite,
a chemical burn, a sunburn, a desiccation, a radiation burn, a
radioactivity burn, smoke inhalation, a torn muscle, a pulled
muscle, a torn tendon, a pulled tendon, a pulled ligament, a torn
ligament, a hyperextension, a torn cartilage, a bone fracture, a
pinched nerve and a gunshot wound; a musculo-skeletal inflammation
such as a muscle inflammation, myositis, a tendon inflammation,
tendinitis, a ligament inflammation, a cartilage inflammation, a
joint inflammation, a synovial inflammation, carpal tunnel syndrome
and a bone inflammation.
[0077] In another embodiment, the locked dimer of the present
invention may also provide for the down regulation of cell surface
expression of CXCR4 without activating the chemotaxis machinery of
said cells. In another embodiment, the locked dimer of the present
invention may enhance the efficacy of CXCR4 receptor
pharmacological antagonists or HIV-1 entry blockers by decreasing
the cell surface expression of CXCR4.
[0078] Kits. In another embodiment, the present invention provides
a kit comprising a pharmaceutical composition according to the
present invention and instructional material. By "instructional
material" we mean a publication, a recording, a diagram, or any
other medium of expression which is used to communicate the
usefulness of the pharmaceutical composition of the invention for
one of the purposes set forth herein in a human. The instructional
material can also, for example, describe an appropriate dose of the
pharmaceutical composition of the invention. The instructional
material of the kit of the invention can, for example, be affixed
to a container which contains a pharmaceutical composition of the
invention or be shipped together with a container which contains
the pharmaceutical composition. Alternatively, the instructional
material can be shipped separately from the container with the
intention that the instructional material and the pharmaceutical
composition be used cooperatively by the recipient.
[0079] The invention may also further comprise a delivery device
for delivering the composition to a subject. By way of example, the
delivery device can be a squeezable spray bottle, a metered-dose
spray bottle, an aerosol spray device, an atomizer, a dry powder
delivery device, a self-propelling solvent/powder-dispensing
device, a syringe, a needle, a tampon, or a dosage-measuring
container. It may be desirable to provide a memory aid on the kit,
e.g., in the form of numbers next to the tablets or capsules
whereby the numbers correspond with the days of the regimen that
the tablets or capsules so specified should be ingested. Another
example of such a memory aid is a calendar printed on the card,
e.g., as follows "First Week, Monday, Tuesday, . . . etc. . . .
Second Week, Monday, Tuesday," etc. Other variations of memory aids
will be readily apparent. A "daily dose" can be a single tablet or
capsule or several pills or capsules to be taken on a given
day.
[0080] The delivery device may comprise a dispenser designed to
dispense the daily doses one at a time in the order of their
intended use is provided. Preferably, the dispenser is equipped
with a memory aid, so as to further facilitate compliance with the
dosage regimen. An example of such a memory aid is a mechanical
counter, which indicates the number of daily doses that have been
dispensed. Another example of such a memory aid is a
battery-powered micro-chip memory coupled with a liquid crystal
readout, or audible reminder signal which, for example, reads out
the date that the last daily dose has been taken and/or reminds one
when the next dose is to be taken.
III. Examples
[0081] The following examples describing materials and methodology
are offered for illustrative purposes only, and are not intended to
limit the scope of the present invention.
[0082] Detection of 2:1 p38: CXCL12-.alpha..sub.2 locked dimer
polypeptide binding. Binding the wild-type CXCL12-.alpha. with p38
requires navigating a complex set of coupled binding equilibria to
permit exchange between complexes with stoichiometries of 1:1, 1:2,
2:1 and 2:2. Because only one set of CXCL12-.alpha..sub.2 locked
dimer signals is observed during the p38 titration and addition of
more than two molar equivalents of p38 induces no further chemical
shift perturbations, the inventors concluded that a symmetric 2:1
p38: CXCL12-.alpha..sub.2 locked dimer complex is formed (see
Example 3).
[0083] Structure analysis of CXCL12-.alpha..sub.2 locked dimer
polypeptide individual sulfotyrosine recognition sites. Structures
of sulfotyrosine-containing protein complexes show that the
sulfonate group typically interact with a positively charged side
chain. Each negatively-charged sulfotyrosine side chains docks into
a unique positively-charged pocket on the CXCL12-.alpha..sub.2
surface (FIG. 4), and the inventors designed a series of amino acid
substitutions in wild-type CXCL12-.alpha. to individually disrupt
those binding sites (see Example 4 and Table 5).
[0084] Structure of CXCL12-.alpha..sub.2 locked dimer polypeptide
bound to sulfated p38 peptides. CXCR4 stabilizes the
CXCL12-.alpha..sub.2 dimer by interacting with both subunits and
recognizing unique features of the CXCL12-.alpha..sub.2 dimer
interface. Near the CXCR4 N-terminus, each p38 peptide crosses the
CXCL12-.alpha..sub.2 dimer interface, such that sY7 and sY12
interact with opposing CXCL12-.alpha. monomers. In the
membrane-proximal portion of the CXCR4 N-terminal domain, P27
inserts between Q59 of one CXCL12-.alpha..sub.2 subunit and L66 of
the opposing subunit, where the C-terminal helices from each
monomer pack against each other (see Example 5).
[0085] Inhibitory Effect of the CXCL12-.alpha..sub.2 locked dimer
polypeptide on cell migration. The inventors data shows that the
CXCL12-.alpha..sub.2 locked dimer inhibits chemotaxis induced by
wild-type CXCL12-.alpha.. The CXCL12-.alpha..sub.2 locked dimer
inhibits chemotaxis induced by wild-type CXCL12-.alpha. with an
IC.sub.50 of approximately 4 nM (FIG. 5D). The inventors' results
show that monomer CXCL12-.alpha. activates cell migration while the
dimeric CXCL12-.alpha..sub.2 halts cell migration. The results also
demonstrate that the CXCL12-.alpha..sub.2 locked dimer acts as a
partial CXCR4 agonist (as evidenced by the detection of the
secondary messenger calcium) and as a selective antagonist that
blocks chemotaxis. This indicates that the CXCL12-.alpha..sub.2
locked dimer is a tool for correlating cellular responses with
different intracellular signaling pathways initiated by CXCR4.
Also, the CXCL12-.alpha..sub.2 locked dimer of the present
invention may be useful in the development of anti-metastatic
agents by preventing CXCL12-.alpha./CXCR4-mediated migration of
circulating cancer cells (see Example 6).
[0086] Physiological relevance of the CXCL12-.alpha.2 locked dimer.
Despite being obtained with a constitutively dimeric chemokine,
CXCL12-.alpha..sub.2:p38 structures correctly identify key elements
of CXCR4 recognition by wild-type CXCL12-.alpha.. The inventors
monitored activation of CXCR4 using THP-1 cells and a similar
Ca.sup.2+-flux assay. Robust CXCR4 activation was observed with
both wild-type CXCL12-.alpha. (EC.sub.50=3.6 nM) and
CXCL12-.alpha..sub.2 (EC.sub.50=12.9 nM) (FIG. 5A). AMD3100, a
small-molecule CXCR4 antagonist, inhibited both proteins with
IC.sub.50 values of 3.3 nM (CXCL12-A) and 3.2 nM
(CXCL12-.alpha..sub.2), demonstrating that the observed calcium
flux responses were mediated by CXCR4. Thus, the inventors data
shows that CXCL12-.alpha..sub.2 binds and activates its cognate
receptor (see Example 7).
[0087] Chemotaxis of the CXCL12-.alpha..sub.2 locked dimer. Results
from Ca.sup.2+-flux assays collectively suggest that CC and CXC
chemokine dimers behave differently. For instance, CC chemokines
form dimers that cannot activate their cognate GPCRs, while CXC
chemokine dimers are functionally indistinguishable from their
monomeric counterparts. In contrast to wild-type CXCL12-.alpha.,
the constitutively dimeric CXCL12-.alpha..sub.2 of the present
invention failed to attract cells in a transwell chemotaxis assay
even at concentrations up to 1 .mu.M (FIG. 5B). Accordingly, the
inventors results demonstrate that at low chemokine concentrations
monomeric CXCL12-.alpha. stimulates chemotaxis, while at higher
concentrations dimeric CXCL12-.alpha..sub.2 halts cell migration
corresponding to the second, downward half of the bell-shaped curve
(see Example 8).
