U.S. patent application number 10/924029 was filed with the patent office on 2005-01-27 for chemokines with amino-terminal modifications.
Invention is credited to Herrmann, Stephen H., Lu, Zhijian, McCoy, John M., Swanberg, Stephen L., Walker, Bruce, Yang, Otto.
Application Number | 20050020528 10/924029 |
Document ID | / |
Family ID | 34107126 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020528 |
Kind Code |
A1 |
Herrmann, Stephen H. ; et
al. |
January 27, 2005 |
Chemokines with amino-terminal modifications
Abstract
This invention provides polynucleotides comprising sequences
encoding amino-terminal-modified chemokines, the encoded
amino-terminal-modified chemokines, and uses thereof.
Inventors: |
Herrmann, Stephen H.;
(Wellesley, MA) ; Lu, Zhijian; (Bedford, MA)
; McCoy, John M.; (Reading, MA) ; Swanberg,
Stephen L.; (Boston, MA) ; Walker, Bruce;
(Charlestown, MA) ; Yang, Otto; (Charlestown,
MA) |
Correspondence
Address: |
JENKENS & GILCHRIST, PC
1445 ROSS AVENUE
SUITE 3200
DALLAS
TX
75202
US
|
Family ID: |
34107126 |
Appl. No.: |
10/924029 |
Filed: |
August 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10924029 |
Aug 23, 2004 |
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09175713 |
Oct 20, 1998 |
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10924029 |
Aug 23, 2004 |
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08808720 |
Feb 28, 1997 |
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6100387 |
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60113672 |
Oct 22, 1997 |
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Current U.S.
Class: |
514/44R ;
536/23.5 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 2319/00 20130101; A61K 2300/00 20130101; C07K 14/522 20130101;
A61K 38/00 20130101; A61K 39/00 20130101; C07K 14/523 20130101;
A61K 39/3955 20130101; C07K 14/521 20130101 |
Class at
Publication: |
514/044 ;
536/023.5 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. A composition comprising an isolated polynudeotide encoding an
amino-terminal-modified chemokine, wherein the chemokine is
selected from the group consisting of SDF-1.alpha., SDF-1.beta.,
IP-10, Mig, GRO.alpha., GRO.beta., GRO.gamma., interleukin-8, PF4,
ENA-78, GCP-2, PBP, CTAP-III, .beta.-thromboglobulin, NAP-2, C10,
DC-CK1, CK.alpha.1, CK.alpha.2, MCP-1, MCP-2, MCP-3, MCP4,
MIP-1.alpha., MIP-1.beta., lymphotactin, ATAC, eotaxin, eotaxin2,
I-309, HCC-1, HCC-2, HCC-3, LARC/MIP-3.alpha., MIP-3.beta., PARC,
TARC, 6Ckine, ELC, SLC, CK.beta.4, CK.beta.6, CK.beta.7, CK.beta.8,
CK.beta.9, CK.beta.11, CK.beta.12, CK.beta.13, and CX3C.
2. The composition of claim 1 wherein the amino-terminal-modified
chemokine comprises at least one methionine residue covalently
attached to the amino terminus of the chemokine.
3. The composition of claim 1 wherein the amino-terminal-modified
chemokine comprises at least one aminooxypentane residue covalently
attached to the amino terminus of the chemokine.
4. The composition of claim 1 wherein the amino-terminal-modified
chemokine comprises at least one GroHEK peptide covalently attached
to the amino terminus of the chemokine.
5. A composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the
amino-terminal-modified chemokine is derived from a chemokine
selected from the group consisting of SDF-1.alpha., SDF-1.beta.,
IP-10, Mig, GRO.alpha., GRO.beta., GRO.gamma., interleukin-8, PF4,
ENA-78, GCP-2, PBP, CTAP-III, .beta.-thromboglobulin, NAP-2, C10,
DC-CK1, CK.alpha.1, CK.alpha.2, MCP-1, MCP-2, MCP-3, MCP-4,
MIP-1.alpha., MIP-1.beta., RANTES, lymphotactin, ATAC, eotaxin,
eotaxin2, I-309, HCC-1, HCC-2, HCC-3, LARC/MIP-3.alpha.,
MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC, CK.beta.4, CK.beta.6,
CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11, CK.beta.12, CK.beta.13
B13, and CX3C.
