U.S. patent application number 12/148143 was filed with the patent office on 2009-06-11 for chimeric polypeptides containing chemokine domains.
This patent application is currently assigned to Genetics Institute Inc.. Invention is credited to Stephen H. Herrmann, Stephen L. Swanberg.
Application Number | 20090148507 12/148143 |
Document ID | / |
Family ID | 25199531 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090148507 |
Kind Code |
A1 |
Herrmann; Stephen H. ; et
al. |
June 11, 2009 |
Chimeric polypeptides containing chemokine domains
Abstract
This invention provides a chimeric DNA molecule comprising a
sequence encoding a chemokine polypeptide covalently attached to a
heterologous polypeptide, the encoded chimeric polypeptide, and
uses thereof.
Inventors: |
Herrmann; Stephen H.;
(Wellesley, MA) ; Swanberg; Stephen L.; (Boston,
MA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
Genetics Institute Inc.
Genetics Institute LLC
|
Family ID: |
25199531 |
Appl. No.: |
12/148143 |
Filed: |
April 16, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10614475 |
Jul 7, 2003 |
7396911 |
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12148143 |
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09467638 |
Dec 20, 1999 |
6730296 |
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10614475 |
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08808720 |
Feb 28, 1997 |
6100387 |
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09467638 |
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Current U.S.
Class: |
424/450 ;
424/134.1; 424/85.1; 435/375 |
Current CPC
Class: |
C07K 14/522 20130101;
A61K 47/6835 20170801; A61P 37/04 20180101; C07K 2319/30 20130101;
A61P 29/00 20180101; A61K 47/6813 20170801; C07K 2319/00 20130101;
A61K 38/00 20130101; A61P 31/18 20180101; C07K 14/523 20130101;
A61K 39/3955 20130101; A61P 43/00 20180101; A61P 37/02 20180101;
A61K 39/3955 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/450 ;
424/134.1; 424/85.1; 435/375 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/395 20060101 A61K039/395; A61K 38/19 20060101
A61K038/19; C12N 5/06 20060101 C12N005/06 |
Claims
1-56. (canceled)
57. A method for altering CXCR4 receptor function in a subject in
need thereof comprising administering to said subject a
therapeutically effective amount of a composition comprising a
chimeric polypeptide and a pharmaceutically acceptable carrier,
wherein said chimeric polypeptide comprises at least one chemokine
polypeptide covalently attached to at least one heterologous
polypeptide, and wherein the heterologous polypeptide is an Fc
polypeptide, wherein the chemokine polypeptide comprises SEQ ID
NO:1 from amino acid 22 to amino acid 328.
58. The method of claim 57, wherein said composition is in the form
of a liposome.
59. The method of claim 57, further comprising administering a
cytokine, a lymphokine, a hematopoietic factor, a thrombolytic
factor, or an anti-thrombolytic factor.
60. The method of claim 57, wherein said composition is
administered orally, topically, by inhalation, or by cutaneous,
subcutaneous, intraperitoneal, parenteral, or intravenous
injection.
61. The method of claim 60, wherein said composition is
administered orally in the form of a tablet, capsule, powder,
solution or elixir.
62. The method of claim 57, wherein said composition comprises
about 0.01 ng to about 100 mg of chimeric polypeptide per kg body
weight.
63. A method for inhibiting binding of a CXCR4 receptor to a CXCR4
receptor ligand comprising contacting a cell expressing said
receptor with a chimeric polypeptide in an amount effective to
inhibit binding of said receptor to said ligand, wherein said
chimeric polypeptide comprises at least one chemokine polypeptide
covalently attached to at least one heterologous polypeptide, and
wherein the heterologous polypeptide is an Fc polypeptide, wherein
the chemokine polypeptide comprises SEQ ID NO:1 from amino acid 22
to amino acid 328.
64. The method of claim 63, wherein said chimeric polypeptide is
administered to a subject in vivo.
65. The method of claim 63, wherein said cell is contacted with
said chimeric polypeptide in vitro.
66. The method of claim 64, wherein said chimeric polypeptide is in
the form of a liposome.
67. The method of claim 64, further comprising administering a
cytokine, a lymphokine, a hematopoietic factor, a thrombolytic
factor, or an anti-thrombolytic factor.
68. The method of claim 64, wherein said chimeric polypeptide is
administered orally, topically, by inhalation, or by cutaneous,
subcutaneous, intraperitoneal, parenteral, or intravenous
injection.
69. The method of claim 68, wherein said chimeric polypeptide is
administered orally in the form of a tablet, capsule, powder,
solution or elixir.
70. The method of claim 64, wherein said chimeric polypeptide
comprises about 0.01 .mu.g to about 100 mg of chimeric polypeptide
per kg body weight.
Description
[0001] The present invention relates generally to chimeric
polypeptides containing chemokine polypeptide domains. More
specifically, the invention relates to the expression in host cells
of recombinant polynucleotide sequences encoding chemokine
polypeptides covalently attached to heterologous polypeptides, and
the use of such chimeric polypeptides as research tools for
identifying chemokine receptors, as vaccine adjuvants, as agents
for the chemotactic recruitment of migratory cells, as agents for
the stimulation or inhibition of angiogenesis, as agents against
autoimmune diseases and inflammation, and as agents to inhibit the
binding of HIV to certain receptors.
[0002] Chemokines (or chemotactic cytokines) are a class of
cytokine molecules capable of chemotactically attracting migratory
cells, and are involved in cell recruitment and activation in
inflammation. Chemokines generally have small molecular weights in
the range of 8-10 kDa and, like other small proteins such as
cytokines, are believed to be rapidly inactivated in vivo,
resulting in relatively short biological half-lives for these
proteins. Most chemokines can be divided into two subgroups, CXC or
CC, on the basis of the spacing of two highly-conserved cysteine
amino acids near the amino terminus of these proteins. Within the
CXC and CC subgroups, chemokines are further grouped into related
families based on amino acid sequence similarity between them. CXC
chemokine families include the IP-10 and Mig family; the
GRO.alpha., GRO.beta., and GRO.gamma. family; the interleukin-8
(IL-8) family; and the PF4 family. CC chemokine families include
the monocyte chemoattractant protein (MCP) family; the family
including macrophage inhibitory protein-1.alpha. (MIP-1.alpha.),
macrophage inhibitory protein-1.beta. (MIP-1.beta.), and regulated
on activation normal T cell expressed (RANTES); and the
lymphotactin family. The chemokines stromal cell-derived factor
1.alpha. (SDF-1.alpha.) and stromal cell-derived factor 1.beta.
(SDF-1.beta.) form a chemokine family that is approximately equally
related by amino acid sequence similarity to the CXC and CC
chemokine subgroups. Individual members of the chemokine families
are known to be bound by at least one chemokine receptor, with CXC
chemokines generally bound by members of the CXCR class of
receptors, and CC chemokines by members of the CCR class of
receptors. For example, SDF-1.alpha. is known to be a ligand for
the CXCR receptor fusin/CXCR4, and MIP-1.alpha. is bound by the CCR
receptors CCR1, CCR4, and CCR5.
[0003] The presence of a chemokine gradient attracts migratory
cells such as lymphocytes, leukocytes, and antigen-presenting cells
(APCs) that may participate in autoimmune reactions, inflammation,
or normal immune responses, or that may release other intercellular
factors to stimulate or inhibit angiogenesis or other cellular
processes. For example, the initiation of autoimmune disease
requires the infiltration or recruitment of lymphocytes able to
respond against self proteins into the organ bearing the antigenic
self proteins. Inflammatory atherosclerotic lesions are due in part
to infiltration of the vascular compartment by leukocytes recruited
to the site. To induce an immune response, antigenic proteins and
glycoproteins must bind to the surface of B lymphocytes to
stimulate antibody production, and must be taken up by
antigen-presenting cells, processed, and represented to T
lymphocytes to mediate a T-lymphocyte response. Migratory cells
that secrete IP10 or IL-8, when attracted by a chemokine gradient
to a particular site, respectively may inhibit or stimulate the
formation of blood vessels at that site. Chemokines may be used to
establish a chemoattractive gradient for migratory cells that are
expressing the appropriate chemokine receptors, or to obscure an
existing chemoattractive gradient.
[0004] Chemokine receptors are also involved in functions other
than chemotaxis, such as interacting with viral proteins. HIV-1 is
known to bind to certain proteins on the surface of cells in order
to gain entrance into these cells and replicate or integrate the
viral gene into the host DNA. The CD4 protein on T lymphocytes and
other cells, including certain antigen presenting cells, has been
shown to be bound by the HIV-1 viral envelope protein gp120. This
is believed to induce in gp120 a conformational change that then
exposes regions of gp120 and perhaps CD4 that subsequently bind to
a chemokine receptor. To date CXCR4 (also known as fusin), CCR5,
and several other chemokine receptors have been identified as
co-receptors for HIV-1. Monocyte-tropic (M-tropic) isolates of
HIV-1 require interaction with CCR5 in order to infect cells, while
T-lymphocyte-tropic (T-tropic) HIV-1 isolates require another
coreceptor, CXCR4, for infection. There is some evidence indicating
that HIV-1 can also use other CCR receptors such as CCR2 and CCR3
to gain entry into cells expressing them. For some HIV-2 isolates,
it appears that certain chemokine receptors such as fusin/CXCR4
alone can provide the cell-surface protein needed for binding and
entrance into the cell.
[0005] HIV-1 infection of cells expressing CD4 and fusin/CXCR4 is
greatly decreased by the addition of purified SDF-1.alpha., which
is bound by fusin/CXCR4. We have found that preincubation of cells
in the presence of purified SDF-1.alpha. for a short period of time
at 37.degree. C. causes a profound down-regulation of the receptor.
This down-regulation of fusin/CXCR4 correlates with a decrease in
the ability of HIV-1 to infect cells.
[0006] There is a continuing requirement for new compositions that
will enhance, alter, or inhibit the effects of chemokine-receptor
interactions, and for methods for their use.
SUMMARY OF THE INVENTION
[0007] Applicants have for the first time constructed novel
chimeric DNA molecules encoding chimeric polypeptides comprising
chemokine polypeptide domains. Chimeric polypeptides expressed from
these constructs have exhibited novel properties, including novel
interactions with cells expressing chemokine receptors.
[0008] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding a
chimeric polypeptide, the chimeric polypeptide comprising at least
one chemokine polypeptide covalently attached to at least one
heterologous polypeptide. Preferably, the chemokine polypeptide is
SDF-1.alpha., MIP-1.alpha., or MIP-1.beta., or is derived from
SDF-1.alpha., MIP-1.alpha., or MIP-1.beta.. Preferably, the
heterologous polypeptide is an Fc polypeptide.