[0088] The activation of CXCR4 in the calcium flux assay by
CXCL12-.alpha..sub.2 and the inhibition of chemotaxis brings up the
question of CXCL12-.alpha..sub.2 CXCR4 stoichiometry. CXCR4 has
been purified as a homodimer, the CXCR4 N-terminus promotes
CXCL12-.alpha. dimer formation and the inventors' structures show
two CXCR4 N-termini bound to CXCL12-.alpha..sub.2. Nevertheless,
the role of homo- and heterodimers in GPCR signaling remains
controversial. Some place a high significance on the role of dimer
formation in signaling, while others discount evidence of
dimerization as an experimental artifact.
[0089] Inspection of the high resolution, dimeric crystal structure
of .beta..sub.2AR, a type A GPCR like CXCR4 suggests that formation
of a 2:2 CXCL12-.alpha.:CXCR4 complex is plausible. The ligand
binding sites of .beta..sub.2 adrenergic receptor monomers are
separated by approximately 42 .ANG., which is the distance between
the N-termini in a CXCL12-.alpha..sub.2 dimer. The N-terminus of
CXCL12-.alpha. activates the CXCR4 receptor and thus is the region
of CXCL12-.alpha. that corresponds to small molecule agonists of
GPCRs like .beta..sub.2AR.
[0090] Additionally, it is reasonable to propose that wild-type
CXCL12 concentrations near the IC.sub.50 for chemotaxis prevention
are physiologically plausible. Here the inventors show that the
locked CXCL12-.alpha..sub.2 dimer of the present invention can
inhibit chemotaxis with an IC.sub.50 of 4 nM. In the presence of
the CXCR4 N-terminus the CXCL12-.alpha. dimer dissociation K.sub.d
is 49 .mu.M, which equates to a 4 nM dimer CXCL12-.alpha..sub.2
concentration at a total CXCL12-.alpha. concentration of 300-400
nM. If interactions with the full-length receptor or
glycosaminoglycans further enhance CXCL12-.alpha. self-association
to yield a K.sub.d of 1 CXCL12-.alpha..sub.2 dimer concentrations
will approach 4 nM when total CXCL12-.alpha. is 50 nM, a
concentration within the physiological range.
[0091] The structure of CXCL12-.alpha. with CXCR4 provides
important knowledge on the role of sulfotyrosine recognition by
chemokines. The inventors' results also continue to highlight the
need to address the role of chemokine oligomers in chemotaxis and
show the importance of investigating each chemokine on an
individual basis, since the CXCL12-.alpha..sub.2 dimer can bind its
receptor while MIP-1.beta. dimer cannot.
[0092] Additionally, both wild-type, preferentially monomeric
CXCL12-.alpha. H25R and dimeric CXCL12-.alpha..sub.2 activate
CXCR4, generating the secondary messenger calcium. However, only
wild-type CXCL12-.alpha. and CXCL12-.alpha. H25R can produce
chemotaxis. This suggests the oligomeric state of CXCL12-.alpha.
controls certain intracellular signals that either lead to or
prevent chemotaxis. Identification of the specific signaling
pathways affected by dimeric CXCL12-.alpha..sub.2 will improve
understanding of cell migration and may suggest intracellular
targets for the prevention of cancer metastasis.
[0093] The dimeric CXCL12 blocks the normal agonistic activity of
CXCL12 but does not necessarily prevent the internalization or
so-called down-regulation of the target CXCR4 receptor. Hence,
CXCL12.alpha..sub.2 locked dimer not only blocks the chemotactic
effect of CXCL12 but also the effective concentration of the CXCR4
receptor on the cell surface decreases. Thus, CXCL12.alpha..sub.2
locked dimer should be expected to display a high degree of
efficacy, even when compared to standard CXCR4 antagonists, which
may block agonist activity, but fail to decrease receptor
number.
Example 1
Materials, Methods and Instrumentation
[0094] The production of sY.sub.1 p38 has been explained and
Y.sub.3 p38 was generated in a similar manner. Samples for
structure elucidation consisted of U-[.sup.15N, .sup.13C]
CXCL12-.alpha..sub.2 with unlabeled peptide and U-[.sup.15N,
.sup.13C] peptide with unlabeled CXCL12-.alpha..sub.2 at a 1:1.25
molar ratio of labeled to unlabeled monomers. Standard NMR
techniques were use for generating chemical shift assignments for
.sup.15N/.sup.13C labeled CXCL12-.alpha..sub.2, p38, sY.sub.1 p38
and sY.sub.1 p38. 3D .sup.15N-edited NOESY-HSQC, .sup.13C-edited
NOESY-HSQC, and .sup.13C(aromatic)-edited NOESY-HSQC spectra
(.tau..sub.mix=80 ms) were used to generate distance constraints. A
3D F1-.sup.13C-filtered/F3-.sup.13C-edited NOESY-HSQC spectrum
(.tau..sub.mix=120 ms) was used for obtaining intermolecular
distance constraints.
[0095] TALOS and the secondary shifts of the .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.13C.sup..beta., .sup.13C', and .sup.15N
nuclei generated backbone phi and psi dihedral angle constraints.
The NOEASSIGN module of the torsion angle dynamics program CYANA
with intermolecular constraints defined was used to calculate the
initial structures in an automated manner. Iterative manual
refinement followed to eliminate constraint violations generating
twenty conformers with the lowest target function. X-PLOR was used
for further refinement, in which physical force field terms and
explicit water solvent molecules were added to the experimental
constraints. Tables 1-4 list the statistics for Procheck-NMR
validation of the final twenty conformers.
TABLE-US-00001 TABLE 1 Statistics for the 20
CXCL12-.sub..alpha..sub.2 L36C A65C conformers Experimental
constraints Distance constraints Long Intra-CXCL12-.alpha. monomer
857 Inter-CXCL12-.alpha. monomers 113 Medium [1 < (i - j)
.ltoreq. 5] 300 Sequential [(i - j) = 1] 312 Intraresidue [i = j]
692 Total 2274 Dihedral angle constraints (.phi. and .psi.) 138
Average atomic R.M.S.D. to the mean structure (.ANG.) Residues
Backbone (C.sup..alpha., C', N) 0.51 .+-. 0.05 Heavy atoms 1.05
.+-. 0.12 Deviations from idealized covalent geometry .sup.a Bond
lengths RMSD (.ANG.) 0.017 Torsion angle violations RMSD (.degree.)
1.4 WHATCHECK quality indicators Z-score -1.52 .+-. 0.23 RMS
Z-score Bond lengths 0.78 .+-. 0.02 Bond angles 0.76 .+-. 0.02
Bumps 0 .+-. 0 Lennard-Jones energy .sup.b (kJ mol.sup.-1) -2927.8
.+-. 108.0 Constraint violations .sup.c, d NOE distance Number >
0.5 .ANG. 0 .+-. 0 NOE distance RMSD (.ANG.) 0.0251 .+-. 0.0013
Torsion angle violations Number > 5.degree. 0.05 .+-. 0.22
Torsion angle violations RMSD (.degree.) 0.8273 .+-. 0.1190
Ramachandran statistics (% of all residues) Most favored 81.74 .+-.
2.59 Additionally allowed 14.91 .+-. 3.05 Generously allowed 1.76
.+-. 1.03 Disallowed 1.56 .+-. 1.23 .sup.a Final X-PLOR force
constants were 250 (bonds), 250 (angles), 300 (impropers), 100
(chirality) and 100 (omega), 50 (NOE constraints), and 200 (torsion
angle constraints). .sup.b Nonbonded energy was calculated in
XPLOR-NIH. .sup.c The largest NOE violation in the ensemble of
structures was 0.355 .ANG.. .sup.d The largest torsion angle
violations in the ensemble of structures was 3.9.degree..