6. The composition of claim 1 wherein the polynucleotide is
selected from the group consisting of: (a) a polynudeotide
comprising the nucleotide sequence of SEQ ID NO:6; (b) a
polynudeotide comprising the nucleotide sequence of the
protein-coding sequence of the polynucleotide encoding
met-hSDF-1.alpha. deposited under accession number ATCC 98506; (c)
a polynucleotide encoding an amino-terminal-modified chemokine
comprising the amino acid sequence of SEQ ID NO:10; (d) a
polynucleotide encoding a protein comprising an amino-terminal
fragment of the of the amino acid sequence of SEQ ID NO:10; (e) a
polynudeotide comprising a nucleotide sequence complementary to any
one of the polynucleotides specified in (a)-(d) above; and (f) a
polynudeotide capable of hybridizing under stringent conditions to
any one of the polynucleotides specified in (a)-(e) above.
7. The composition of claim 1 wherein the polynucleotide is
selected from the group consisting of: (a) a polynudeotide
comprising the nucleotide sequence of SEQ ID NO:7; (b) a
polynucleotide comprising the nucleotide sequence of the
protein-coding sequence of the polynucleotide encoding
met-hSDF-1.beta. deposited under accession number ATCC 98507; (c) a
polynucleotide encoding an amino-terminal-modified chemokine
comprising the amino acid sequence of SEQ ID NO:11; (d) a
polynucleotide encoding a protein comprising an amino-terminal
fragment of the of the amino acid sequence of SEQ ID NO:11; (e) a
polynucleotide comprising a nucleotide sequence complementary to
any one of the polynucleotides specified in (a)-(d) above; and (f)
a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(e) above.
8. The composition of claim 1 wherein the polynucleotide is
selected from the group consisting of: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:8; (b) a
polynucleotide comprising the nucleotide sequence of the
protein-coding sequence of the polynucleotide encoding
GroHEK/hSDF-1.alpha. deposited under accession number ATCC 98508;
(c) a polynucleotide encoding an amino-terminal-modified chemokine
comprising the amino acid sequence of SEQ ID NO:12; (d) a
polynucleotide encoding a protein comprising an amino-terminal
fragment of the of the amino acid sequence of SEQ ID NO:12; (e) a
polynucleotide comprising a nucleotide sequence complementary to
any one of the polynucleotides specified in (a)-(d) above; and (f)
a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(e) above.
9. The composition of claim 1 wherein the polynucleotide is
selected from the group consisting of: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:9; ~ (b) a
polynucleotide comprising the nucleotide sequence of the
protein-coding sequence of the polynucleotide encoding
GroHEK/hSDF-1.beta. deposited under accession number ATCC 98509;
(c) a polynucleotide encoding an amino-terminal-modified chemokine
comprising the amino acid sequence of SEQ ID NO:13; (d) a
polynucleotide encoding a protein comprising an amino-terminal
fragment of the of the amino acid sequence of SEQ ID NO:13; (e) a
polynudeotide comprising a nucleotide sequence complementary to any
one of the polynucleotides specified in (a)-(d) above; and (f) a
polynudeotide capable of hybridizing under stringent conditions to
any one of the polynucleotides specified in (a)-(e) above.
10. A composition of claim 1 wherein the polynucleotide is operably
linked to an expression control sequence.
11. The composition of claim 10 wherein the polynucleotide is
further operably linked to a sequence directing secretion of the
expressed amino-terminal-modified chemokine.
12. A host cell transformed with a composition of claim 10.
13. The host cell of claim 12, wherein the cell is a mammalian
cell.
14. A process for producing an amino-terminal-modified chemokine,
which comprises: (a) growing a culture of the host cell of claim 12
in a suitable culture medium; and (b) purifying the
amino-terminal-modified chemokine from the culture.