[0009] Another embodiment provides a composition comprising an
isolated polynucleotide encoding a chimeric polypeptide, wherein a
heterologous polypeptide is covalently attached to the amino
terminus of a chemokine polypeptide, preferably by a linker
polypeptide.
[0010] Another embodiment provides a composition comprising an
isolated polynucleotide encoding a chimeric polypeptide, wherein a
heterologous polypeptide is covalently attached to the carboxyl
terminus of a chemokine polypeptide, preferably by a linker
polypeptide.
[0011] In another embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding a
chimeric polypeptide, wherein the polynucleotide is selected from
the group consisting of: [0012] (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:2 from nucleotide 12 to nucleotide
1213; [0013] (b) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:2 from nucleotide 69 to nucleotide 1213;
[0014] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:2 from nucleotide 72 to nucleotide 1213; [0015] (d) a
polynucleotide comprising the nucleotide sequence of SEQ ID NO:2
from nucleotide 75 to nucleotide 1213; [0016] (e) a polynucleotide
comprising a fragment of the nucleotide sequence of SEQ ID NO:2;
[0017] (f) a polynucleotide comprising the nucleotide sequence of
the full-length protein-coding sequence of clone S1-3 deposited
under accession number ATCC XXXXX; [0018] (g) a polynucleotide
comprising the nucleotide sequence of the mature protein-coding
sequence of clone S1-3 deposited under accession number ATCC XXXXX;
[0019] (h) a polynucleotide encoding a chimeric polypeptide
comprising the amino acid sequence of SEQ ID NO:1; [0020] (i) a
polynucleotide encoding a chimeric polypeptide comprising the amino
acid sequence of SEQ ID NO:1 from amino acid 20 to amino acid 328;
[0021] (k) a polynucleotide encoding a chimeric polypeptide
comprising the amino acid sequence of SEQ ID NO:1 from amino acid
22 to amino acid 328; [0022] (k) a polynucleotide encoding a
chimeric polypeptide comprising a fragment of the amino acid
sequence of SEQ ID NO:1; [0023] (l) a polynucleotide comprising a
nucleotide sequence complementary to any one of the polynucleotides
specified in (a)-(k) above; and
[0024] (m) a polynucleotide capable of simultaneously hybridizing
under stringent conditions to sequences encoding the chemokine
polypeptide and to sequences encoding the heterologous polypeptide
in any one of the polynucleotides specified in (a)-(l) above.
[0025] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:2 from nucleotide 12 to nucleotide 1213; the
nucleotide sequence of the full-length protein-coding sequence of
clone S1-3 deposited under accession number ATCC XXXXX; or the
nucleotide sequence of the mature protein-coding sequence of clone
S1-3 deposited under accession number ATCC XXXXX.
[0026] In a further embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding a
chimeric polypeptide, wherein the polynucleotide is selected from
the group consisting of: [0027] (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:4 from nucleotide 12 to nucleotide
1207; [0028] (b) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:4 from nucleotide 69 to nucleotide 1207;
[0029] (c) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:4; [0030] (d) a polynucleotide comprising the
nucleotide sequence of the full-length protein-coding sequence of
clone SK2-2 deposited under accession number ATCC XXXXX; [0031] (e)
a polynucleotide comprising the nucleotide sequence of the mature
protein-coding sequence of clone SK2-2 deposited under accession
number ATCC XXXXX; [0032] (f) a polynucleotide encoding a chimeric
polypeptide comprising the amino acid sequence of SEQ ID NO:3;
[0033] (g) a polynucleotide encoding a chimeric polypeptide
comprising the amino acid sequence of SEQ ID NO:3 from amino acid
20 to amino acid 326; [0034] (h) a polynucleotide encoding a
chimeric polypeptide comprising a fragment of the amino acid
sequence of SEQ ID NO:3; [0035] (i) a polynucleotide comprising a
nucleotide sequence complementary to any one of the polynucleotides
specified in (a)-(h) above; and [0036] (j) a polynucleotide capable
of simultaneously hybridizing under stringent conditions to
sequences encoding the chemokine polypeptide and to sequences
encoding the heterologous polypeptide in any one of the
polynucleotides specified in (a)-(i) above.
[0037] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:4 from nucleotide 12 to nucleotide 1207; the
nucleotide sequence of the full-length protein-coding sequence of
clone SK2-2 deposited under accession number ATCC XXXXX; or the
nucleotide sequence of the mature protein-coding sequence of clone
SK2-2 deposited under accession number ATCC XXXXX.
[0038] In another embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding a
chimeric polypeptide, wherein the polynucleotide is selected from
the group consisting of: [0039] (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:6 from nucleotide 15 to nucleotide
1225; [0040] (b) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:6 from nucleotide 81 to nucleotide 1225;
[0041] (c) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:6; [0042] (d) a polynucleotide comprising the
nucleotide sequence of the full-length protein-coding sequence of
clone MP-1 deposited under accession number ATCC XXXXX; [0043] (e)
a polynucleotide comprising the nucleotide sequence of the
full-length protein-coding sequence of clone MP-2 deposited under
accession number ATCC XXXXX; [0044] (f) a polynucleotide comprising
the nucleotide sequence of the full-length protein-coding sequence
of clone MP-6 deposited under accession number ATCC XXXXX; [0045]
(g) a polynucleotide comprising the nucleotide sequence of the
mature protein-coding sequence of clone MP-1 deposited under
accession number ATCC XXXXX; [0046] (h) a polynucleotide comprising
the nucleotide sequence of the mature protein-coding sequence of
clone MP-2 deposited under accession number ATCC XXXXX; [0047] (i)
a polynucleotide comprising the nucleotide sequence of the mature
protein-coding sequence of clone MP-6 deposited under accession
number ATCC XXXXX; [0048] (j) a polynucleotide encoding a chimeric
polypeptide comprising the amino acid sequence of SEQ ID NO:5;
[0049] (k) a polynucleotide encoding a chimeric polypeptide
comprising the amino acid sequence of SEQ ID NO:5 from amino acid
23 to amino acid 331; [0050] (l) a polynucleotide encoding a
chimeric polypeptide comprising a fragment of the amino acid
sequence of SEQ ID NO:5; [0051] (m) a polynucleotide comprising a
nucleotide sequence complementary to any one of the polynucleotides
specified in (a)-(l) above; and [0052] (n) a polynucleotide capable
of simultaneously hybridizing under stringent conditions to
sequences encoding the chemokine polypeptide and to sequences
encoding the heterologous polypeptide in any one of the
polynucleotides specified in (a)-(m) above.
[0053] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:6 from nucleotide 15 to nucleotide 1225; the
nucleotide sequence of the full-length protein-coding sequence of
clones MP-1, MP-2, and MP-6 deposited under accession numbers ATCC
XXXXX, ATCC XXXXX, and ATCC XXXXX, respectively; or the nucleotide
sequence of the mature protein-coding sequence of clones MP-1,
MP-2, and MP-6 deposited under accession numbers ATCC XXXXX, ATCC
XXXXX, and ATCC XXXXX, respectively.
[0054] In a further embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding a
chimeric polypeptide, wherein the polynucleotide is selected from
the group consisting of: [0055] (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:8 from nucleotide 16 to nucleotide
1226; [0056] (b) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:8 from nucleotide 85 to nucleotide 1226;
[0057] (c) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:8; [0058] (d) a polynucleotide comprising the
nucleotide sequence of the full-length protein-coding sequence of
clone MPB-X deposited under accession number ATCC XXXXX; [0059] (e)
a polynucleotide comprising the nucleotide sequence of the mature
protein-coding sequence of clone MPB-X deposited under accession
number ATCC XXXXX; [0060] (f) a polynucleotide encoding a chimeric
polypeptide comprising the amino acid sequence of SEQ ID NO:7;
[0061] (g) a polynucleotide encoding a chimeric polypeptide
comprising the amino acid sequence of SEQ ID NO:7 from amino acid
24 to amino acid 331; [0062] (h) a polynucleotide encoding a
chimeric polypeptide comprising a fragment of the amino acid
sequence of SEQ ID NO:7; [0063] (i) a polynucleotide comprising a
nucleotide sequence complementary to any one of the polynucleotides
specified in (a)-(h) above; and [0064] (j) a polynucleotide capable
of simultaneously hybridizing under stringent conditions to
sequences encoding the chemokine polypeptide and to sequences
encoding the heterologous polypeptide in any one of the
polynucleotides specified in (a)-(i) above.
[0065] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:8 from nucleotide 16 to nucleotide 1226; the
nucleotide sequence of the full-length protein-coding sequence of
clone MPB-X deposited under accession number ATCC XXXXX; or the
nucleotide sequence of the mature protein-coding sequence of clone
MPB-X deposited under accession number ATCC XXXXX.
[0066] In certain preferred embodiments, the polynucleotide is
operably linked to an expression control sequence. The invention
also provides a host cell, preferably a mammalian cell, transformed
with such polynucleotide compositions.
[0067] Processes are also provided for producing a chimeric
polypeptide, which comprise: [0068] (a) growing a culture of the
host cell transformed with such polynucleotide compositions in a
suitable culture medium; and [0069] (b) purifying the protein from
the culture. The polypeptide produced according to such methods is
also provided by the present invention. Preferred embodiments
include those in which the polypeptide produced by such process is
a mature form of the polypeptide.
[0070] In other embodiments, the present invention provides a
composition comprising a chimeric polypeptide, the chimeric
polypeptide comprising at least one chemokine polypeptide
covalently attached to at least one heterologous polypeptide.
Preferably, the chemokine polypeptide is SDF-1.alpha.,
MIP-1.alpha., or MIP-1.beta., or is derived from SDF-1.alpha.,
MIP-1.alpha., or MIP-1.beta.. Preferably, the heterologous
polypeptide is an Fc polypeptide.
[0071] A further embodiment provides a composition comprising a
chimeric polypeptide, wherein a heterologous polypeptide is
covalently attached to the amino terminus of a chemokine
polypeptide, preferably by a linker polypeptide.
[0072] Another embodiment provides a composition comprising a
chimeric polypeptide, wherein a heterologous polypeptide is
covalently attached to the carboxyl terminus of a chemokine
polypeptide, preferably by a linker polypeptide.