TABLE-US-00002 TABLE 2 Statistics for the 20
CXCL12-.sub..alpha..sub.2 L36C A65C with CXCR4 P38 C28A conformers
Experimental constraints Distance constraints Long Intra-subunit
444 Inter-CXCL12-.alpha. monomers 110 Intermolecular
(CXCL12-.alpha. to peptide) 92 Medium [1 < (i - j) .ltoreq. 5]
238 Sequential [(i - j) = 1] 384 Intraresidue [i = j] 744 Total
2012 Dihedral angle constraints (.phi. and .psi.) 128 Average
atomic R.M.S.D. to the mean structure (.ANG.) Residues .sup.a
Chemokine Backbone (C.sup..alpha., C', N) 0.71 .+-. 0.07 Chemokine
Heavy atoms 1.24 .+-. 0.10 Total Backbone (C.sup..alpha., C', N)
1.80 .+-. 0.24 Total Heavy atoms 2.26 .+-. 0.20 Deviations from
idealized covalent geometry .sup.b Bond lengths RMSD (.ANG.) 0.017
Torsion angle violations RMSD (.degree.) 1.5 WHATCHECK quality
indicators Z-score -3.47 .+-. 0.32 RMS Z-score Bond lengths 0.81
.+-. 0.03 Bond angles 0.79 .+-. 0.03 Bumps 0 .+-. 0 Lennard-Jones
energy .sup.c (kJ mol.sup.-1) -4620.1 .+-. 142.7 Constraint
violations .sup.d NOE distance Number > 0.5 .ANG. 0 .+-. 0 NOE
distance RMSD (.ANG.) 0.0276 .+-. 0.0017 Torsion angle violations
Number > 5.degree. 0.1 .+-. 0.45 Torsion angle violations RMSD
(.degree.) 0.8400 .+-. 0.1693 Ramachandran statistics (% of all
residues) Most favored 71.30 .+-. 3.37 Additionally allowed 23.25
.+-. 3.08 Generously allowed 3.10 .+-. 1.09 Disallowed 2.38 .+-.
1.34 .sup.a 20 structure in the ensemble were aligned using
residues 9-43 and 47-66 of the chemokine and 11-27 or the peptide.
Chemokine RMSD includes residues 9-43, 47-66. Total RMSD includes
the chemokine residues plus peptide residues 11-27. .sup.b Final
X-PLOR force constants were 250 (bonds), 250 (angles), 300
(impropers), 100 (chirality) and 100 (omega), 50 (NOE constraints),
and 200 (torsion angle constraints). .sup.c Nonbonded energy was
calculated in XPLOR-NIH. .sup.d The largest NOE violation in the
ensemble of structures is .ANG..
TABLE-US-00003 TABLE 3 Statistics for the 20
CXCL12-.sub..alpha..sub.2 L36C A65C with sY21 CXCR4 P38 C28A
conformers Experimental constraints Distance constraints Long
Intra-subunit 418 Inter-CXCL12-.alpha. monomers 116 Intermolecular
(CXCL12-.alpha. to peptide) 92 Medium [1 < (i - j) .ltoreq. 5]
246 Sequential [(i - j) = 1] 470 Intraresidue [i = j] 728 Total
2070 Dihedral angle constraints (.phi. and .psi.) 128 Average
atomic R.M.S.D. to the mean structure (.ANG.) Residues .sup.a
Chemokine Backbone (C.sup..alpha., C', N) 0.60 .+-. 0.10 Chemokine
Heavy atoms 1.05 .+-. 0.11 Total Backbone (C.sup..alpha., C', N)
0.82 .+-. 0.09 Total Heavy atoms 1.37 .+-. 0.10 Deviations from
idealized covalent geometry .sup.b Bond lengths RMSD (.ANG.) 0.018
Torsion angle violations RMSD (.degree.) 1.6 WHATCHECK quality
indicators Z-score -3.34 .+-. 0.21 RMS Z-score Bond lengths 0.83
.+-. 0.02 Bond angles 0.83 .+-. 0.03 Bumps 0 .+-. 0 Lennard-Jones
energy .sup.c (kJ mol.sup.-1) -4640.2 .+-. 199.2 Constraint
violations .sup.d NOE distance Number > 0.5 .ANG. 0 .+-. 0 NOE
distance RMSD (.ANG.) 0.0324 .+-. 0.0016 Torsion angle violations
Number > 5.degree. 0.0 .+-. 0.0 Torsion angle violations RMSD
(.degree.) 0.7907 .+-. 0.1300 Ramachandran statistics (% of all
residues) Most favored 70.21 .+-. 2.72 Additionally allowed 24.29
.+-. 2.80 Generously allowed 3.71 .+-. 1.18 Disallowed 1.74 .+-.
1.02 .sup.a 20 structure in the ensemble were aligned using
residues 9-43 and 47-66 of the chemokine and 11-27 or the peptide.
Chemokine RMSD includes residues 9-43, 47-66. Total RMSD includes
the chemokine residues plus peptide residues 11-27. .sup.b Final
X-PLOR force constants were 250 (bonds), 250 (angles), 300
(impropers), 100 (chirality) and 100 (omega), 50 (NOE constraints),
and 200 (torsion angle constraints). .sup.c Nonbonded energy was
calculated in XPLOR-NIH. .sup.d The largest NOE violation in the
ensemble of structures was 0.47 .ANG..
TABLE-US-00004 TABLE 4 Statistics for the 20
CXCL12-.sub..alpha..sub.2 L36C A65C with sY7-12-21 CXCR4 P38 C28A
conformers. Experimental constraints Distance constraints Long
Intra-subunit 420 Inter-CXCL12-.alpha. monomers 116 Intermolecular
(CXCL12-.alpha. to peptide) 86 Medium [1 < (i - j) .ltoreq. 5]
238 Sequential [(i - j) = 1] 456 Intraresidue [i = j] 722 Total
2092 Dihedral angle constraints (.phi. and .psi.) 128 Average
atomic R.M.S.D. to the mean structure (.ANG.) Residues .sup.a
Chemokine Backbone (C.sup..alpha., C', N) 0.64 .+-. 0.07 Chemokine
Heavy atoms 1.10 .+-. 0.09 Total Backbone (C.sup..alpha., C', N)
1.01 .+-. 0.16 Total Heavy atoms 1.56 .+-. 0.017 Deviations from
idealized covalent geometry .sup.b Bond lengths RMSD (.ANG.) 0.017
Torsion angle violations RMSD (.degree.) 1.5 WHATCHECK quality
indicators Z-score -3.60 .+-. 0.25 RMS Z-score Bond lengths 0.78
.+-. 0.02 Bond angles 0.80 .+-. 0.02 Bumps 0 .+-. 0 Lennard-Jones
energy .sup.c (kJ mol.sup.-1) -4766.8 .+-. 145.4 Constraint
violations .sup.d NOE distance Number > 0.5 .ANG. 0 .+-. 0 NOE
distance RMSD (.ANG.) 0.0260 .+-. 0.0017 Torsion angle violations
Number > 5.degree. 0 .+-. 0 Torsion angle violations RMSD
(.degree.) 0.7380 .+-. 0.1314 Ramachandran statistics (% of all
residues) Most favored 74.03 .+-. 2.65 Additionally allowed 22.13
.+-. 2.70 Generously allowed 2.15 .+-. 1.01 Disallowed 1.72 .+-.
1.22 .sup.a 20 structure in the ensemble were aligned using
residues 9-43 and 47-66 of the chemokine and 11-27 or the peptide.
Chemokine RMSD includes residues 9-43, 47-66. Total RMSD includes
the chemokine residues plus peptide residues 11-27. .sup.b Final
X-PLOR force constants were 250 (bonds), 250 (angles), 300
(impropers), 100 (chirality) and 100 (omega), 50 (NOE constraints),
and 200 (torsion angle constraints). .sup.c Nonbonded energy was
calculated in XPLOR-NIH. d The largest NOE violation in the
ensemble of structures was 0.407 .ANG..
[0096] The Protein Data Bank (PDB), under accession numbers 2K01,
2K04, 2K03 and 2K05 contain coordinates for these structural
models. Restraints employed for structure determination have been
deposited in the Biological Magnetic Resonance Bank, accession
numbers 15633, 15636, 15635 and 15637. Standard calcium flux assays
were used for testing CXCR4 activation and transwell chemotaxis
assays were used to compare the chemotactic response of THP-1 cells
towards wild-type CXCL12-.alpha., CXCL12-.alpha. H25R and
CXCL12-.alpha..sub.2. THP-1 cells are a CXCR4-expressing monocyte
leukemia cell line and were obtained from ATCC.
Example 2
Preparing the CXCL12-.alpha..sub.2 Locked Dimer Polypeptide
[0097] In this Example, the inventors prepared the
CXCL12-.alpha..sub.2 locked dimer structure. Guided by the
CXCL12-.alpha. crystal structure, the inventors identified L36 and
A65 as residues at the dimer interface that could be replaced with
intermolecular disulfide bonds (FIG. 1A). The CXCL12-.alpha.