15. A polypeptide produced according to the process of claim
14.
16. A process for producing an amino-terminal-modified chemokine in
a host, which comprises: (a) isolating stem cells from the host;
(b) transforming the stem cells with the composition of claim 10;
and (c) reintroducing the transformed stem cells into the host,
wherein the transformed stem cells will express the
amino-terminal-modified chemokine.
17. A composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the chemokine binds the
fusin/CXCR4 chemokine receptor.
18. A composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the
amino-terminal-modified chemokine is a more effective inhibitor of
HIV infection than the corresponding unmodified chemokine.
19. A composition comprising an amino-terminal-modified chemokine,
wherein the chemokine is selected from the group consisting of
SDF-1.alpha., SDF-1.beta., IP-10, Mig, GRO.alpha., GRO.beta.,
GRO.gamma., interleukin-8, PF4, ENA-78, GCP-2, PBP, CTAP-III,
.beta.-thromboglobulin, NAP-2, C10, DC-CK1, CK.alpha.1, CK.alpha.2,
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.alpha., MIP-1.beta.,
lymphotactin, ATAC, eotaxin, eotaxin2, I-309, HCC-1, HCC-2, HCC-3,
LARC/MIP-3.alpha., MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC,
CK.beta.4, CK.beta.6, CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11,
CK.beta.12, CK.beta.13, and CX3C.
20. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises at least one methionine residue covalently
attached to the amino terminus of the chemokine.
21. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises at least one aminooxypentane residue covalently
attached to the amino terminus of the chemokine.
22. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises at least one GroHEK peptide covalently attached
to the amino terminus of the chemokine.
23. A composition comprising an amino-terminal-modified chemokine,
wherein the amino-terminal-modified chemokine is derived from a
chemokine selected from the group consisting of SDF-1.alpha.,
SDF-1.beta., IP-10, Mig, GRO.alpha., GRO.beta., GRO.gamma.,
interleukin-8, PF4, ENA-78, GCP-2, PBP, CTAP-III,
.beta.-thromboglobulin, NAP-2, C10, DC-CK1, CK.alpha.1, CK.alpha.2,
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.alpha., MIP-1.beta., RANTES,
lymphotactin, ATAC, eotaxin, eotaxin2, 1-309, HCC-1, HCC-2, HCC-3,
LARC/MIP-3.alpha., MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC,
CK.beta.4, CK.beta.6, CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11,
CK.beta.12, CK.beta.13, and CX3C.
24. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of SEQ ID NO:10; (b) the
amino add sequence of the protein encoded by the met-hSDF-1.alpha.
polynucleotide deposited under accession number ATCC 98506; (c)
amino-terminal fragments of the amino acid sequence of SEQ ID
NO:10; and (d) amino-terminal fragments of the amino acid sequence
of the protein encoded by the met-hSDF-1.alpha. polynucleotide
deposited under accession number ATCC 98506.
25. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of SEQ ID NO:11; (b) the
amino acid sequence of the protein encoded by the met-hSDF-1.beta.
polynucleotide deposited under accession number ATCC 98507; (c)
amino-terminal fragments of the amino acid sequence of SEQ ID
NO:11; and (d) amino-terminal fragments of the amino acid sequence
of the protein encoded by the met-hSDF-1.beta. polynucleotide
deposited under accession number ATCC 98507.
26. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of SEQ ID NO:12; (b) the
amino acid sequence of the protein encoded by the
GroHEK/hSDF-1.alpha. polynudeotide deposited under accession number
ATCC 98508; (c) amino-terminal fragments of the amino acid sequence
of SEQ ID NO:12; and (d) amino-terminal fragments of the amino acid
sequence of the protein encoded by the GroHEK/hSDF-1.alpha.
polynucleotide deposited under accession number ATCC 98508.