[0073] In another embodiment, the present invention provides a
composition comprising a chimeric polypeptide, wherein the chimeric
polypeptide comprises an amino acid sequence selected from the
group consisting of: [0074] (a) the amino acid sequence of SEQ ID
NO:1; [0075] (b) the amino acid sequence of SEQ ID NO:1 from amino
acid 20 to amino acid 328; [0076] (c) the amino acid sequence of
SEQ ID NO:1 from amino acid 21 to amino acid 328; [0077] (d) the
amino acid sequence of SEQ ID NO:1 from amino acid 22 to amino acid
328; and [0078] (e) fragments of the amino acid sequence of SEQ ID
NO:1.
[0079] Preferably, such chimeric polypeptide comprises the amino
acid sequence of SEQ ID NO:1.
[0080] In a further embodiment, the present invention provides a
composition comprising a chimeric polypeptide, wherein the chimeric
polypeptide comprises an amino acid sequence selected from the
group consisting of: [0081] (a) the amino acid sequence of SEQ ID
NO:3; [0082] (b) the amino acid sequence of SEQ ID NO:3 from amino
acid 20 to amino acid 326; and [0083] (c) fragments of the amino
acid sequence of SEQ ID NO:3.
[0084] Preferably, such chimeric polypeptide comprises the amino
acid sequence of SEQ ID NO:3.
[0085] In another embodiment, the present invention provides a
composition comprising a chimeric polypeptide, wherein the chimeric
polypeptide comprises an amino acid sequence selected from the
group consisting of: [0086] (a) the amino acid sequence of SEQ ID
NO:5; [0087] (b) the amino acid sequence of SEQ ID NO:5 from amino
acid 23 to amino acid 331; and [0088] (c) fragments of the amino
acid sequence of SEQ ID NO:5.
[0089] Preferably, such chimeric polypeptide comprises the amino
acid sequence of SEQ ID NO:5.
[0090] In a further embodiment, the present invention provides a
composition comprising a chimeric polypeptide, wherein the chimeric
polypeptide comprises an amino acid sequence selected from the
group consisting of: [0091] (a) the amino acid sequence of SEQ ID
NO:7; [0092] (b) the amino acid sequence of SEQ ID NO:7 from amino
acid 24 to amino acid 331; and [0093] (c) fragments of the amino
acid sequence of SEQ ID NO:7.
[0094] Preferably, such chimeric polypeptide comprises the amino
acid sequence of SEQ ID NO:7.
[0095] Polypeptide compositions of the present invention may
further comprise a pharmaceutically acceptable carrier.
Compositions comprising an antibody which specifically reacts with
such polypeptide are also provided by the present invention.
[0096] Methods are also provided for preventing, treating or
ameliorating a medical condition which comprises administering a
therapeutically effective amount of a composition comprising a
polypeptide of the present invention and a pharmaceutically
acceptable carrier.
[0097] The present invention also provides methods for identifying
molecules capable of interacting with a chimeric polypeptide which
comprise: [0098] (a) combining a composition of claim 23 with a
composition comprising molecules to be tested for interaction,
forming a first mixture; [0099] (b) combining the first mixture
with a composition comprising indicator molecules, so that the
indicator molecules are capable of being altered by the first
mixture; and [0100] (c) detecting the presence of altered indicator
molecules.
[0101] Methods are also provided for attracting migratory cells to
a region of an organism which comprises administering
therapeutically effective amounts of at least one composition
comprising a chimeric polypeptide.
[0102] Methods for stimulating or inhibiting angiogenesis, which
comprise administering therapeutically effective amounts of at
least one composition comprising a chimeric polypeptide, are also
provided.
[0103] Methods are also provided for preventing, treating, or
ameliorating an inflammatory or an autoimmune condition, which
comprise administering therapeutically effective amounts of at
least one composition comprising a chimeric polypeptide.
[0104] Methods for enhancing antigen-presenting cell activity,
which comprise administering therapeutically effective amounts of
at least one composition comprising a chimeric polypeptide, wherein
at least one chimeric polypeptide comprises antigen molecules, are
also provided.
[0105] Methods are provided for inducing an immune response which
comprise administering a vaccine and therapeutically effective
amounts of at least one composition comprising a chimeric
polypeptide.
[0106] Methods for altering receptor function which comprise
causing a receptor to bind at least one chimeric polypeptide, and
for decreasing receptor function which comprise causing a receptor
to bind at least one chimeric polypeptide, resulting in a decrease
in the number of functional receptor molecules, are provided.
[0107] Methods are provided for preventing, treating, or
ameliorating HIV infection which comprise administering
therapeutically effective amounts of at least one composition
comprising a chimeric polypeptide. Preferably, the chemokine
polypeptide of the chimeric polypeptide comprises SDF-1.alpha.,
MIP-1.alpha., or MIP-1.beta..
[0108] Other aspects and advantages of the present invention will
be apparent upon consideration of the following detailed
description of preferred embodiments thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0109] FIG. 1 shows the expression of chimeric polypeptides,
described in Example 2.
[0110] FIG. 2 shows chimeric SDF-1.alpha. polypeptide binding to
cells expressing the fusin/CXCR4 receptor, as described in Example
3.
DETAILED DESCRIPTION OF THE INVENTION
[0111] The present inventors have for the first time constructed
novel chimeric polypeptides comprising a chemokine polypeptide
covalently attached to a heterologous polypeptide. These chimeric
polypeptides interact with chemokine receptors and have novel
properties.
[0112] As used herein, "chemokine" includes all molecules with
chemotactic activity or derived from molecules with chemotactic
activity by any kind of alteration, addition, insertion, deletion,
mutation, substitution, replacement, or modification. Chemotactic
activity for a particular cell population is the direct or indirect
stimulation of the directed orientation or movement of such cell
population. Preferably, the cell population comprises circulating
blood cells, bone marrow stem cells. More preferably, the cell
population may include monocytes, B cells, T cells, basophils,
eosinophils, neutrophils, natural killer (NK) cells, and bone
marrow stem cells. Most preferably, the cell population may include
monocytes, T cells, basophils, and bone marrow stem cells.
Preferably, the chemokine has the ability to directly stimulate
directed movement of cells. Whether a particular polypeptide has
chemotactic activity for a population of cells can be readily
determined by employing the polypeptide in any known assay for cell
chemotaxis. Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce the
adhesion of one cell population to another cell population.
Suitable assays for movement and adhesion include, without
limitation, those described in: Current Protocols in Immunology,
Ed. by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.
Shevach, W. Strober, Pub. by Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28); Taub et al., J. Clin. Invest.
95:1370-1376, 1995; Lind et al., APMIS 103:140-146, 1995; Muller et
al., Eur. J. Immunol. 25: 1744-1748; Gruber et al., J. of Immunol.
152:5860-5867, 1994; Johnston et al., J. of Immunol. 153:
1762-1768, 1994; all of which are incorporated herein by
reference.
[0113] As used herein, "covalently attached" means the attachment
of molecules to each other by covalent chemical bonds, either
directly or through a linker molecule that is itself covalently
attached to said molecules.
[0114] As used herein, "heterologous polypeptides" include all
polypeptides that can be covalently attached to a chemokine
polypeptide, including without limitation chemokines, cytokines,
immunoglobulins, antigens, antibody-binding tags such as His, Flag,
or myc, lectin-binding domains, toxins, kinases, proteases, other
enzymes, structural proteins; polypeptides derived from the
foregoing by any form of alteration, addition, insertion, deletion,
mutation, substitution, replacement, or modification; but excluding
thioredoxin. For example, chemokine polypeptides can be attached
through "linker" sequences to the Fc portion of an immunoglobulin.
For a bivalent form of the chemokine, such a fusion could be to the
Fc portion of an IgG molecule. Other immunoglobulin isotypes may
also be used to generate such fusions. For example, a chemokine-IgM
fusion would generate a decavalent form of the chemokine. In
addition, it is possible to create a multivalent form of a chimeric
polypeptide by connecting the chimeric polypeptide through a
P.sub.i linkage to the phosphatidyl inositol present in micellular
preparations.
[0115] Fragments of chimeric chemokine polypeptides are also
encompassed by the present invention. Preferably, such fragments
retain the desired activity of the polypeptide or modify it to
create a desired activity. Fragments of polypeptides may be in
linear form or they may be cyclized using known methods, for
example, as described in H. U. Saragovi, et al., Bio/Technology 10,
773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc.
114, 9245-9253 (1992), both of which are incorporated herein by
reference. The polypeptides provided herein also include
polypeptides characterized by amino acid sequences similar to those
of purified proteins but into which modification are naturally
provided or deliberately engineered. For example, modifications in
the polypeptide or DNA sequences can be made by those skilled in
the art using known techniques. Modifications of interest in the
polypeptide sequences may include the alteration, addition,
insertion, deletion mutation, substitution, replacement, or
modification of, a selected amino acid residue in the coding
sequence. As one example, one or more of the cysteine residues may
be deleted or replaced with another amino acid to alter the
conformation of the molecule. As another example, an additional
amino acid may be added to the N-terminus of the polypeptide. Also,
the amino acid sequence of the polypeptide may be altered using
random mutation techniques. It is also possible to attach to
polypeptides other moieties, including without limitation
carbohydrates, lipids, or polyethylene glycol, or to remove or
alter such moieties. Techniques for such alterations, additions,
insertions, deletions, mutations, substitutions, replacements, or
modifications are well known to those skilled in the art (see,
e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration,
addition, insertion, deletion, mutation, substitution, replacement,
or modification retains the desired activity of the polypeptide or
modifies it to create a desired activity.
[0116] Other fragments and derivatives of the sequences of
polypeptides which would be expected to retain polypeptide activity
and may thus be useful for screening or other immunological
methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the present invention.
[0117] The present invention also provides both full-length and
mature forms of chimeric chemokine polypeptides. The full-length
form of such polypeptides is identified in the sequence listing by
translation of the protein-coding region, excluding introns, of the
nucleotide sequence of each disclosed construct. The mature form of
such polypeptides may be obtained by expression of the disclosed
full-length polynucleotide (preferably those deposited with ATCC)
in a suitable mammalian cell, preferably CHO or COS cell, or other
host cell. The sequence of the mature form of the polypeptide may
also be determinable from the amino acid sequence of the
full-length form.
[0118] Chimeric chemokine polypeptides including chemokine
polypeptides that are species homologs of disclosed polypeptides
are also provided by the present invention. Species homologs may be
isolated and identified by making suitable probes or primers from
the sequences provided herein and screening a suitable nucleic acid
source from the desired species. The invention also encompasses
allelic variants of the disclosed chemokine polypeptides or
chemokine-encoding polynucleotides; that is, naturally-occurring
alternative forms of the isolated polynucleotide which also encode
polypeptides which are identical, homologous or related to that
encoded by the polynucleotides.