(L36C/A65C) double mutant was expressed and purified from E. coli
as previously described for wild-type CXCL12-.alpha., and migrated
as a stable dimer in non-reducing SDS-PAGE (FIG. 1B). Pulsed-field
gradient NMR diffusion measurements indicated that CXCL12-.alpha.
(L36C/A65C) is dimeric, even in solution conditions that favor the
monomeric state (FIG. 1C).
[0098] The inventors confirmed the presence of disulfide bonds
linking the two monomers and solved the structure of the
CXCL12-.alpha..sub.2 (L36C/A65C) by NMR. The covalently-locked,
symmetric CXCL12-.alpha. (L36C/A65C) dimer (CXCL12-.alpha..sub.2)
is superimposable with the wild-type CXCL12-.alpha. dimer observed
crystallographically (FIG. 2A). Table 1 lists refinement statistics
for the CXCL12-.alpha..sub.2 structure ensemble.
CXCL12-.alpha..sub.2 also displays the canonical chemokine fold in
which a flexible N-terminus is connected by the N-loop to a
three-stranded antiparallel .beta.-sheet and a C-terminal
.alpha.-helix.
Example 3
Detection of 2:1 p38: CXCL12-.alpha..sub.2 Binding by NMR Chemical
Shift Mapping
[0099] In this Example, the inventors determined if the NMR
broadening arises from exchange between different
CXCL12-.alpha.:p38 complexes. Previously, the inventors noted that
binding of p38 (FIG. 5D) to .sup.15N labeled CXCL12-.alpha. induced
chemical shift perturbations attributable to a combination of
CXCL12-.alpha. dimer formation and peptide binding. Titration of
.sup.15N p38 with CXCL12-.alpha. showed extreme line broadening.
Based on the inventors' studies of the CXCL12-.alpha. monomer-dimer
equilibrium, the inventors investigated that the NMR broadening
arises from exchange between different CXCL12-.alpha.:p38
complexes.
[0100] When both CXCL12-.alpha. dimerization and p38 binding are
considered, a complex set of coupled binding equilibria permits
exchange between complexes with stoichiometries of 1:1, 1:2, 2:1
and 2:2. Because the locked dimer reduces the number of accessible
states, interpretation of NMR spectra of CXCL12-.alpha..sub.2 upon
p38 binding is straightforward. Titration of .sup.15N labeled
CXCL12-.alpha..sub.2 with p38 (FIG. 2B) perturbs NMR signals for
N-loop residues but not the dimer interface (FIG. 2C), thus
identifying likely CXCR4-CXCL12-.alpha. binding determinants.
Because only one set of CXCL12-.alpha..sub.2 signals is observed
during the p38 titration and addition of more than two molar
equivalents of p38 induces no further chemical shift perturbations,
the inventors concluded that a symmetric 2:1 p38:
CXCL12-.alpha..sub.2 complex was formed.
Example 4
Structure Analysis of CXCL12-.alpha..sub.2 Locked Dimer
Sulfotyrosine Recognition Sites
[0101] In this Example, the inventors measured the EC.sub.50 of
each protein using a Ca.sup.2+-flux assay on CXCR4-expressing THP-1
cells to assess the relative contribution of each sulfotyrosine to
CXCL12-.alpha.:CXCR4 binding. The CXCL12-.alpha.:p38 interaction
contributes only to binding affinity and receptor specificity, but
not to CXCR4 activation. In contrast, a peptide consisting of
CXCL12-.alpha. residues 1-8 can fully activate CXCR4 at micromolar
concentrations. Since each CXCL12-.alpha. variant retains the
native N-terminus, the EC.sub.50 value reflects its affinity for
CXCR4. Consequently, an amino acid substitution that alters the
Ca.sup.2+-flux EC.sub.50 relative to wild-type CXCL12-.alpha.
(3.6.+-.1.4 nM) has necessarily disrupted an interaction between
the chemokine and the N-terminus or extracellular loops of CXCR4.
Overall, mutations in wild-type CXCL12-.alpha. that alter
interactions observed in the CXCL12-.alpha..sub.2:p38 complexes
resulted in higher EC.sub.50 values for CXCR4 activation
corresponding to a loss of CXCL12-.alpha. binding affinity (Table
5). However, comparison of the results for each binding site
reveals a hierarchy among CXCR4 sulfotyrosines.
[0102] NOE constraints from valine 23 of one CXCL12-.alpha..sub.2
subunit position the sY7 O-sulfonate to form a favorable
electrostatic interaction with a positively-charged arginine side
chain (FIG. 4B), but replacing R20 with alanine in wild-type
CXCL12-.alpha. produced no change in EC.sub.50 (Table 5).
TABLE-US-00005 TABLE 5 CXCR4 activation by CXCL12-.alpha. mutants.
Fold EC.sub.50 (nM) Folded Increase p38 contact CXCL12-.alpha. 3.6
.+-. 1.4 + R20A 4.3 .+-. 0.6 + 1.2 + V23A NA - NA + H25R 5.1 .+-.
0.9 + 1.4 - K27A 10.1 .+-. 2.9 + 2.8 + K27E 16.8 .+-. 1.1 + 4.7 +
V39A 27.1 .+-. 0.2 + 7.5 + R41A 4.3 .+-. 0.9 + 1.2 - R47A 14.1 .+-.
0.6 + 3.9 + R47E 654 .+-. 93 + 181.7 + V49A 8.6 .+-. 2.4 + 2.4 +
E60A 4.1 .+-. 0.1 + 1.1 - E63A 3.7 .+-. 0.8 + 1.0 - K64A 5.0 .+-.
1.1 + 1.4 -
[0103] In a similar fashion, NOEs connect sY12 to P10 and L29 of
the other CXCL12-.alpha..sub.2 subunit and place the sulfotyrosine
within approximately 3 .ANG. of the positively charged amino group
of K27 (FIG. 4C). Substitutions of alanine and glutamic acid at
this position in wild-type CXCL12-.alpha. increased the EC.sub.50
to 10.1 and 16.8 nM, respectively. Alanine substitution of a
structurally adjacent valine residue (V39A) increased the EC.sub.50
to 27.1 nM.
[0104] Residues connecting the N-terminal CXC motif with .beta.1 of
CXCL12-.alpha. (the `N loop`), particularly the RFFESH motif
consisting of residues 12-17, were predicted from mutagenic studies
to interact with the CXCR4 N-terminus. The inventors observed
intermolecular NOEs between .sup.1H.sup.N of F14 in
CXCL12-.alpha..sub.2 and the .sup.1H.sup..alpha. of G19 from CXCR4
and from V18 in the chemokine to sY21. NOEs also link sY21 with
V49, located in the .beta.3 strand of CXCL12-.alpha..sub.2, and
position the sY21 O-sulfonate <5 .ANG. from the R47 guanidinium
(FIG. 4D), consistent with our earlier measurement of
sulfotyrosine-specific chemical shift perturbations. CXCL12-.alpha.
R47A has an EC.sub.50 of 14.1 nM, and replacement of the positive
arginine side chain with a negatively charged glutamic acid
drastically alters CXCL12-.alpha. binding (R47E EC.sub.50=654 nM)
relative to wild-type CXCL12-.alpha. (EC.sub.50=3.6 nM).
[0105] The level of sulfation for each CXCR4 tyrosine has not been
characterized in THP-1 cells, but Farzan et al. suggested that
CXCR4 Y21 is sulfated to higher degree than Y7 or Y12 and that sY21
contributes the most to CXCL12-.alpha. binding affinity. This is
consistent with the results herein which suggest the sY7 and sY12
binding sites contribute only modestly to the overall interaction.
The binding pocket for sY21 in CXCL12-.alpha. appears to be well
conserved within the CXC chemokine family with 8 out of 16 CXC
chemokines showing high conservation or identity at CXCL12-.alpha.
positions 18, 47 and 49. With the exception of CXCR6, a tyrosine
corresponding to sulfotyrosine 21 of CXCR4 appear to be present in
all receptors of the CXC family. Neither sY7, sY12 nor their
putative binding sites are conserved in the CXC ligands or
receptors.
Example 5
Structure of CXCL12-.alpha..sub.2 Locked Dimer Bound to Sulfated
p38 Peptides
[0106] In this Example, the inventors solved structures of
unsulfated, selectively sulfated and fully sulfated CXCR4 peptides
bound to the CXCL12-.alpha..sub.2 locked dimer polypeptide to
understand the role of sulfotyrosine in CXCL12-a-CXCR4 binding.