27. The composition of claim 19 wherein the amino-terminal-modified
chemokine comprises an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of SEQ ID NO:13; (b) the
amino acid sequence of the protein encoded by the
GroHEK/hSDF-1.beta. polynudeotide deposited under accession number
ATCC 98509; (c) amino-terminal fragments of the amino acid sequence
of SEQ ID NO:13; and (d) amino-terminal fragments of the amino acid
sequence of the protein encoded by the GroHEK/hSDF-1.beta.
polynucleotide deposited under accession number ATCC 98509.
28. The composition of claim 19, further comprising a
pharmaceutically acceptable carrier.
29. A composition comprising an antibody which reacts with the
amino-terminal-modified chemokine of claim 19, but does not react
with the unmodified chemokine.
30. A method for identifying molecules capable of interacting with
an amino-terminal-modified chemokine which comprises: (a) combining
a composition of claim 19 with an indicator molecule and with a
composition comprising molecules to be tested for interaction; and
(b) detecting the presence of altered indicator molecules.
31. A method for altering receptor function which comprises causing
a receptor to bind at least one amino-terminal-modified chemokine
of claim 19.
32. A method for inhibiting the interaction between a chemokine
receptor and a ligand of the receptor which comprises causing the
receptor to bind at least one amino-terminal-modified chemokine of
claim 19.
33. A method for decreasing receptor function which comprises
causing a receptor to bind at least one amino-terminal-modified
chemokine of claim 19, resulting in a decrease in the number of
functional receptor molecules.
34. A method for preventing, treating, or ameliorating HIV
infection which comprises administering therapeutically effective
amounts of at least one composition of claim 19.
35. The method of claim 34, wherein the compositions administered
comprise: (a) an amino-terminal-modified chemokine comprising a
chemokine selected from the group consisting of SDF-1.alpha. and
SDF-1.beta.; and (b) an amino-terminal-modified chemokine
comprising a chemokine selected from the group consisting of
MIP-1.alpha. and MIP-1.beta..
36. A method for identifying amino-terminal-modified chemokines
capable of inhibiting the interaction of HI with an HIV receptor
which comprises: (a) combining a composition of claim 19 with a
composition comprising HIV receptor molecules, forming a first
mixture; (b) combining the first mixture with a composition
comprising HIV molecules, forming a second mixture; (c) combining a
composition comprising HIV receptor molecules with a composition
comprising HIV molecules, forming a control mixture; (d)
determining the amount of interaction between the HIV molecules and
the HIV receptor molecules in the second mixture and in the control
mixture; and (e) comparing the amount of interaction between the
HIV molecules and the HIV receptor molecules in the second mixture
with the amount of interaction between the HIV molecules and the
HIV receptor molecules in the control mixture, wherein the
amino-terminal-modified chemokine inhibits the interaction of HIV
with the HIV receptor when the amount of interaction between the
HIV molecules and the HIV receptor molecules is less in the second
mixture than in the control mixture.
37. A method for identifying amino-terminal-modified chemokines
capable of inhibiting the infection by HIV of cells susceptible to
HIV infection which comprises: (a) combining a composition of claim
19 with a composition comprising cells susceptible to HIV
infection, forming a first mixture; (b) combining the first mixture
with a composition comprising HIV particles, forming a second
mixture; (c) combining a composition comprising cells susceptible
to HIV infection with a composition comprising HIV particles,
forming a control mixture; (d) determining the amount of infection
of the susceptible cells by HIV in the second mixture and in the
control mixture; and (e) comparing the amount of infection of the
susceptible cells by HIV in the second mixture with the amount of
infection of the susceptible cells by HIV in the control mixture,
wherein the amino-terminal-modified chemokine inhibits the
infection of the susceptible cells by HIV when the amount of
infection of the susceptible cells by HIV is less in the second
mixture than in the control mixture.
38. A method for attracting migratory cells to a region of an
organism which comprises administering therapeutically effective
amounts of at least one composition of claim 19.
39. A method for stimulating angiogenesis which comprises
administering therapeutically effective amounts of at least one
composition of claim 19.