[0119] The present invention also includes polynucleotides capable
of hybridizing under stringent conditions, preferably highly
stringent conditions, to polynucleotides described herein. Highly
stringent conditions include, for example, 0.2.times.SSC at
65.degree. C.; stringent conditions include, for example,
4.times.SSC at 65.degree. C. or 50% formamide and 4.times.SSC at
42.degree. C. Preferably, such hybridizing polynucleotides are at
least 70% homologous by sequence identity (more preferably, at
least 80% homologous; most preferably 90% or 95% homologous) with
the polynucleotide of the present invention to which they
hybridize.
Preferred Chimeric Polypeptides And Polynucleotides Encoding
them
[0120] Amino acid sequences of chimeric chemokine polypeptides are
set forth below, along with the sequences of polynucleotides
encoding them. In these chimeric polypeptides, the chemokine has
been linked to an Fc polypeptide by a [Gly-Ser].sub.5 linker
peptide. The polynucleotides encoding these chimeric polypeptides
were derived from chemokine cDNA sequences and genomic Fc
sequences, as described in Example 1 below.
[0121] The sequence of a polynucleotide encoding one such chimeric
polypeptide including an SDF-1.alpha. domain is set forth in SEQ ID
NO:2, with the protein-coding sequence (including introns)
extending from nucleotide 12 to 1213. This polynucleotide has been
identified as S1-2 or S1-3, the DNA sequences of these two
constructs appearing to be identical. The amino acid sequence of
the chimeric polypeptide encoded by S1-2 and S1-3 is set forth in
SEQ ID NO:1. The chimeric polypeptide encoded by S1-2 and S1-3 is
328 amino acids in length, with the mature polypeptide produced by
cleavage of the secretory leader sequence beginning at amino acid
20, 21, or 22 of SEQ ID NO:1, depending on how the polypeptide is
processed. The polynucleotide construct S1-3 was deposited with the
American Type Culture Collection on Feb. 28, 1997 and given the
accession number ATCC XXXXX.
[0122] The sequence of a polynucleotide encoding another such
chimeric polypeptide that includes a domain derived from
SDF-1.alpha. is set forth in SEQ ID NO:4, with the protein-coding
sequence (including introns) extending from nucleotide 12 to 1207.
This polynucleotide has been identified as SK2-2. The amino acid
sequence of the chimeric polypeptide encoded by SK2-2 is set forth
in SEQ ID NO:3. The chimeric polypeptide encoded by SK2-2 is 326
amino acids in length, with the mature polypeptide produced by
cleavage of the secretory leader sequence beginning at amino acid
20 of SEQ ID NO:3. The polypeptide encoded by SK2-2 differs from
that encoded by S1-2 and S1-3 in that two amino acids have been
deleted from the SK2-2 sequence so that cleavage of the secretory
leader sequence is predicted to always produce a product beginning
at amino acid 20 of SEQ ID NO:3. The polynucleotide construct SK2-2
was deposited with the American Type Culture Collection on Feb. 28,
1997 and given the accession number ATCC XXXXX.
[0123] The sequence of a polynucleotide encoding a chimeric
polypeptide that includes an MIP-1.alpha. domain is set forth in
SEQ ID NO:6, with the protein-coding sequence (including introns)
extending from nucleotide 15 to 1225. This polynucleotide is
identified as MP-1. The DNA sequence of MP-1 has been determined,
and while the DNA sequences of MP-2 and MP-6 are anticipated to be
identical to that of MP-1, these clones may contain some
PCR-generated DNA sequence alterations. The amino acid sequence of
the chimeric polypeptide encoded by MP-1, and presumably encoded by
MP-2 and MP-6, is set forth in SEQ ID NO:5. The chimeric
polypeptide encoded by MP-1 is 331 amino acids in length, with the
mature polypeptide produced by cleavage of the secretory leader
sequence beginning at amino acid 23 of SEQ ID NO:5. The
polynucleotide constructs MP-1, MP-2, and MP-6 were deposited with
the American Type Culture Collection on Feb. 28, 1997 and given the
accession numbers ATCC XXXXX, XXXXX, and XXXXX.
[0124] The sequence of a polynucleotide encoding a chimeric
polypeptide that includes an MIP-1.beta. domain is set forth in SEQ
ID NO:8, with the protein-coding sequence (including introns)
extending from nucleotide 16 to 1226. This polynucleotide is
identified as MPB-X. The amino acid sequence of the chimeric
polypeptide encoded by MPB-X is set forth in SEQ ID NO:7. The
chimeric polypeptide encoded by MPB-X is predicted to be 331 amino
acids in length, with the mature polypeptide produced by cleavage
of the secretory leader sequence beginning at amino acid 24 of SEQ
ID NO:7.
Expression and Purification of Chimeric Polypeptides
[0125] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
14, 4485-4490 (1991), in order to produce the protein
recombinantly. Many suitable expression control sequences are known
in the art. General methods of expressing recombinant proteins are
also known and are exemplified in R. Kaufman, Methods in Enzymology
185, 537-566 (1990). As defined herein "operably linked" means that
the isolated polynucleotide of the invention and an expression
control sequence are situated within a vector or cell in such a way
that the protein is expressed by a host cell which has been
transformed (transfected) with the ligated
polynucleotide/expression control sequence.
[0126] A number of types of cells may act as suitable host cells
for expression of the protein. Mammalian host cells include, for
example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human
kidney 293 cells, human epidermal A431 cells, human Colo205 cells,
3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells.
[0127] Alternatively, it may be possible to produce the protein in
lower eukaryotes such as yeast or in prokaryotes such as bacteria.
Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing heterologous proteins. If
the protein is made in yeast or bacteria, it may be necessary to
modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the
functional protein. Such covalent attachments may be accomplished
using known chemical or enzymatic methods.
[0128] The protein may also be produced by operably linking the
isolated polynucleotide of the invention to suitable control
sequences in one or more insect expression vectors, and employing
an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially
available in kit form from, e.g., Invitrogen, San Diego, Calif.,
U.S.A. (the MaxBac.RTM. kit), and such methods are well known in
the art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present invention is "transformed."
[0129] The protein of the invention may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by
somatic or germ cells containing a nucleotide sequence encoding the
is protein.
[0130] Alternatively, the protein of the invention may also be
expressed in a form which will facilitate purification. For
example, it may be expressed as a fusion protein, such as those of
maltose binding protein (MBP), glutathione-5-transferase (GST) or
thioredoxin (TRX). Kits for expression and purification of such
fusion proteins are commercially available from New England BioLabs
(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen,
respectively. The protein can also be tagged with an epitope and
subsequently purified by using a specific antibody directed to such
epitope. One such epitope ("Flag") is commercially available from
Kodak (New Haven, Conn.).
[0131] The protein of the invention may be prepared by culturing
transformed host cells under culture conditions suitable to express
the recombinant protein. The resulting expressed protein may then
be purified from such culture (i.e., from culture medium or cell
extracts) using known purification processes, such as gel
filtration and ion exchange chromatography. The purification of the
protein may also include an affinity column containing agents which
will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, Heparin-Toyopearl.RTM.
or Cibacrom blue 3GA Sepharose.RTM.; one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl
ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
[0132] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a substantially homogeneous isolated
recombinant protein. The protein thus purified is substantially
free of other mammalian proteins and is defined in accordance with
the present invention as an "isolated protein."
[0133] The protein may also be produced by known conventional
chemical synthesis. Methods for constructing the proteins of the
present invention by synthetic means are known to those skilled in
the art. The synthetically-constructed protein sequences, by virtue
of sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins may possess biological
properties in common therewith, including protein activity. Thus,
they may be employed as biologically active or immunological
substitutes for natural, purified proteins in screening of
therapeutic compounds and in immunological processes for the
development of antibodies.
Uses of Chimeric Polypeptides
[0134] Chimeric chemokine polypeptides can be used as tools for
identifying cells expressing receptor for the chemokine, or for
studying binding of chemokine to isolated receptor molecules. The
construct when incubated with cells expressing a receptor for the
chemokine will bind to these cells and can be indicated using a
commercially available fluorescently tagged antibody, or other
protein, able to bind to the heterologous polypeptide domain, such
as the Fc region of human immunoglobulin, of the chimeric
polypeptide. This will indicate cells having a surface receptor for
a given chemokine as well as the density of this receptor on the
cell surface.
[0135] Interactions between chimeric chemokine polypeptides and
chemokine receptors can also be detected directly by measuring
changes in surface plasmon resonance using a Biacore sensor
(Pharmacia). The chemokine receptor or the chimeric polypeptide can
be covalently immobilized to different flow cells on the Biacore
sensor chip as recommended by the manufacturer. Molecules to be
tested for interaction are then injected across the flow cells and
binding is detected as a change in resonance units, a reflection of
the mass of protein bound to the sensor chip surface.
[0136] Other suitable assays for receptor-ligand activity include
without limitation those described in: Current Protocols in
Immunology, edited by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, published by Greene
Publishing Associates and Wiley-Interscience (Chapter 7.28,
Measurement of Cellular Adhesion under static conditions
7.28.1-7.28.22); Takai et al., Proc. Natl. Acad. Sci. USA
84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156,
1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg
et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell
80:661-670, 1995; all of which are incorporated herein by
reference.
[0137] Chimeric chemokine polypeptides can also be used as vaccine
adjuvants. Proteins and glycoproteins injected to induce an immune
response must bind to surface of B lymphocytes to stimulate
antibody production and must be taken up by antigen presenting
cells, processed, and represented to T lymphocytes to mediate a T
lymphocyte response. By including with the antigen injection a
chimeric chemokine-Fc polypeptide the infiltration of the necessary
APCs and lymphocytes will be induced by the chemoattractive
presence of the chemokine. One advantage of including an Fc domain
in the chimeric polypeptide is that the chimeric polypeptide will
have a longer biological half life than the chemokine alone would
have. Also, by including in the chimeric polypeptide an Fc domain
able to bind to existing Fc-receptors on cells at the site of
injection, the chemokine activity will be concentrated at the site,
much like a depot so that the chemokine gradient could be
maintained over a long enough period of time to ensure the
infiltration of the necessary responding cell populations.
[0138] Chimeric chemokine polypeptides can also be used to enhance
the activity of antigen-presenting cells (APCs). The presence of
the chemokine domain of the chimeric polypeptide would
chemotactically attract APCs. Additionally, an antigenic molecule
could be included in the chimeric polypeptide for delivery to the
APC. When such an antigen-containing chimeric polypeptide binds to
the surface of an APC and is internalized, and the chimeric
polypeptide is degraded within the APC, the antigenic portion of
the chimeric polypeptide would be freed for interaction with MHC
proteins and presentation on the surface of the APC.