[0107] Tyrosine sulfation in the CXCR4 N-terminal domain
contributes substantially to CXCL12-.alpha. binding. The inventors
showed previously that sulfation of Tyr 21 enhances the affinity of
p38 for CXCL12-.alpha. by approximately 3-fold, and the inventors
observed that fully-sulfated p38-sY.sub.3 binds approximately
20-fold more tightly than the unsulfated peptide (apparent
K.sub.d=0.2.+-.0.2 .mu.M).
[0108] Recombinant [U-.sup.15N, .sup.13C]-labeled CXCR4 peptide
(p38) was modified using purified tyrosyl protein sulfotransferase
to contain sulfotyrosine at position 21 (p38-sY.sub.1) or positions
7, 12 and 21 (p38-sY.sub.3) (FIG. 1D). For each complex, NOEs
between CXCL12-.alpha..sub.2 and the (sulfo)tyrosine side chains of
CXCR4 unambiguously defined the same arrangement of both p38
peptides on the chemokine as shown in FIG. 3 with representative
intermolecular NOEs in FIG. 1B.
[0109] Two p38 molecules bind in equivalent orientations with each
peptide wrapping around the symmetric CXCL12-.alpha..sub.2 dimer in
an extended conformation (FIG. 2D). When mapped onto the
CXCL12-.alpha..sub.2 surface, p38-induced chemical shift
perturbations (FIG. 2D, green surface) correlate strongly with the
observed binding interface. In contrast, residues of the flexible
N-terminus and C-terminal a-helix of CXCL12-.alpha..sub.2 were
unperturbed by p38 binding and do not interact with the CXCR4
N-terminus.
[0110] CXCR4 stabilizes the CXCL12-.alpha. dimer by interacting
with both subunits and recognizing unique features of the dimer
interface. Near the CXCR4 N-terminus, each p38 peptide crosses the
CXCL12-.alpha..sub.2 dimer interface, such that sY7 and sY12
interact with opposing CXCL12-.alpha. monomers. In the
membrane-proximal portion of the CXCR4 N-terminal domain, P27
inserts between Q59 of one CXCL12-.alpha..sub.2 subunit and L66 of
the opposing subunit, where the C-terminal helices from each
monomer pack against each other.
Example 6
Inhibitory Effect of CXCL12-.alpha..sub.2 Locked Dimer on Cell
Migration
[0111] In this example, the inventors conducted chemotaxis assays
using a C-CXCL12-.alpha. mutant that remains monomeric at higher
concentrations than wild-type CXCL12-.alpha.. Since the dimer
K.sub.d of CXCL12-.alpha. (H25R) is approximately 10-fold higher
than wild-type, it should resist inactivation due to dimerization
and maintain a chemotactic response at higher concentrations where
the wild-type CXCL12-.alpha. loses activity. Both proteins induce a
dose-dependent chemotactic response from 1-30 nM, but
CXCL12-.alpha.(H25R) promotes cell migration much more strongly
than the wild-type chemokine at higher concentrations (70-100 nM)
before returning to baseline levels (FIG. 5C).
[0112] The inventors data shows that CXCL12-.alpha..sub.2 inhibits
chemotaxis induced by wild-type CXCL12-.alpha.. FIG. 5D shows
CXCL12-.alpha..sub.2 inhibits chemotaxis induced by wild-type
CXCL12-.alpha. with an IC.sub.50 of approximately 4 nM. The
inventors' results show that monomer CXCL12-.alpha. activates cell
migration while dimer CXCL12-.alpha. halts cell migration. The
results also demonstrate that CXCL12-.alpha..sub.2 acts as a
partial CXCR4 agonist (as evidenced by the detection of the
secondary messenger calcium) and as a selective antagonist that
blocks chemotaxis. This indicates CXCL12-.alpha..sub.2 can serve as
a tool for correlating cellular responses with different
intracellular signaling pathways initiated by CXCR4. Also,
CXCL12-.alpha..sub.2 may be useful in the development of
anti-metastatic agents by preventing CXCL12-.alpha./CXCR4-mediated
migration of circulating cancer cells.
Example 7
Physiological Relevance of the CXCL12-.alpha..sub.2 Locked
Dimer
[0113] In this Example, the inventors next investigated whether
CXCL12-.alpha..sub.2 locked dimers participate in CXCR4 signaling.
Like most chemokines, CXCL12-.alpha. self-association occurs well
above the concentrations required for receptor binding and
activation (1-10 nM). Consequently, chemokine dimers are considered
relevant mainly in the context of glycosaminoglycan binding for
immobilization in the extracellular matrix. Debate over the
functional role of chemokine dimers in vivo is complicated by
conflicting results obtained on a variety of the >40 different
chemokine proteins.
[0114] The inventors monitored activation of CXCR4 using THP-1
cells and a similar Ca.sup.2+-flux assay. Robust CXCR4 activation
was observed with both wild-type CXCL12-.alpha. (EC.sub.50=3.6 nM)
and CXCL12-.alpha..sub.2 (EC.sub.50=12.9 nM) (FIG. 5A). AMD3100, a
small-molecule CXCR4 antagonist, inhibited both proteins with
IC.sub.50 values of 3.3 nM (CXCL12-A) and 3.2 nM
(CXCL12-.alpha..sub.2), demonstrating that the observed calcium
flux responses were mediated by CXCR4. Thus, the inventors data
shows that CXCL12-.alpha..sub.2 binds and activates its cognate
receptor.
Example 8
Chemotactic Response of the CXCL12-.alpha..sub.2 Locked Dimer
Polypeptide
[0115] In this Example, the inventors used standard transwell
chemotaxis assays to compare the chemotactic response of THP-1
cells towards wild-type CXCL12-.alpha. and the CXCL12-.alpha..sub.2
locked dimer polypeptide of the present invention. As expected, the
THP-1 cells responded chemotactically when exposed to wild-type
CXCL12-.alpha. in the 1-30 nM range, but migration decreases and
ultimately ceases at higher chemokine concentrations (FIG. 5B).
[0116] In contrast to wild-type CXCL12-.alpha., the constitutively
dimeric CXCL12-.alpha..sub.2 of the present invention failed to
attract cells in a transwell chemotaxis assay even at
concentrations up to 1 .mu.M (FIG. 5B). Accordingly, the inventors
results demonstrate that at low chemokine concentrations monomeric
CXCL12-.alpha. stimulates chemotaxis, while at higher
concentrations CXCL12-.alpha..sub.2 halts cell migration
corresponding to the second, downward half of the bell-shaped curve
(see Example 8).
Example 9
Prophetic Example on the Inhibitory Effect of the
CXCL12-.alpha..sub.2 Locked Dimer on Tumor Growth
[0117] In this Example, the inventors describe how one would used
the inhibitory effect of the CXCL12-.alpha..sub.2 locked dimer of
the present invention to affect tumor growth. Primary tumors are
easier to treat as compared to cancer which has spread through the
body and formed secondary tumors or metastases. It has long been
observed that secondary tumors in metastatic cancer patients form
preferentially in a subset of tissues, including bone marrow, lymph
nodes, liver, and lungs. At least three theories have been proposed
to explain these observations, including: certain tissues are
better environments for metastatic cancer cell survival; the
vasculature of some tissues express adhesion molecules that bind
metastatic cells better than others; or there is active recruitment
of metastatic cells out of the blood stream or lymphatic system
only to certain locations.
[0118] Patterns of CXCR4 and CXCL12 expression and signaling
suggest that metastatic cancer cells are actively recruited to
tissues producing CXCL12. An increase in CXCR4 expression
accompanies the transition from a primary tumor cell to a
metastatic cancer cell, and CXCR4 levels have been correlated with
metastasis and poor patient outcomes in many different cancer
types. These metastatic, CXCR4-expressing cancer cells break away
from the primary tumor and enter the circulation where they
systematically target tissues constitutively expressing CXCL12, the
only natural ligand for CXCR4, like bone marrow, lymph nodes,
liver, and lungs. It is thought that this targeting occurs in a
manner analogous to the recruitment of a circulating leukocyte to
an infection site.
[0119] CXCL12- and CXCR4-directed localization of metastatic cancer
cells has been implicated in a broad range of over twenty cancer
types, including: breast, prostate, colon, myeloma, melanoma,
tongue, ovarian, small and non-small cell lung cancers, pancreatic,
esophageal, head and neck, bladder, osteosarcoma, neuroblastoma,
and leukemia.