40. A method for inhibiting angiogenesis which comprises
administering therapeutically effective amounts of at least one
composition of claim 19.
41. A method for preventing, treating,.or ameliorating an
inflammatory condition which comprises administering
therapeutically effective amounts of at least one composition of
claim 19.
42. A method for preventing, treating, or ameliorating an
autoimmune condition which comprises administering therapeutically
effective amounts of at least one composition of claim 19.
43. A method for inducing an immune response which comprises
administering a vaccine and therapeutically effective amounts of at
least one composition of claim 19.
44. A composition comprising an amino-terminal-modified chemokine,
wherein the chemokine binds the fusin/CXCR4 chemokine receptor.
45. A composition comprising an amino-terminal-modified chemokine,
wherein the amino-terminal-modified chemokine is a more effective
inhibitor of HIV infection than the corresponding unmodified
chemokine.
46. A method for preventing, treating, or ameliorating HIV
infection of a host which comprises: (a) isolating stem cells from
the host; (b) transforming the stem cells with at least one
composition of claim 10; and (c) reintroducing the transformed stem
cells into the host, wherein the transformed stem cells will
express at least one amino-terminal-modified chemokine.
47. The method of claim 46, wherein the transformed stem cells
express an amino-terminal-modified chemokine comprising a chemokine
selected from the group consisting of SDF-1.alpha. and SDF-1.beta.;
and an amino-terminal-modified chemokine comprising a chemokine
selected from the group consisting of MIP-1.alpha. and MIP-1.beta..
Description
[0001] This application is a continuation-in-part of application
Ser. No. 08/808,720, filed Feb. 28, 1997, which is a continuation
of Ser. No. 60/______ (converted to a provisional application from
non-provisional application Ser. No. 08/955,826), filed Oct. 22,
1997, all of which are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to
amino-terminal-modified (N-terminal-modified) chemokines and the
use of such chemokines to inhibit the interaction between chemokine
receptors and naturally occurring ligands of those receptors. More
specifically, the invention relates to the expression in host cells
of recombinant polynudeotide sequences encoding chemokines having
additional amino acids or other chemical groups attached to their
amino termini, and the use of such N-terminal-modified chemokines
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 and the
infection of susceptible cells by HIV.
[0003] 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
(alpha chemokines) or CC (beta chemokines), 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
platelet factor 4 (PF4) family; other CXC chemokines that have been
identified are: C10, DC-CK1, CK.alpha.1, CK.alpha.2, ENA-78, GCP-2,
and platelet basic protein (PBP) and its derivatives CTAPIII,
.beta.-thromboglobulin, and NAP-2. CC chemokine families include
the monocyte chemoattractant protein (MCP) family including MCP-1
to MCP-4; 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) protein; and the lymphotactin family; other
CC chemokines that have been identified are: ATAC, eotaxin,
eotaxin2, I-309, HCC-1, HCC-2, HCC-3, LARC/MIP-3.alpha.,
MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC, CK.beta.4, CK.beta.6,
CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11, CK.beta.12,
CK.beta.13. CX.sub.3C (or CX3C) is a recently identified member of
a new class of chemokines. 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 (CXCR1-CXCR4), and CC chemokines by members of the CCR
class of receptors (CCR1-CCR8). 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.
[0004] Other chemokine receptors that have been identified are:
BLR1, MDR15, EBI-1, CMKBRL1, HCMV-US28, HSV-ECRF3, and Duffy
antigen (DARC).
[0005] 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, bone resorption, 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 IP-10 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.
[0006] 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. 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.
[0007] There is a continuing requirement for new compositions that
will enhance, alter, or inhibit chemokine-receptor interactions,
and for methods for their use.
SUMMARY OF THE INVENTION
[0008] Applicants have for the first time constructed novel
polynucleotides encoding certain amino-terminal-modified chemokines
comprising chemokines or polypeptides derived from chemokines.
Amino-terminal-modified chemokines expressed from these constructs
have exhibited novel and unexpected properties, including novel
interactions with cells expressing chemokine receptors.