[0139] Chimeric chemokine polypeptides can also be used to affect
the chemotactic recruitment of migratory cells. Chimeric chemokines
may be used to establish a chemoattractive gradient for migratory
cells that are expressing the appropriate chemokine receptors, or
to obscure an existing chemoattractive gradient. By including a
large or particularly stable heterologous polypeptide in the
chimeric polypeptide, the chimeric polypeptide will have a longer
biological half life and will be able to establish a longer lasting
chemoattractive gradient, and will be more effective in obscuring a
preexisting gradient. Also, a heterologous polypeptide domain may
be selected that, by binding to particular molecules or cells, will
target the chimeric chemokine polypeptide to a particular site in
order to establish a chemoattractive gradient at that site. By
altering chemoattractive gradients, chimeric chemokine polypeptides
can be used to treat inflammatory and autoimmune disorders that
require the recruitment of migratory cells. Also, by attracting to
a particular site migratory cells that produce other intercellular
factors such as IL-8 or IP-10, chimeric chemokine polypeptides may
for example be used to stimulate angiogenesis at that site (if, for
example, the recruited migratory cells were secreting IL-8) or to
inhibit angiogenesis at that site (if, for example, the recruited
migratory cells were secreting IP-10). In addition, by establishing
a gradient of chimeric chemokine polypeptide within the bone marrow
of a bone marrow transplant recipient, the chimeric chemokine
polypeptide could be used to recruit the transplanted bone marrow
cells to the bone marrow where they are needed. Similarly, other
cellular processes could be affected by chimeric chemokine
polypeptides, by using them to attract particular classes of
migratory cells secreting determined factors.
[0140] Chimeric chemokine polypeptides can also be used to affect
the nature of chemokine-receptor interactions, and may block the
binding of endogenous molecules to their receptors. By binding to a
receptor, chimeric chemokines may deliver a signal similar to that
received via the normal ligand. When the heterologous polypeptide
is an Fc polypeptide, because of its bivalent nature this signal
may be delivered at a lower molar concentration of ligand. The
signal delivered by binding the chimeric polypeptide may have some
properties different from that of the normal ligand because of the
structure of the chimeric polypeptide. This could include prolonged
triggering/activation or decreased activation. The chimeric
polypeptides, because of their larger size or the nature of the
structure of the heterologous polypeptide domain, will have a
longer half life in vivo compared to monomeric ligand, possibly
leading to prolonged signaling/activation. Also the larger size of
the chimeric polypeptide will cause some stearic hindrance which
may block the binding of the natural ligand. A chimeric chemokine
polypeptide may desensitize a receptor's response to normal ligand
by binding and inactivating further signaling through the same
receptor. In the case where a receptor has more than one signaling
function, the chimeric chemokine polypeptide may inhibit one form
of signaling while enhancing or altering another. Also, a chimeric
chemokine polypeptide may bind to a receptor and cause down
regulation and/or internalization of the receptor. Additionally, a
chimeric chemokine polypeptide may bind to a receptor and cause the
internalization and destruction of the receptor, thus preventing it
from recycling to the membrane surface. Also, by binding to one
receptor a chimeric polypeptide may cause another receptor or
membrane protein to become desensitized or unable to carry out its
normal function.
[0141] Chimeric chemokine polypeptides can also be used to prevent
infection of cells by HIV or other viruses by blocking the binding
of virus to chemokine receptors. The chimeric chemokine polypeptide
including SDF-1.alpha. and Fc polypeptides has been shown to bind
to cells expressing the fusin/CXCR4 receptor. This binding will
block HIV-1 isolates that are T-tropic from infecting
fusin-positive cells in multiple ways: competing with HIV for
existing chemokine receptors, down-regulation of the chemokine
receptors by internalization, as well as desensitization of
receptors required by HIV for infection. In a similar manner the
construct consisting of an MIP-1.alpha. or MIP-1.beta. polypeptide
and an Fc polypeptide will bind to cells expressing the CCR5
receptor. This binding will block HIV-1 isolates that are M-tropic
from infecting CCR5-positive cells in multiple ways: competing with
HIV for existing chemokine receptors, down-regulation of the
chemokine receptors by internalization, as well as desensitization
of receptors required by HIV for infection. Alterations of the
chimeric polypeptide, such as additions of amino acids at the
N-terminus of the chemokine domain, may result in enhanced binding
with loss of signaling, resulting in strong antagonism. By making
chimeric chemokine polypeptides with several different chemokines a
wide range of chemokine receptors can be inhibited or desensitized,
thus blocking viral isolates that have mutated to infect cells
using other chemokine receptors. It is also possible to modify a
chemokine sequence so that it will bind to a wider array of
receptors; thus, one construct could bind to CCR5 as well as other
CCR receptors and another construct could bind to CXCR4 as well as
a variety of other CXCR receptors. By simultaneously administering
a combination of chimeric chemokine polypeptides, the greatest
number of chemokine receptor types could be protected from binding
by HIV or other viral isolates.
Administration and Dosing
[0142] A chimeric polypeptide of the present invention (from
whatever source derived, including without limitation from
recombinant and non-recombinant sources) may be used in a
pharmaceutical composition when combined with a pharmaceutically
acceptable carrier. Such a composition may also contain (in
addition to polypeptide and a carrier) diluents, fillers, salts,
buffers, stabilizers, solubilizers, and other materials well known
in the art. The term "pharmaceutically acceptable" means a
non-toxic material that does not interfere with the effectiveness
of the biological activity of the active ingredient(s). The
characteristics of the carrier will depend on the route of
administration. The pharmaceutical composition of the invention may
also contain cytokines, chemokines, lymphokines, or other
hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem cell factor, and erythropoietin. The
pharmaceutical composition may further contain other agents which
either enhance the activity of the polypeptide or compliment its
activity or use in treatment. Such additional factors and/or agents
may be included in the pharmaceutical composition to produce a
synergistic effect with protein of the invention, or to minimize
side effects. Conversely, polypeptides of the present invention may
be included in formulations of the particular cytokine, lymphokine,
other hematopoietic factor, thrombolytic or anti-thrombotic factor,
or anti-inflammatory agent to minimize side effects of the
cytokine, lymphokine, other hematopoietic factor, thrombolytic or
anti-thrombotic factor, or anti-inflammatory agent.
[0143] A polypeptide of the present invention may be active in
multimers (e.g., heterodimers or homodimers) or complexes with
itself or other proteins. As a result, pharmaceutical compositions
of the invention may comprise a polypeptide of the invention in
such multimeric or complexed form.
[0144] The pharmaceutical composition of the invention may be in
the form of a complex of the polypeptide(s) of present invention
along with protein or peptide antigens. The protein and/or peptide
antigen will deliver a stimulatory signal to both B and T
lymphocytes. B lymphocytes will respond to antigen through their
surface immunoglobulin receptor. T lymphocytes will respond to
antigen through the T cell receptor (TCR) following presentation of
the antigen by MHC proteins. MHC and structurally related proteins
including those encoded by class I and class II MHC genes on host
cells will serve to present the peptide antigen(s) to T
lymphocytes. The antigen components could also be supplied as
purified MHC-peptide complexes alone or with co-stimulatory
molecules that can directly signal T cells. Alternatively
antibodies able to bind surface immunoglobulin and other molecules
on B cells as well as antibodies able to bind the TCR and other
molecules on T cells can be combined with the pharmaceutical
composition of the invention.
[0145] The pharmaceutical composition of the invention may be in
the form of a liposome in which protein of the present invention is
combined, in addition to other pharmaceutically acceptable
carriers, with amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals,
or lamellar layers in aqueous solution. Suitable lipids for
liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal
formulations is within the level of skill in the art, as disclosed,
for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728;
U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which
are incorporated herein by reference.
[0146] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that is sufficient to show a
meaningful patient benefit, i.e., treatment, healing, prevention or
amelioration of the relevant medical condition, or an increase in
rate of treatment, healing, prevention or amelioration of such
conditions. When applied to an individual active ingredient,
administered alone, the term refers to that ingredient alone. When
applied to a combination, the term refers to combined amounts of
the active ingredients that result in the therapeutic effect,
whether administered in combination, serially or
simultaneously.
[0147] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of polypeptide of the
present invention is administered to an organism, preferably a
mammal, having a condition to be treated. Chimeric polypeptides of
the present invention may be administered in accordance with the
method of the invention either alone or in combination with other
therapies such as treatments employing cytokines, lymphokines or
other hematopoietic factors. When co-administered with one or more
cytokines, lymphokines or other hematopoietic factors, polypeptides
of the present invention may be administered either simultaneously
with the cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors, or sequentially. If
administered sequentially, the attending physician will decide on
the appropriate sequence of administering protein of the present
invention in combination with cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
[0148] Administration of polypeptides of the present invention used
in the pharmaceutical composition or to practice the method of the
present invention can be carried out in a variety of conventional
ways, such as oral ingestion, inhalation, topical application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous
injection. Intravenous administration to the patient is
preferred.
[0149] When a therapeutically effective amount of polypeptide of
the present invention is administered orally, polypeptide of the
present invention will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% polypeptide
of the present invention, and preferably from about 25 to 90%
polypeptide of the present invention. When administered in liquid
form, a liquid carrier such as water, petroleum, oils of animal or
plant origin such as peanut oil, mineral oil, soybean oil, or
sesame oil, or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition
contains from about 0.5 to 90% by weight of polypeptide of the
present invention, and preferably from about 1 to 50% polypeptide
of the present invention.
[0150] When a therapeutically effective amount of polypeptide of
the present invention is administered by intravenous, cutaneous or
subcutaneous injection, polypeptide of the present invention will
be in the form of a pyrogen-free, parenterally acceptable aqueous
solution. The preparation of such parenterally acceptable
polypeptide solutions, having due regard to pH, isotonicity,
stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to chimeric
polypeptide of the present invention, an isotonic vehicle such as
Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,
Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or other vehicle as known in the art. The pharmaceutical
composition of the present invention may also contain stabilizers,
preservatives, buffers, antioxidants, or other additives known to
those of skill in the art.
[0151] The amount of polypeptide of the present invention in the
pharmaceutical composition of the present invention will depend
upon the nature and severity of the condition being treated, and on
the nature of prior treatments which the patient has undergone.