[0120] The inventors have shown that the CXCL12-.alpha..sub.2
locked dimer of the present invention is a potent inhibitor of
CXCL12/CXCR4 mediated chemotaxis of a THP1 cells (a leukemia cell
line) (FIG. 5E). This CXCL12/CXCR4 mediated chemotaxis or cell
migration is required for CXCL12/CXCR4 directed cancer metastasis.
Based on the potent inhibition of CXCL12 induced THP1 cell
chemotaxis by CXCL12-.alpha..sub.2, the inventors predict that the
CXCL12-.alpha..sub.2 locked dimer of the present invention will
prevent CXCL12-induced chemotaxis (or cell migration) and thus
metastasis of cancer cells that express CXCR4. The inventors
therefore predict that blockade of the CXCL12/CXCR4-directed
metastasis with the CXCL12-.alpha..sub.2 locked dimer of the
present invention will prevent or reduce the formation of secondary
tumors or metastases. For example, a person diagnosed with breast
cancer could be treated with CXCL12-.alpha..sub.2 to prevent
metastasis before and after surgical removal of the primary tumor.
Continued CXCL12-.alpha..sub.2 administration could improve the
efficacy of subsequent chemotherapy or radiation treatments by
preventing circulating cancer cells from migrating to tissues and
organs that would normally serve as a preferred location for
metastatic cancer growth. The inventors predict that fewer
recurrences or metastases would occur after a successful initial
treatment of breast cancer. Pancreatic cancer has a high mortality
rate and kills quickly, largely because pancreatic cancer
metastasizes rapidly in a CXCL12/CXCR4 dependant manner. In a
manner analogous to breast cancer the inventors predict that
treatment with CXCL12-.alpha..sub.2 would increase the life
expectancy of a patient with pancreatic cancer by slowing the
spread of metastatic disease.
Example 10
Prophetic Example on the Inhibitory Effect of the
CXCL12-.alpha..sub.2 Locked Dimer on Angiogenisis
[0121] In this example, the inventors show how one would use the
inhibitory effect of the CXCL12-.alpha..sub.2 locked dimer of the
present invention to inhibit angiogenesis. Angiogenesis is the
formation of new blood vessels that penetrate a tissue and supply
that tissue with oxygen and nutrients while also removing waste.
Usually the formation of new blood vessels results from expansion
or growth of existing blood vessels through the growth of vascular
endothelial cells. The vascular endothelial cells that line blood
vessels rarely divide but there are cues that can induce or inhibit
growth. When the expansion cues outweigh the inhibiting cues,
angiogenesis occurs.
[0122] CXCL12 expression is increased in tissues that are hypoxic
(or lack oxygen) due to a lack of vascularization (or lack of blood
vessels and blood supply). CXCL12 is also a chemoattractant that
functions to attract the newly forming blood vessels into the
hypoxic tissue. Angiogenesis is important for the progression of
numerous disease states and inhibition of angiogenesis by
CXCL12.alpha..sub.2 may be therapeutically useful in the prevention
or progression of diseases conditions including but not limited to
diabetic retinopathy, macular degeneration, rheumatoid arthritis,
inflammatory bowel disease, cancer, psoriasis, osteoarthritis,
ulcerative colitis, Crohn's disease and coronary thrombosis.
Example 11
Prophetic Example on the Inhibitory Effect of the
CXCL12-.alpha..sub.2 Locked Dimer on Autoimmune Diseases
[0123] In this example, the inventors show how one would use the
inhibitory effect of the CXCL12-.alpha..sub.2 locked dimer of the
present invention to inhibit autoimmune diseases.
[0124] Autoimmune diseases occur when a body's immune system
attacks its own cells and tissues in addition to or instead of
things foreign. CXCL12 is a chemokine and like other chemokines is
involved in regulating immune cell trafficking and the immune
system in general. As such, CXCL12 plays roles in recruiting immune
cells during an autoimmune reaction. Rheumatoid arthritis is an
example of such an autoimmune disease. In rheumatoid arthritis
CXCL12 recruits T-cells to joints where the recruited T-cells
orchestrate the generation of immunologically driven inflammation.
Therefore, preventing the migration and recruitment of T-cells to
the synovium of joints with the CXCL12.alpha..sub.2 locked dimer of
the present invention may prevent or reduce the inflammation
associated with rheumatoid arthritis.
Example 12
Prophetic Example on the Inhibitory Effect of the
CXCL12-.alpha..sub.2 Locked Dimer on HIV/AIDS
[0125] In this example, the inventors show how one would use the
inhibitory effect of the CXCL12-.alpha..sub.2 locked dimer of the
present invention to inhibit HIV/AIDS. In this example, the
inventors show how one would also evaluate the effect of
combination therapies using the inhibitory effect of the
CXCL12-.alpha..sub.2 locked dimer of the present invention to treat
HIV/AIDS.
[0126] Human immunodeficiency virus (HIV) is a retrovirus that can
lead to acquired immunodeficiency syndrome (AIDS), a condition in
humans in which the immune system begins to fail, leading to
life-threatening opportunistic infections. Infection with HIV
occurs by the transfer of contaminated bodily fluids such as blood,
semen, vaginal fluid, pre-ejaculate, or breast milk. Within these
bodily fluids, HIV is present as both free virus particles and
virus within infected immune cells. CXCR4 and/or CCR5 along with
CD4 are coreceptors that HIV uses when infecting cells. There are
different strains of HIV. R5 strains use CCR5 and CD4 as
coreceptors to infect macrophages. X4 strains use CXCR4 and CD4 to
gain entrance into T cells while dual tropic HIV strains can use
either CXCR4 or CCR5 along with CD4 as coreceptors when infecting
cells.
[0127] HIV primarily infects vital cells in the human immune system
such as helper T cells (specifically CD4.sup.+ T cells),
macrophages and dendritic cells. HIV infection leads to low levels
of CD4.sup.+ T cells through three main mechanisms: firstly, direct
viral killing of infected cells; secondly, increased rates of
apoptosis in infected cells; and thirdly, killing of infected
CD4.sup.+ T cells by CD8 cytotoxic lymphocytes that recognize
infected cells. When CD4.sup.+ T cell numbers decline below a
critical level, cell-mediated immunity is lost, and the body
becomes progressively more susceptible to opportunistic infections.
If untreated, eventually most HIV-infected individuals develop AIDS
(Acquired Immunodeficiency Syndrome) and die; however about one in
ten remains healthy for many years, with no noticeable symptoms.
Treatment with anti-retrovirals, where available, increases the
life expectancy of people infected with HIV.
[0128] Based on the inhibitory effect of the CXCL12-.alpha..sub.2
locked dimer of the present invention, the inventors predict that
the CXCL12-.alpha..sub.2 locked dimer would be effective in
blocking the entry of HIV-1 into human T cells. Certain strains of
HIV (X4 or dual tropic) utilize CXCR4 as a co-receptor, and this
interaction is critical for fusion of the viral and T cell
membranes in the process of HIV entry. By binding to CXCR4 and
preventing it from serving as a viral coreceptor, the wild-type
CXCL12-.alpha. chemokine inhibits HIV entry with an IC50 of 79 nM
(9, 38). Accordingly, the inventors predict that the
CXCL12-.alpha..sub.2 locked dimer of the present invention will be
more effective at lower concentrations since it should block the
CXCR4 receptor more completely than the monomeric, wild-type
chemokine. Additionally, the inventers predict the
CXCL12-.alpha..sub.2 locked dimer will work synergistically or
additively with HAART, a combination therapy, or other current
HIV/AIDS treatments.
Example 13
Prophetic Example of the Inhibitory Effect of the
CXCL12.alpha..sub.2 Locked Dimer on Blood Cancers
[0129] In this example, the inventors show how one would use the
inhibitory effect of the CXCL12-.alpha..sub.2 locked dimer of the
present invention to treat blood cancers.
[0130] Blood cancers cells such as leukemia, lymphoma and myeloma
that express CXCR4 are trafficked throughout the body in response
to CXCL12. Blocking CXCR4 signaling can help prevent this
trafficking, thus exposing these cancers to treatments, like
chemotherapy, resulting in an enhanced effectiveness of the current
therapy. CXCL12 traffics these cancers to the bone marrow, which
provides a protective environment that enhances proliferation and
anti-apoptotic signals that can result in the cancers being less
sensitive to the therapies currently used. The inventors have shown
that the CXCL12.alpha..sub.2 locked dimer of the present invention
inhibits migration of THP-1 cells, a leukemia cell line, toward
CXCL12 (FIG. 5). Therefore, adding the CXCL12.alpha..sub.2 locked
dimer of the present invention to the current treatment of blood
cancers, like leukemia, lymphoma and myeloma, will prevent the
migration of these cancers into the bone marrow in response to the
CXCL12 that is produced there. Thus, the cancer cells will not
enter the protective bone marrow and will be more sensitive and
responsive to the current cytotoxic therapeutics.