[0009] In one embodiment, the present invention provides a
composition comprising an isolated polynudeotide encoding an
amino-terminal-modified chemokine, wherein the chemokine is
selected from the group consisting of SDF-1.alpha., SDF-1.beta.,
IP-10, Mig, GRO.alpha., GRO.beta., GRO.gamma., interleukin-8, PF4,
ENA-78, GCP-2, PBP, CTAP-III, .beta.-thromboglobulin, NAP-2, C10,
DC-CK1, CK.alpha.1, CK.alpha.2, MCP-1, MCP-2, MCP-3, MCP4,
MIP-1.alpha., MIP-1.beta., lymphotactin, ATAC, eotaxin, eotaxin2,
I-309, HCC-1, HCC-2, HCC-3, LARC/MIP-3.alpha., MIP-3.beta., PARC,
TARC, 6Ckine, ELC, SLC, CK.beta.4, CK.beta.6, CK.beta.7, CK.beta.8,
CK.beta.9, CK.beta.11, CK.beta.12, CK.beta.13, and CX3C.
Preferably, the amino-terminal-modified chemokine comprises at
least one methionine residue covalently attached to the amino
terminus of the chemokine, or at least one aminooxypentane residue
covalently attached to the amino terminus of the chemokine, or at
least one GroHEK peptide covalently attached to the amino terminus
of the chemokine. In certain preferred embodiments, the
polynucleotide is operably linked to an expression control
sequence, or is further operably linked to a sequence directing
secretion of the expressed amino-terminal-modified chemokine. The
invention also provides a host cell, preferably a mammalian cell,
transformed with such polynudeotide compositions.
[0010] Processes are also provided for producing an
amino-terminal-modified chemokine, which comprise:
[0011] (a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
[0012] (b) purifying the amino-terminal-modified chemokine from the
culture.
[0013] The polypeptide produced according to such methods is also
provided by the present invention.
[0014] Processes are also provided for producing an
amino-terminal-modified chemokine in a host, which comprise:
[0015] (a) isolating stem cells from the host;
[0016] (b) transforming the stem cells with such polynucleotide
compositions; and
[0017] (c) reintroducing the transformed stem cells into the host,
wherein the transformed stem cells will express the
amino-terminal-modified chemokine.
[0018] Another embodiment provides a composition comprising an
isolated polynucleotide encoding an amino-terminal-modified
chemokine, wherein the amino-terminal-modified chemokine is derived
from a chemokine selected from the group consisting of
SDF-1.alpha., SDF-1.beta., IP-10, Mig, GRO.alpha., GRO.beta.,
GRO.gamma., interleukin-8, PF4, ENA-78, GCP-2, PBP, CTAP-III,
.beta.-thromboglobulin, NAP-2, C10, DC-CK1, CK.alpha.1, CK.alpha.2,
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.alpha., MIP-1.beta., RANTES,
lymphotactin, ATAC, eotaxin, eotaxin2, I-309, HCC-1, HCC-2, HCC-3,
LARC/MIP-3.alpha., MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC,
CK.beta.4, CK.beta.6, CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11,
CK.beta.12, CK.beta.13, and CX3C.
[0019] In another embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the polynucleotide is
selected from the group consisting of:
[0020] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:6;
[0021] (b) a polynucleotide comprising the nucleotide sequence of
the protein-coding sequence of the polynucleotide encoding
met-hSDF-1.alpha. deposited under accession number ATCC 98506;
[0022] (c) a polynucleotide encoding an amino-terminal-modified
chemokine comprising the amino acid sequence of SEQ ID NO:10;
[0023] (d) a polynucleotide encoding a protein comprising an
amino-terminal fragment of the of the amino acid sequence of SEQ ID
NO:10;
[0024] (e) a polynucleotide comprising a nucleotide sequence
complementary to any one of the polynucleotides specified in
(a)-(d) above; and
[0025] (f) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(e)
above.