Ultimately, the attending physician will decide the amount of
chimeric polypeptide of the present invention with which to treat
each individual patient. Initially, the attending physician will
administer low doses of polypeptide of the present invention and
observe the patient's response. Larger doses of polypeptide of the
present invention may be administered until the optimal therapeutic
effect is obtained for the patient, and at that point the dosage is
not increased further. It is contemplated that the various
pharmaceutical compositions used to practice the method of the
present invention should contain about 0.01 ng to about 100 mg
(preferably about 0.1 .mu.g to about 10 mg, more preferably about
0.1 .mu.g to about 1 mg) of polypeptide of the present invention
per kg body weight.
[0152] The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the
severity of the disease being treated and the condition and
potential idiosyncratic response of each individual patient. It is
contemplated that the duration of each application of the
polypeptide of the present invention will be in the range of 12 to
24 hours of continuous intravenous administration. Ultimately the
attending physician will decide on the appropriate duration of
intravenous therapy using the pharmaceutical composition of the
present invention.
[0153] Polypeptide of the invention may also be used to immunize
animals to obtain polyclonal and monoclonal antibodies which
specifically react with the chimeric polypeptide. Such antibodies
may be obtained using either the entire chimeric polypeptide or
fragments thereof as an immunogen, the fragments preferably
comprising portions of both the chemokine and heterologous
polypeptide domains. The peptide immunogens additionally may
contain a cysteine residue at the carboxyl terminus, and are
conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
Methods for synthesizing such peptides are known in the art, for
example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154
(1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).
Monoclonal antibodies binding to the polypeptide of the invention
may be useful diagnostic agents for the immunodetection of the
polypeptide. Neutralizing monoclonal antibodies binding to the
chimeric polypeptide may also be useful therapeutics for both
conditions associated with the chemokine corresponding to the
chemokine domain of the chimeric polypeptide and also in the
treatment of some forms of cancer where abnormal expression of that
chemokine is involved. In the case of cancerous cells or leukemic
cells, neutralizing monoclonal antibodies against the chimeric
polypeptide may be useful in detecting and preventing the
metastatic spread of the cancerous cells, which may be mediated by
the chemokine corresponding to the chemokine domain of the chimeric
polypeptide.
[0154] For compositions of the present invention which are useful
for bone, cartilage, tendon, or ligament regeneration, the
therapeutic method includes administering the composition
topically, systematically, or locally as an implant- or device.
When administered, the therapeutic composition for use in this
invention is, of course, in a pyrogen-free, physiologically
acceptable form. Further, the composition may desirably be
encapsulated or injected in a viscous form for delivery to the site
of bone, cartilage, or tissue damage. Topical administration may be
suitable for wound healing and tissue repair. Therapeutically
useful agents other than a polypeptide of the invention which may
also optionally be included in the composition as described above,
may alternatively or additionally be administered simultaneously or
sequentially with the composition in the methods of the invention.
Preferably for bone and/or cartilage formation, the composition
would include a matrix capable of delivering the
polypeptide-containing composition to the site of bone and/or
cartilage damage, providing a structure for the developing bone and
cartilage and optimally capable of being resorbed into the body.
Such matrices may be formed of materials presently in use for other
implanted medical applications.
[0155] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance, and
interface properties. The particular application of the
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium 20 wt %, preferably 1-10 wt % based on total
formulation weight, which represents the amount necessary to
prevent desorption of the chimeric polypeptide from the polymer
matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from
infiltrating the matrix, thereby providing the polypeptide the
opportunity to assist the osteogenic activity of the progenitor
cells.
[0156] In further compositions, polypeptides of the invention may
be combined with other agents beneficial to the treatment of the
bone and/or cartilage defect, wound, or tissue in question. These
agents include various growth factors such as epidermal growth
factor (EGF), platelet derived growth factor (PDGF), transforming
growth factors (TGF-.alpha. and TGF-.beta.), and insulin-like
growth factor (IGF).
[0157] The therapeutic compositions are also presently valuable for
veterinary applications. Particularly domestic animals and
thoroughbred horses, in addition to humans, are desired patients
for such treatment with polypeptides of the present invention.
[0158] The dosage regimen of a polypeptide-containing
pharmaceutical composition to be used in tissue regeneration will
be determined by the attending physician considering various
factors which modify the action of the chimeric polypeptides, e.g.,
amount of tissue weight desired to be formed, the site of damage,
the condition of the damaged tissue, the size of a wound, type of
damaged tissue (e.g., bone), the patient's age, sex, and diet, the
severity of any infection, time of administration, and other
clinical factors. The dosage may vary with the type of matrix used
in the reconstitution and with inclusion of other polypeptides in
the pharmaceutical composition. For example, the sulfate,
tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid, and polyanhydrides. Other potential materials are
biodegradable and biologically well-defined, such as bone or dermal
collagen. Further matrices are comprised of pure proteins or
extracellular matrix components. Other potential matrices are
nonbiodegradable and chemically defined, such as sintered
hydroxapatite, bioglass, aluminates, or other ceramics. Matrices
may be comprised of combinations of any of the above mentioned
types of material, such as polylactic acid and hydroxyapatite or
collagen and tricalciumphosphate. The bioceramics may be altered in
composition, such as in calcium-aluminate-phosphate and processing
to alter pore size, particle size, particle shape, and
biodegradability.
[0159] Presently preferred is a 50:50 (mole weight) copolymer of
lactic acid and glycolic acid in the form of porous particles
having diameters ranging from 150 to 800 microns. In some
applications, it will be useful to utilize a sequestering agent,
such as carboxymethyl cellulose or autologous blood clot, to
prevent the chimeric polypeptide compositions from disassociating
from the matrix.
[0160] A preferred family of sequestering agents is cellulosic
materials such as alkylcelluloses (including
hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most
preferred being cationic salts of carboxymethylcellulose (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium
alginate, poly(ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-addition of other known
growth factors, such as IGF I (insulin like growth factor I), to
the final composition, may also effect the dosage. Progress can be
monitored by periodic assessment of tissue/bone growth and/or
repair, for example, X-rays, histomorphometric determinations, and
tetracycline labeling.
[0161] Polynucleotides of the present invention can also be used
for gene therapy. Such polynucleotides can be introduced either in
vivo or ex vivo into cells for expression in a mammalian subject.
Polynucleotides of the invention may also be administered by other
known methods for introduction of nucleic acid into a cell or
organism (including, without limitation, in the form of viral
vectors or naked DNA).
[0162] Cells may also be cultured ex vivo in the presence of
chimeric polypeptides of the present invention in order to
proliferate or to produce a desired effect on or activity in such
cells. Treated cells can then be introduced in vivo for therapeutic
purposes.
[0163] Patent and literature references cited herein are
incorporated by reference as if fully set forth.
[0164] The following examples illustrate embodiments of the present
invention, but are not intended to limit the scope of the
disclosure.
EXAMPLE 1
Construction of Plasmids Encoding Chimeric Polypeptides
[0165] Plasmids containing chimeric gene constructions were created
by ligating together four DNA fragments: a chemokine-encoding
fragment, a fragment containing a linker and part of the Fc portion
of the IgG4 gene, a fragment containing the rest of the Fc portion
of the IgG4 gene, and a vector fragment. The resulting plasmid
includes a chemokine-encoding sequence joined in-frame to a
[Glycine-Serine].sub.5 linker sequence that is joined in-frame to
the first codon for the hinge region of human-IgG4. The Fc region
of this chimeric gene is comprised of the hinge, CH2, and CH3
regions of human IgG4, including introns, and several bases
downstream from the IgG4 stop codon. The Fc portion of this
chimeric gene construct also includes two amino acid changes which
result in reduced Fc-receptor binding and complement fixation.
Chemokine-Encoding Fragments:
[0166] The SDF1-.alpha. fragment of clones S1-2 and S1-3 was
generated using PCR with human SDF-1.alpha. cDNA as template, and
with PCR primers adding NotI and BamHI sites to the upstream and
downstream ends of the SDF-1.alpha. sequence, respectively. The
SDF1-.alpha. fragment of clones S1-2 and S1-3 consists of eleven
bases upstream of the initiating ATG of the signal sequence,
through the final codon of the mature protein sequence. The DNA
sequences of clones S1-2 and S1-3 appear to be identical. The
SDF1-.alpha. fragment of clone SK2-2 was constructed similarly to
that of S1-2 and S1-3, except that the upstream primer adding the
NotI site extended through the signal sequence and into the mature
protein coding sequence, with a deletion of the six nucleotides for
amino acids 20 and 21 of the protein encoded by clones S1-2 and
S1-3. The MIP1-.alpha. fragment of clones MP-1, MP-2, and MP-6 was
generated using PCR with human MIP-1.alpha. cDNA (the HUMCYTNEWA
allele) as template, and with PCR primers adding NotI and BamHI
sites to the upstream and downstream ends of the MIP-1.alpha.
sequence, respectively. The protein sequence for MIP-1.alpha.
sequence is that derived from HUMCYTNEWA (SEQ ID NO:9); there is
another MIP-1.alpha. allele which is not present in all humans, but
HUMCYTNEWA is present in all humans. Some nucleotides were changed
by the 3' MIP-1.alpha. primer in the PCR, but these nucleotide
changes do not alter the amino acid sequence. The DNA sequence of
MP-1 has been determined and while the DNA sequences of MP-2 and
MP6 are anticipated to be identical to that of MP-1, these clones
may contain some PCR-generated DNA sequence alterations. The
MIP-1.alpha. fragment of clones MP-1, MP-2, and MP-6 consists of
fourteen bases upstream of the initiating ATG of the signal
sequence, through the final codon of the mature protein
sequence.
[0167] The MIP1-.beta. fragment of clone MPB-X is generated using
PCR with human PHA-stimulated T-cell cDNA as template, and with PCR
primers adding NotI and BamHI sites to the upstream and downstream
ends of the MIP-1.beta. sequence, respectively. The nucleotide and
protein sequences for MIP-1.beta. sequence are derived from HUMACTA
(SEQ ID NO:10). The MIP-1.beta. fragment is predicted to consist of
fifteen bases upstream of the initiating ATG of the signal
sequence, through the final codon of the mature protein
sequence.
Linker and Partial IgG4 Fc Fragment
[0168] All of the chimeric gene constructions use the same DNA
fragment encoding a [Glycine-Serine].sub.5 linker sequence and a
portion of the Fc region of human IgG4. Mutations were introduced
into the IgG4 sequence, so that two amino acids in the CH2 region
were changed from wild-type (in SEQ ID NO:1, 116 L has been changed
to E, and 118 G has been changed to A, with corresponding
nucleotide changes). The IgG4 sequence in this fragment contains an
intron (nucleotides 346 to 463 in SEQ ID NO:2). The linker/partial
Fc fragment was generated using PCR from the mutated human IgG4
sequence, with plasmid G081 (phhcd28.2higg4mcys) as template, and
with one PCR primer adding a BamHI site and the Gly-Ser linker
region to the 5' end and the other PCR primer adding a SacII site
to the 3' end.