Example 14
Prophetic Example of the Inhibitory Effect of the
CXCL12.alpha..sub.2 Locked Dimer on IBD
[0131] In this example, the inventors show how one would use the
inhibitory effect of the CXCL12-.alpha..sub.2 locked dimer of the
present invention to treat gastrointestinal inflammation associated
with IBD.
[0132] By "gastrointestinal inflammation" we mean inflammation of a
mucosal layer of the gastrointestinal tract, and encompass acute
and chronic inflammatory conditions. Acute inflammation is
generally characterized by a short time of onset and infiltration
or influx of neutrophils. Chronic inflammation is generally
characterized by a relatively longer period of onset and
infiltration or influx of mononuclear cells. By "chronic
gastrointestinal inflammatory conditions" (also referred to as
"chronic gastrointestinal inflammatory diseases") we mean, but are
not necessarily limited to, inflammatory bowel disease (IBD),
colitis induced by environmental insults (e.g., gastrointestinal
inflammation (e.g., colitis) caused by or associated with (e.g., as
a side effect) a therapeutic regimen, such as administration of
chemotherapy, radiation therapy, and the like), colitis in
conditions such as chronic granulomatous disease, celiac disease,
celiac sprue (a heritable disease in which the intestinal lining is
inflamed in response to the ingestion of a protein known as
gluten), food allergies, gastritis, infectious gastritis or
enterocolitis (e.g., Helicobacter pylori-infected chronic active
gastritis) and other forms of gastrointestinal inflammation caused
by an infectious agent, and other like conditions.
[0133] By "inflammatory bowel disease" or "IBD" we mean any of a
variety of diseases characterized by inflammation of all or part of
the intestines. Examples of IBD include, but are not limited to,
Crohn's disease and ulcerative colitis. Reference to IBD throughout
the specification is often referred to in the specification as
exemplary of gastrointestinal inflammatory conditions, and is not
meant to be limiting.
[0134] Clinical and experimental evidence suggest that the
pathogenesis of IBD is multifactorial involving susceptibility
genes and environmental factors. The interaction of these factors
with the immune system leads to intestinal inflammation and
dysregulated mucosal immunity against commensal bacteria, various
microbial products (e.g., LPS) or antigens. Animal models of
colitis have highlighted the prominent role of CD4+ T cells in the
regulation of intestinal inflammation. Cytokine imbalance, and the
production of inflammatory mediators have been postulated to play
an important role in the pathogenesis of both experimental colitis
and IBD. In particular, dysregulated CD4+ T cell responses play a
pivotal role in the pathogenesis of experimental colitis.
Therefore, adding the CXCL12.alpha..sub.2 locked dimer of the
present invention to the current treatment of IBD will prevent the
migration of these inflammatory mediators.
[0135] The invention provides a new and potent therapeutic
advantage that is effective across species in a variety of animal
models of chronic and/or acute gastrointestinal inflammation,
particularly in animal models of IBD, which animal models are
regarded in the field as models of disease in humans. In use, the
composition and methods of the present invention will reduce
disease activity, e.g., diarrhea, rectal bleeding and weight loss,
reduce colon weight and colon lesions, as well as reduce colonic
inflammation, as measured by, for example, anti-neutrophil
cytoplasmic antibodies (ANCA), colonic myclo-peroxidase activity,
or other conventional indicator of gastrointestinal
inflammation.
Example 15
Prophetic Example of the Effect of Combination Therapies Using the
CXCL12.alpha..sub.2 Locked Dimer on Cancer, Inflammation,
Auto-Immune Diseases and/or HIV/AIDS
[0136] In this example, the inventors show how one would evaluate
the effect of combination therapies using the inhibitory effect of
the CXCL12-.alpha..sub.2 locked dimer of the present invention to
treat cancer, inflammation, auto-immune diseases and/or
HIV/AIDS.
[0137] For instance, the locked dimer of the present invention may
be used as an agonist or antagonist in combination with other known
anti-inflammatory, HIV, autoimmune or cancer therapies. By
"agonist" we mean a ligand that stimulates the receptor the ligand
binds to in the broadest sense. An "agonist" or an "antagonist" is
a compound or composition that, respectively, either detectably
increases or decreases the activity of a receptor, an enzyme or
another biological molecule, which can lead to increased or
decreased transcription or mRNA levels of a regulated gene or to
another measurable effect such as altered level of activity of the
gene product or protein. The increase or decrease in a receptor's
or enzyme's activity will be an increase or a decrease of at least
about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or a
range about between any two of these values, for one or more
measurable activities. Receptors, their accessory factors and
associated transcription factors can modulate transcription of
their target gene(s) by detectably increasing or decreasing
transcription or mRNA levels. Biological activities of receptors
may also include modulating biological responses such as signal
transduction within a cell or ion flux, e.g., sodium, potassium or
calcium, across cell or organelle membranes, e.g., across
mitochondria.
[0138] The locked dimer of the present invention may also be used
as a super-agonist. By "super-agonist", we mean a type of agonist
that binds permanently to a receptor in such a manner that the
receptor is permanently activated. It is distinct from a mere
agonist in that the association of an agonist to a recepter is
reversible, whereas the binding of an super-agonist to a receptor
is, at least in theory, irreversible.
[0139] In use, the locked dimer of the present invention allows
b-arrestin mediated receptor internalization and down-regulation.
This is a major advantage of the dimer since the combination of
antagonism with respect to CXCL12 induced migration AND ongoing
CXCL12 dimer induced receptor internalization is profoundly
synergistic. Further, the dimer of the present invention may also
be synergistic when used with other CXCR4 pharmacophores.
A. Inflammation.
[0140] The anti-inflammatory activity of the combination therapies
of invention can be determined by using various experimental animal
models of inflammatory arthritis known in the art and described in
Crofford L. J. and Wilder R. L., "Arthritis and Autoimmunity in
Animals", in Arthritis and Allied Conditions: A Textbook of
Rheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger,
1993). Experimental and spontaneous animal models of inflammatory
arthritis and autoimmune rheumatic diseases can also be used to
assess the anti-inflammatory activity of the combination therapies
of invention.
[0141] The principle animal models for arthritis or inflammatory
disease known in the art and widely used include: adjuvant-induced
arthritis rat models, collagen-induced arthritis rat and mouse
models and antigen-induced arthritis rat, rabbit and hamster
models, all described in Crofford L. J. and Wilder R. L.,
"Arthritis and Autoimmunity in Animals", in Arthritis and Allied
Conditions: A Textbook of Rheumatology, McCarty et al. (eds.),
Chapter 30 (Lee and Febiger, 1993), incorporated herein by
reference in its entirety.
[0142] The anti-inflammatory activity of the combination therapies
of invention can be assessed using a carrageenan-induced arthritis
rat model. Carrageenan-induced arthritis has also been used in
rabbit, dog and pig in studies of chronic arthritis or
inflammation. Quantitative histomorphometric assessment is used to
determine therapeutic efficacy. The methods for using such a
carrageenan-induced arthritis model is described in Hansra P. et
al., "Carrageenan-Induced Arthritis in the Rat," Inflammation,
24(2): 141-155, (2000). Also commonly used are zymosan-induced
inflammation animal models as known and described in the art.
[0143] The anti-inflammatory activity of the combination therapies
of invention can also be assessed by measuring the inhibition of
carrageenan-induced paw edema in the rat, using a modification of
the method described in Winter C. A. et al., "Carrageenan-Induced
Edema in Hind Paw of the Rat as an Assay for Anti-inflammatory
Drugs" Proc. Soc. Exp. Biol Med. 111, 544-547, (1962). This assay
has been used as a primary in vivo screen for the anti-inflammatory
activity of most NSAIDs, and is considered predictive of human
efficacy. The anti-inflammatory activity of the test prophylactic
or therapeutic agents is expressed as the percent inhibition of the
increase in hind paw weight of the test group relative to the
vehicle dosed control group. Additionally, animal models for
inflammatory bowel disease can also be used to assess the efficacy
of the combination therapies of invention.