[0026] In a further embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the polynucleotide is
selected from the group consisting of:
[0027] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:7;
[0028] (b) a polynucleotide comprising the nucleotide sequence of
the protein-coding sequence of the polynucleotide encoding
met-hSDF-1.beta. deposited under accession number ATCC 98507;
[0029] (c) a polynucleotide encoding an amino-terminal-modified
chemokine comprising the amino acid sequence of SEQ ID NO:11;
[0030] (d) a polynucleotide encoding a protein comprising an
amino-terminal fragment of the of the amino acid sequence of SEQ ID
NO:11;
[0031] (e) a polynucleotide comprising a nucleotide sequence
complementary to any one of the polynucleotides specified in
(a)-(d) above; and
[0032] (f) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(e)
above.
[0033] In another embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the polynucleotide is
selected from the group consisting of:
[0034] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:8;
[0035] (b) a polynucleotide comprising the nucleotide sequence of
the protein-coding sequence of the polynudeotide encoding
GroHEK/hSDF-1.alpha. deposited under accession number ATCC
98508;
[0036] (c) a polynucleotide encoding an amino-terminal-modified
chemokine comprising the amino acid sequence of SEQ ID NO:12;
[0037] (d) a polynucleotide encoding a protein comprising an
amino-terminal fragment of the of the amino acid sequence of SEQ ID
NO:12;
[0038] (e) a polynudeotide comprising a nucleotide sequence
complementary to any one of the polynucleotides specified in
(a)-(d) above; and
[0039] (f) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(e)
above.
[0040] In a further embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the polynucleotide is
selected from the group consisting of:
[0041] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:9;
[0042] (b) a polynudeotide comprising the nucleotide sequence of
the protein-coding sequence of the polynucleotide encoding
GroHEK/hSDF-1.beta. deposited under accession number ATCC
98509;
[0043] (c) a polynudeotide encoding an amino-terminal-modified
chemokine comprising the amino acid sequence of SEQ ID NO:13;
[0044] (d) a polynucleotide encoding a protein comprising an
amino-terminal fragment of the of the amino acid sequence of SEQ ID
NO:13;
[0045] (e) a polynudeotide comprising a nucleotide sequence
complementary to any one of the polynucleotides specified in
(a)-(d) above; and
[0046] (e) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(e)
above.
[0047] In a further embodiment, the present invention provides a
composition comprising an isolated polynucleotide encoding an
amino-terminal-modified chemokine, wherein the chemokine binds the
fusin/CXCR4 chemokine receptor.
[0048] The present invention also provides a composition comprising
an isolated polynudeotide encoding an amino-terminal-modified
chemokine, wherein the amino-terminal-modified chemokine is a more
effective inhibitor of HIV infection than the corresponding
unmodified chemokine.
[0049] In other embodiments, the present invention provides a
composition comprising an amino-terminal-modified chemokine,
wherein the chemokine is selected from the group consisting of
SDF-1.alpha., SDF-1.beta., IP-10, Mig, GRO.alpha., GRO.beta.,
GRO.gamma., interleukin-8, PF4, ENA-78, GCP-2, PBP, CTAP-III,
.beta.-thromboglobulin, NAP-2, C10, DC-CK1, CK.alpha.1, CK.alpha.2,
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.alpha., MIP-1.beta.,
lymphotactin, ATAC, eotaxin, eotaxin2, I-309, HCC-1, HCC-2, HCC-3,
LARC/MIP-3.alpha., MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC,
CK.beta.4, CK.beta.6, CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11,
CK.beta.12, CK.beta.13, and CX3C. Preferably, the
amino-terminal-modified chemokine comprises at least one methionine
residue covalently attached to the amino terminus of the chemokine,
or at least one aminooxypentane residue covalently attached to the
amino terminus of the chemokine, or at least one GroHEK peptide
covalently attached to the amino terminus of the chemokine.