Remainder of IgG4 Fc Fragment
[0169] This DNA fragment was generated by restriction enzyme
digestion with SacII and EcoRI from the plasmid G022 encoding human
IgG4 (CD28-IgG4), and purified. The IgG4 sequence in this fragment
contains an intron (nucleotides 794 to 890 in SEQ ID NO:2). In the
human IgG4 sequence of this fragment, a base-pair change from
wild-type IgG4 sequence (in SEQ ID NO:2, base 832, C has been
changed to T) was found in the intron (non-coding) region, which is
expected to have no effect on expression or composition of the gene
product encoded by the chimeric gene construct.
Vector Fragment
[0170] This fragment was derived from the pED Fc vector by
digestion with NotI and EcoRI to remove the human IgG1 insert,
resulting in a vector fragment with COS and CHO mammalian
expression sequences that is similar to the pED vector.
EXAMPLE 2
Expression And Purification of Chimeric Polypeptides
[0171] Chimeric polypeptides S1-3, SK2-2, MP1, MP2, and MP6,
encoded by chimeric plasmid constructs, were expressed by transient
expression in COS cells and released into the cell culture medium.
COS 1 (clone M6) cells were transiently transfected with the
appropriate plasmid, using Lipofectamine.TM. Reagent (GibcoBRL) and
following the procedure given in the product insert, with the
following modifications. COS cells are seeded into 100-mm tissue
culture dishes 16-24 hours prior to transfection, at about
1-1.5.times.10.sup.6 cells per plate, in complete DME medium (DME
plus 10% fetal bovine serum, 2 mM glutamine, and 100 units each
penicillin and streptomycin). All incubations of COS cells were at
37 degrees C. in 10% CO.sub.2. For each culture dish of cells, 8
.mu.g plasmid DNA and 48 .mu.l Lipofectamine.TM. Reagent are mixed
in 0.8 ml DME. After 30 minutes at room temperature, 3.2 ml DME
(plus 2 mM glutamine and 100 units each penicillin and
streptomycin) are added to the DNA-Lipofectamine.TM. Reagent
mixture, mixed, and layered on top of the DME-washed COS cells.
After 18 to 24 hours of incubation, this medium is replaced with
complete DME medium. After an additional 2 to 4 hours incubation,
the COS plates are washed twice with 5-10 ml DME, and 10 ml DME
medium without serum (plus 2 mM glutamine) is added. After an
additional 36 to 48 hours incubation the medium is collected, with
any COS cells removed by centrifugation. The chimeric polypeptide
MPB-X can also be expressed in a similar fashion.
[0172] The secreted chimeric polypeptide can be purified from this
medium, or the medium can be used in various assays after
quantitation of the amount of chimeric polypeptide by ELISA, using
human IgG4 Kappa of known concentration to generate a standard
curve.
[0173] Concentrations of the expressed chimeric polypeptides
secreted into the cell culture medium were determined by ELISA
using human IgG4 as a standard, and the results are shown in the
table below.
TABLE-US-00001 TABLE 1 Concentration (.mu.g/ml): Plasmid Construct:
Experiment 1: Experiment 2: S1-2 0.8 not done S1-3 1.5 5.0 SK2-2
1.0 5.5
[0174] Chimeric polypeptides were purified from cell culture
supernatants by immunoprecipitation using Protein A Sepharose.RTM.
(Phamacia CL4B). For example, chimeric polypeptides can be purified
from 75 ml of conditioned medium by the following method. Adjust
the conditioned medium to 50 mM Tris, pH 7.5. Add 100 mg Protein A
Sepharose suspended in about 1 ml PBS. Incubate with rotation at 4
degrees C. overnight. Sodium azide may be added. Collect Protein A
Sepharose by centrifugation, and transfer it to a BioRad
Poly-Prep.RTM. column. Wash the Sepharose with 10 to 20 ml PBS.
Elute the chimeric polypeptide with 12 mM HCl, and immediately
neutralize the eluant by adjusting it to 50 mM Tris, pH 7.5. Elute
in steps by suspending the Sepharose in 2 ml 12 mM HCl and
collecting 1 ml fractions. The amount of chimeric polypeptide in
the fractions can be quantitated by ELISA.
[0175] FIG. 1, panels A-D, depicts SDS-PAGE gels stained with
Coomasie Blue demonstrating the expression of chimeric chemokine
polypeptides in mammalian COS cells. The chimeric polypeptides were
purified using protein-A, then electrophoresed on SDS-PAGE gels
under reducing and non-reducing conditions. The SDF1-.alpha.-Fc
chimeric polypeptides S1-3 and SK2-2 and the MIP-1.alpha.-Fc
chimeric polypeptides MP-1, MP-2, and MP-6 migrated as bands with a
M.sub.r of .about.40 kD under reducing conditions and .about.80 kD
under non-reducing conditions.
EXAMPLE 3
Binding of Chimeric Polypeptides to Cells Expressing Receptors
[0176] Several human cell lines have been stained using the SDF-Fc
chimeric polypeptides, demonstrating binding of the chimeric
polypeptides to receptors expressed by these cells. A typical
binding assay is described below. Cells were incubated on ice for a
short period of time (15-60 minutes) in media containing of 2-10%
FCS, 0-0.02% BSA, 0-0.02% rabbit serum, and 0.02-0.1% azide. The
SDF-Fc chimeric polypeptide was added at concentrations of 0.5-2
.mu.g/ml. After incubation with occasional mixing samples were
washed with 5-6 mls of the above media. In parallel cells were
stained with a mouse monoclonal antibody (12G5, IgG2a) specific for
fusin/CXCR4, added at 5-20 .mu.g/ml. For negative controls a human
IgG4 or a mouse IgG2a were used at 5-20 .mu.g/ml. The cells were
then incubated for a short period of time with 100 .mu.l of a 1:100
dilution of the second or detecting antibody. The detecting
antibody used was a goat anti-human IgG F(ab)'2 antibody (for the
human IgG4 controls and the SDF-Fc samples) or a goat anti-mouse
IgG F(ab')2 antibody (for the mouse Ig controls, murine anti-human
fusin, and murine anti-human cell-surface proteins or CD3) that was
labeled with PE fluorescence. After another 15-60 minutes on ice
with occasional mixing followed by an extensive wash with 5-6 ml of
staining media, the cells were resuspended in 400 .mu.l and
analyzed using a FACSCAN (BD) fluorescence-activated cell
analyzer.
[0177] Table 2 shows the results of staining Jurkat and U937 cells
by binding anti-fusin antibody or the chimeric SDF-1.alpha.
chemokine polypeptides S1-3 or SK2-2 to them. Detection of
fusin/CXCR4 expression by a human T cell line and a human monocyte
line using a fusin-specific mAb (12G5) is comparable to detection
with SDF-Fc constructs SK2-2 or S1-3. Jurkat cells, derived from a
patient with acute T cell leukemia, and U937, a macrophage-like
cell line derived from a patient with histiocytic lymphoma, were
used. About 5.times.10.sup.5 cells were added to 12.times.75 mm
plastic tubes in 50 .mu.l of staining buffer consisting of
RPMI-1640 (phenol red free with 10 mM HEPES) or PBS containing 2%
FCS, 2% rabbit serum, and 0.1% azide. Anti-fusin staining controls
consisted of either media only or a mouse IgG2a control antibody
and were equivalent in staining. The anti-fusin mAb 12G5 was added
at a final concentration of 16 .mu.g/ml (Exp. 1) or 20 .mu.g/ml
(Exp. 2) diluted in staining buffer. After 30 minutes on ice with
mixing the cells were washed with 5 ml of staining buffer and 100
.mu.l of a 1:100 dilution of Goat anti-mouse Ig PE (Southern
Biotech) was added to detect cell-bound mouse antibody. For SDF-Fc
staining the control consisted of either media only or human IgG4
antibody. The SDF-Fc constructs were added at a final concentration
of 0.5 .mu.g/ml for the SK2-2 in Exp. 1 or at 1 .mu.g/ml for the
S1-3 in Exp. 2. After 30 minutes on ice with mixing the cells were
washed with 5 ml of staining buffer and 100 .mu.l of a 1:100
dilution of goat anti-human Ig PE was added to detect cell-bound
SDF-Fc. The control antibodies gave no increase over the second
antibody only. The staining with anti-fusin (12G5) was equivalent
to that seen with the SDF-Fc constructs, indicating that all human
cells that were expressing the fusin receptor, as shown by
anti-fusin antibody binding, also bound the SDF-Fc chimeric
polypeptides. Human RPMI 8866 cells that do not express fusin (as
indicated by absence of staining) did not bind SDF-Fc chimeric
polypeptides (data not shown).
[0178] The data shown for Exp. 1 in Table 2 corresponds to the
results shown graphically as histograms in FIG. 2. The x axis of
the histograms (see FIG. 2A) was divided into three regions:
M1=channel 1-11; M2=channel 11-123; and M3=channel 123-1370. The
data is expressed as the percentage of appropriately gated cells in
each of these regions. Also given in Table 2 is the peak channel
and the median channel. The peak channel is the channel containing
the highest distribution of cells. The median is the channel where
50% of the cells are to the right or left of this point. FIG. 2,
panels A-D represent the histograms for Exp. 1 comparing the
anti-fusin antibody 12G5 to the chimeric polypeptide SK2-2. The
thinner line is that for the control while the thicker line is that
for the 12G5 (FIGS. 2A and 2C) or for SK2-2 (FIG. 2B and 2D). FIGS.
2A and 2B indicate staining of Jurkat cells. FIGS. 2C and 2D
indicate staining of U937 cells.