[0144] Animal models for asthma can also be used to assess the
efficacy of the combination therapies of invention. An example of
one such model is the murine adoptive transfer model in which
aeroallergen provocation of TH1 or TH2 recipient mice results in TH
effector cell migration to the airways and is associated with an
intense neutrophilic (TH1) and eosinophilic (TH2) lung mucosal
inflammatory response (Cohn et al., 1997, J. Exp. Med. 186,
1737-1747).
B. Auto-Immune Disorders.
[0145] Animal models for autoimmune disorders can also be used to
assess the efficacy of the combination therapies of invention.
Animal models for autoimmune disorders such as type 1 diabetes,
thyroid autoimmunity, systemic lupus eruthematosus, and
glomerulonephritis have been developed. Further, any assays known
to those skilled in the art can be used to evaluate the
prophylactic and/or therapeutic utility of the combinatorial
therapies disclosed herein for autoimmune and/or inflammatory
diseases.
[0146] Animal models for autoimmune and/or intestinal inflammation
can also be used to test the efficacy of the combination therapies
of the invention. An example of one such model is the murine
dextran sodium sulfate colitis model as described in Wirtz S. et
al., "Chemically induced mouse models of intestinal inflammations"
Nature Protocols 2, 541-546, (2007).
C. Cancer.
[0147] The anti-cancer activity of the therapies used in accordance
with the present invention can also be determined by using various
experimental animal models for the study of cancer such as the SCID
mouse model or transgenic mice or nude mice with human xenografts,
animal models, such as hamsters, rabbits, etc. known in the art and
described in Relevance of Tumor Models for Anticancer Drug
Development (1999, eds. Fiebig and Burger); Contributions to
Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991,
eds. Boven and Winograd); and Anticancer Drug Development Guide
(1997 ed. Teicher), herein incorporated by reference in their
entireties.
[0148] Toxicity and efficacy of the prophylactic and/or therapeutic
protocols of the instant invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50%
of the population). The dose ratio between toxic and therapeutic
effects is the therapeutic index and it can be expressed as the
ratio LD50/ED50. Prophylactic and/or therapeutic agents that
exhibit large therapeutic indices are preferred. While prophylactic
and/or therapeutic agents that exhibit toxic side effects may be
used, care should be taken to design a delivery system that targets
such agents to the site of affected tissue in order to minimize
potential damage to uninfected cells and, thereby, reduce side
effects.
[0149] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of the
prophylactic and/or therapeutic agents for use in humans. The
dosage of such agents lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For any
agent used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC 50 (i.e., the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0150] The protocols and compositions of the invention are
preferably tested in vitro, and then in vivo, for the desired
therapeutic or prophylactic activity, prior to use in humans.
Therapeutic agents and methods may be screened using cells of a
tumor or malignant cell line. Many assays standard in the art can
be used to assess such survival and/or growth; for example, cell
proliferation can be assayed by a variety of methods known to the
art, including by direct cell count, by detecting changes in
transcriptional activity of known genes such as proto-oncogenes or
cell cycle markers; cell viability can be assessed by trypan blue
staining, differentiation can be assessed visually based on changes
in morphology, decreased growth and/or colony formation in soft
agar or tubular network formation in three-dimensional basement
membrane or extracellular matrix preparation, etc.
[0151] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to in rats, mice, chicken, cows, monkeys, rabbits,
hamsters, etc., for example, the animal models described above. The
compounds can then be used in the appropriate clinical trials.
Further, any assays known to those skilled in the art can be used
to evaluate the prophylactic and/or therapeutic utility of the
combinatorial therapies disclosed herein for treatment or
prevention of cancer, inflammatory disorder, or autoimmune
disease.
D. HIV Infection
[0152] The Human Immunodeficiency Virus (HIV) infects millions of
people globally. Cases are reported from nearly every country
amounting to 40 million adults and children living with HIV/AIDS
worldwide. In 2001, 5 million people were newly infected with HIV,
and there were 3 million adult and child deaths due to HIV/AIDS. A
full third of those people living with AIDS are aged 15-24 (World
Health Organization, 2001).
[0153] The typical human immune system response, killing the
invading virion, is taxed because the virus infects and kills the
immune system's T cells. In addition, viral reverse transcriptase,
the enzyme used in making a new virion particle, is not very
specific, and causes transcription mistakes that result in
continually changed glycoproteins on the surface of the viral
protective coat. This lack of specificity decreases the immune
system's effectiveness because antibodies specifically produced
against one glycoprotein may be useless against another, hence
reducing the number of antibodies available to fight the virus. The
virus continues to reproduce while the immune response system
continues to weaken. Eventually, the HIV largely holds free reign
over the body's immune system, allowing opportunistic infections to
set in and, without the administration of antiviral agents,
immunomodulators, or both, death may result.
[0154] While treatments for HIV/AIDS exist, the drugs currently
used in treatment modalities exhibit numerous side effects, require
prolonged treatment that often induces drug resistance, and do not
result in complete eradication of the virus from the body. For
example, nucleoside analogs, such as 3'-azido-3'-deoxythymidine
(AZT), 2',3'-dideoxycytidine (ddC), 2',3'-dideoxythymidinene (d4T),
2',3'-dideoxyinosine (ddI), and 2',3'-dideoxy-3'-thia-cytidine
(3TC) have been shown to be relatively effective in halting HIV
replication at the reverse transcriptase (RT) stage. Even with the
current success of reverse transcriptase inhibitors, it has been
found that HIV patients can become resistant to a single inhibitor.
Thus, it is desirable to develop compounds for use in combination
with other known HIV treatments to further combat HIV infection and
inhibit the replication of drug resistant strains of HIV.
[0155] In use, the locked dimer of the present invention may be
administered in combination with one or more other compound having
activity against HIV disease or HIV-related disease. By "HIV
disease or HIV-related disease" we mean a disease state which is
marked by HIV infection. Such disorders associated with HIV
infection include, but are not limited to, AIDS, Kaposi's sarcoma,
opportunistic infections such as those caused by Pneumocystis
carinii and Mycobacterium tuberculosis; oral lesions, including
thrush, hairy leukoplakia, and aphthous ulcers; generalized
lymphadenopathy, shingles, thrombocytopenia, aseptic meningitis,
and neurologic disease such as toxoplasmosis, cryptococcosis, CMV
infection, primary CNS lymphoma, and HIV-associated dementia,
peripheral neuropathies, seizures and myopathy.
[0156] Standard tissue culture models of HIV infection can be used
to determine the efficacy of CXCL12.alpha..sub.2 locked dimer as an
HIV entry inhibitor in combination with current HIV entry
inhibitors. Suitable therapeutic agents for use in combination with
the compounds of the present invention include, but are not limited
to, protease inhibitors, non-nucleoside reverse transcriptase
inhibitors, nucleoside reverse transcriptase inhibitors,
antiretroviral nucleosides, entry inhibitors as well as other
anti-viral agents effective to inhibit or treat HIV infection.
Further examples of suitable therapeutic agents include, but are
not limited to, zidovudine, didanosine, stavudine, interferon,
lamivudine, adefovir, nevirapine, delaviridine, loviride,
saquinavir, indinavir and AZT. Other suitable therapeutic agents
include, but are not limited to, antibiotics or other anti-viral
agents, e.g., acyclovir. Other combination therapies known to those
of skill in the art can be used in conjunction with the
compositions and methods of the present invention.
[0157] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration from the
specification and practice of the invention disclosed herein. All
references cited herein for any reason, including all journal
citations and U.S./foreign patents and patent applications, are
specifically and entirely incorporated herein by reference for all
purposes.
[0158] It is understood that the invention is not confined to the
specific reagents, formulations, reaction conditions, etc., herein
illustrated and described, but embraces such modified forms thereof
as come within the scope of the following claims.
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Sequence CWU 1
1
2168PRTHomo sapiens 1Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys
Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His
Leu Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys Ala Cys Gln Ile Val
Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro
Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60 Cys Leu Asn
Lys 65 238PRTHomo sapiens 2Met Glu Gly Ile Ser Ile Tyr Thr Ser Asp
Asn Tyr Thr Glu Glu Met 1 5 10 15 Gly Ser Gly Asp Tyr Asp Ser Met
Lys Glu Pro Ala Phe Arg Glu Glu 20 25 30 Asn Ala Asn Phe Asn Lys
35
* * * * *