TABLE-US-00002 TABLE 2 Jurkat and U937 cell staining by anti-Fusin
mAb and SDF-Fc constructs % of Cells in Each Channel Range Peak
Median M1 M2 M3 Channel Channel Channel Range: 1-11 11-123 123-1370
Exp. 1 Jurkat Control 97.5% 2.5% 0% 3 3 Jurkat anti-Fusin 0.5%
21.4% 78.2% 281 225 Jurkat Control 98.1% 1.9% 0% 3 3 Jurkat SK2-2
2.4% 42.3% 56.1% 145 132 U937 Control 97.8% 2.2% 0% 3 3 U937
anti-Fusin 0.3% 71.8% 28.4% 81 95 U937 Control 99.6% 0.5% 0% 3 3
U937 SK2-2 2.3% 72.6% 25.5% 47 63 Exp. 2 Jurkat Control 91.6% 8.5%
0.1% 5 5 Jurkat anti-Fusin 0.4% 48.1% 51.9% 121 134 Jurkat Control
86.3% 13.9% 0% 6 6 Jurkat S1-3 6.5% 51.3% 42.8% 139 106 U937
Control 98.9% 1.1% 0% 4 4 U937 anti-Fusin 0.2% 53% 47.5% 114 118
U937 Control 98% 2.0% 0% 5 5 U937 S1-3 1.0% 77.3% 22.1% 62 78
TABLE-US-00003 TABLE 3 Lymphocyte and dendritic cell staining by
anti-Fusin mAb and SDF-Fc constructs % of Cells in Each Channel
Range Peak Median M1 M2 M3 Channel Channel Channel Range: 1-11
11-123 123-1370 T lymphocytes Control* 74.8% 2.3% 0% 1 2 anti-CD3
2.8% 4.2% 88.7% 610 523 anti-Fusin 39.3% 56.9% 3.2% 1 18 IgG4
Control** 79.8% 1.8% 0.2% 1 2 SDF-Fc SK2-2 27.2% 70.5% 2.5% 37 21
SDF-Fc S1-2 31.5% 66.4% 2.3% 31 20 Dendritic Cells and other
Adherent Cells Control* 38.6% 60.5% 1.5% 13 13 anti-Fusin 16.1%
59.0% 25.1% 12 31 IgG4 Control 53.1% 47.3% 0.1% 8 11 SDF-Fc SK2-2
18.6% 77.6% 4.3% 10 23 *Mouse gamma 2a control + Goat anti-Mouse PE
second step **Human IgG4 control + Goat anti-Human PE second
step
[0179] Table 3 shows the results of staining T lymphocytes isolated
from peripheral blood, and dendritic and other adherent cells
isolated from human bone marrow (following culture in media
containing IL4 and GMSF then TNF), by binding anti-fusin antibody
or the chimeric SDF-1.alpha.chemokine polypeptides S1-3 or SK2-2 to
them. These results indicate that a variety of cells expressing the
fusin receptor bind the chimeric SDF-1.alpha. chemokine
polypeptides.
TABLE-US-00004 TABLE 4 Effect of adding Human SDF-1.beta. during
staining of U937 cells by Anti-Fusin mAb and chimeric SDF-Fc
constructs % of Cells in Each Channel Range Peak Median M1 M2 M3
Channel Channel Channel Range: 1-11 11-123 123-1370 Control* 93.5%
5.7% 0.8% 3 3 anti-Fusin 0% 41.3% 58.8% 157 136 50 ng/ml Human SDF-
0% 47.1% 52.9% 106 127 1.beta. + anti-Fusin 500 ng/ml Human 0%
70.0% 30.0% 95 95 SDF-1.beta. + anti-Fusin IgG4 Control** 99.0%
1.0% 0% 3 3 SDF-Fc SK2-2 2.0% 97.0% 1.5% 47 47 50 ng/ml Human SDF-
1.6% 97% 1.6% 45 46 1.beta. + SDF-Fc SK2-2 500 ng/ml Human 8.4%
91.1% 0.7% 29 30 SDF-1.beta. + SDF-Fc SK2-2 SDF-Fc S1-3 1.4% 96.7%
1.9% 48 47 50 ng/ml Human SDF- 2.0% 96.7% 1.9% 42 46 1.beta. +
SDF-Fc S1-2 500 ng/ml Human 11.0% 88.7% 0.3% 30 26 SDF-1.beta. +
SDF-Fc S1-2 *Mouse gamma 2a control + Goat anti-Mouse PE second
step **Human IgG4 control + Goat anti-Human PE second step
[0180] For the experiment shown in Table 4, purified human
SDF-1.beta. chemokine, prepared in E. coli and containing an
N-terminal methionine residue, was mixed with either anti-fusin
antibody or chimeric SDF-1.alpha. polypeptide, then incubated with
cells on ice in the presence of azide. The results shown in Table 4
indicate that a 10-fold increase in the amount of SDF-1.beta.
chemokine eliminates some anti-fusin antibody binding to cells. but
does not reduce the amount of chimeric SDF-Fc polypeptide binding
to cells. This suggests that the affinity of the chimeric SDF-Fc
polypeptide for its binding site on cells. presumably the fusin
receptor, is sufficiently high that it cannot be competed off by
addition of excess SDF-1.beta. chemokine.
[0181] Binding of the MIP-1.alpha.-Fc and MIP-1.beta.-Fc chimeric
polypeptides to cells is determined by a cell-staining assay
analogous to that described above.
EXAMPLE 4
Alteration or Inhibition of Calcium Flux by Chimeric
Polypeptides
[0182] When chemokines bind to receptors present within the
membranes of cells, a calcium flux may be induced. When chimeric
chemokine polypeptides bind to these receptors, the duration,
intensity, or other properties of the calcium flux may be altered,
or the calcium flux may be inhibited. This calcium flux may be
measured using the following protocol, and the effects of chemokine
and chimeric chemokine polypeptide binding to receptors
compared.
[0183] Harvest the cells, wash twice in first wash buffer (10 mM
MOPS or HEPES at about pH 7.2, 1 mM CaCl.sub.2, 1 mM glucose, 140
mM NaCl), adjust to 10.sup.7 cells per ml, resuspend in
loading/FACS buffer (10 mM MOPS or HEPES at about pH7.2, 1 mM
CaCl.sub.2, 1 mM glucose, 140 mM NaCl, 0.2% BSA). Dissolve 50 .mu.g
vial of FLUO-3 ester (Molecular Probes, cat. #F-1242) in 50 .mu.l
DMSO right before use. Add 5 .mu.l FLUO-3 ester (approximately 5
.mu.M, different concentrations may be needed for different cell
types) for each ml of cells. Incubate for 20-30 minutes at room
temperature. Wash twice in medium (for example, RPMI with fetal
calf serum). Resuspend cells at 10.sup.7 per ml in medium (or
loading/FACS buffer). Store on ice until ready to use (or store at
room temperature). To test for calcium flux, dilute cells into
loading/FACS buffer, 100 .mu.l of cells per 1 ml buffer. Using a
FACSCAN (BD) fluorescence-activated cell analyzer, determine the
background reading for the loaded cells (use FL1 channel; set
maximum signal at about 200). Stimulate appropriately (with one or
more reagents, sequentially) and read on FACS for 3-15 minutes or
more, watching for an increase in fluorescence due to calcium flux.
The ionophore ionomycin can be used as a positive control to
demonstrate that the cells being tested are capable of
demonstrating a calcium flux.
EXAMPLE 5
Stimulation of Chemotaxis by Chimeric Polypeptides
[0184] Chimeric chemokine polypeptides can be tested for their
ability to stimulate chemotaxis by any of the following assays for
chemotactic activity. These assays (which will identify proteins
that induce or prevent chemotaxis) measure the ability of a protein
to induce the migration of cells across a membrane as well as the
ability of a protein to induce the adhesion of one cell population
to another cell population. Suitable assays for movement and
adhesion include, without limitation, those described in: Current
Protocols in Immunology, Ed. by J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E. M. Shevach, W. Strober, Pub. by Greene Publishing
Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and beta Chemokines 6.12.1-6.12.28); Taub et al., J. Clin.
Invest. 95:1370-1376, 1995; Lind et al., APMIS 103:140-146, 1995;
Muller et al., Eur. J. Immunol. 25: 1744-1748; Gruber et al., J. of
Immunol. 152:5860-5867, 1994; Johnston et al., J. of Immunol. 153:
1762-1768, 1994; all of which are incorporated herein by
reference.
EXAMPLE 6
Down-Modulation of Receptor by Chimeric Poly-Peptide Binding
[0185] The ability of the chimeric SDF-Fc polypeptides to
down-modulate chemokine receptors was compared with that of human
SDF-1.beta.. Jurkat cells were incubated for 3 hours or 15 hours at
37 degrees C. with either human SDF-1.beta. or chimeric SDF-Fc
polypeptide, followed by a wash of the cells and staining with
anti-fusin antibody as described in Example 3. Mock experiments
involved incubating cells with COS cell supernatant containing
neither SDF-1.beta. nor chimeric SDF-Fc polypeptide. The results of
these experiments are shown in Table 5.
TABLE-US-00005 TABLE 5 Down-Modulation of Fusin/CXCR4 by Incubation
with Human SDF-1.beta. or chimeric SDF-FC % of Cells in Each
Channel Range Peak Median M1 M2 M3 Channel Channel Channel Range:
1-11 11-123 123-1370 Jurkat 3-hour Incubation Media control 97.5%
2.5% 0% 3 3 Anti-fusin Media 0.4% 20.2% 79.6% 281 231 500 ng/ml
Human 14.1% 83.2% 3.1% 20 22.3 SDF1.beta. Mock 0.4% 12.3% 87.2% 276
302 70 ng/ml SDF-Fc S1-2 6.1% 91.7% 2.5% 23 26 140 ng/ml SDF-Fc
15.5% 82.5% 2.4% 13 20 S1-3 90 ng/ml SDF-Fc 11.3% 86.8% 2.4% 23 22
SK2-2 Jurkat 15-hour Incubation Media control 97.5% 2.5% 0% 3 3
Anti-fusin Media 0.4% 20.2% 79.6% 281 231 500 ng/ml Human 1.7%
77.7% 20.9% 66 75 SDF1.beta. 70 ng/ml SDF-FC S-2 5.9% 91.7% 2.7% 35
34 140 ng/ml SDF-FC 9.2% 90.0% 1.1% 26 25 S1-3 90 ng/ml SDF-FC 7.0%
91.7% 1.5% 38 28 SK2-2
[0186] The apparent down-modulation of fusin receptor by human
SDF-1.beta. is not simply due to blocking of staining by the
anti-fusin antibody by the binding of SDF-1.beta. to fusin, since
the results shown in Table 4 above indicate that the presence of
human SDF-1.beta. does not prevent anti-fusin binding to the extent
observed here. The down-modulation by the chimeric SDF-Fc
polypeptide is demonstrated by the failure of anti-fusin antibody
to bind after incubation with this chimeric polypeptide (Table 5)
and the weak staining of these cells with PE-labeled goat
anti-human Ig to detect chimeric SDF-Fc polypeptide remaining after
the 3- or 15-hour incubation (data not shown).
[0187] Down-regulation of receptor by binding of MIP-1.alpha.-Fc
and MIP-1.beta.-Fc chimeric polypeptides to cells is determined by
an assay for receptor down-regulation analogous to that described
above.
Sequence CWU 1
1
* * * * *