[0050] Another embodiment provides a composition comprising an
amino-terminal-modified chemokine, wherein the
amino-terminal-modified chemokine is derived from a chemokine
selected from the group consisting of SDF-1.alpha., SDF-1.beta.,
IP-10, Mig, GRO.alpha., GRO.beta., GRO.gamma., interleukin-8, PF4,
ENA-78, GCP-2, PBP, CTAP-III, .beta.-thromboglobulin, NAP-2, C10,
DC-CK1, CK.alpha.1, CK.alpha.2, MCP-1, MCP-2, MCP-3, MCP-4,
MIP-1.alpha., MIP-1.beta., RANTES, lymphotactin, ATAC, eotaxin,
eotaxin2, I-309, HCC-1, HCC-2, HCC-3, LARC/MIP-3.alpha.,
MIP-3.beta., PARC, TARC, 6Ckine, ELC, SLC, CK.beta.4, CK.beta.6,
CK.beta.7, CK.beta.8, CK.beta.9, CK.beta.11, CK.beta.12,
CK.beta.13, and CX3C.
[0051] In another embodiment, the present invention provides a
composition comprising an amino-terminal-modified chemokine wherein
the amino-terminal-modified chemokine comprises an amino acid
sequence selected from the group consisting of:
[0052] (a) the amino acid sequence of SEQ ID NO:10;
[0053] (b) the amino add sequence of the protein encoded by the
met-hSDF-1.alpha. polynucleotide deposited under accession number
ATCC 98506;
[0054] (c) amino-terminal fragments of the amino acid sequence of
SEQ ID NO:10; and
[0055] (d) amino-terminal fragments of the amino acid sequence of
the protein encoded by the met-hSDF-1.alpha. polynucleotide
deposited under accession number ATCC 98506.
[0056] In a further embodiment, the present invention provides a
composition comprising an amino-terminal-modified chemokine wherein
the amino-terminal-modified chemokine comprises an amino acid
sequence selected from the group consisting of:
[0057] (a) the amino acid sequence of SEQ ID NO:11;
[0058] (b) the amino acid sequence of the protein encoded by the
met-hSDF-1.beta. polynucleotide deposited under accession number
ATCC 98507;
[0059] (c) amino-terminal fragments of the amino acid sequence of
SEQ ID NO:11; and
[0060] (d) amino-terminal fragments of the amino acid sequence of
the protein encoded by the met-hSDF-1.beta. polynucleotide
deposited under accession number ATCC 98507.
[0061] In another embodiment, the present invention provides a
composition comprising an amino-terminal-modified chemokine wherein
the amino-terminal-modified chemokine comprises an amino acid
sequence selected from the group consisting of:
[0062] (a) the amino acid sequence of SEQ ID NO:12;
[0063] (b) the amino add sequence of the protein encoded by the
GroHEK/hSDF-1.alpha. polynucleotide deposited under accession
number ATCC 98508;
[0064] (c) amino-terminal fragments of the amino acid sequence of
SEQ ID NO:12; and
[0065] (d) amino-terminal fragments of the amino acid sequence of
the protein encoded by the GroHEK/hSDF-1.alpha. polynucleotide
deposited under accession number ATCC 98508.
[0066] In a further embodiment, the present invention provides a
composition comprising an amino-terminal-modified chemokine wherein
the amino-terminal-modified chemokine comprises an amino acid
sequence selected from the group consisting of:
[0067] (a) the amino acid sequence of SEQ ID NO:13;
[0068] (b) the amino acid sequence of the protein encoded by the
GroHEK/hSDF-1.beta. polynudeotide deposited under accession number
ATCC 98509;
[0069] (c) amino-terminal fragments of the amino acid sequence of
SEQ ID NO:13; and
[0070] (d) amino-terminal fragments of the amino acid sequence of
the protein encoded by the GroHEK/hSDF-1.beta. polynudeotide
deposited under accession number ATCC 98509.
[0071] Compositions comprising amino-terminal-modified chemokines
of the present invention may further comprise a pharmaceutically
acceptable carrier. Compositions comprising an antibody which
reacts with an amino-terminal-modified chemokine but does not react
with the unmodified chemokine are also provided by the present
invention.
[0072] The present invention also provides methods for identifying
molecules capable of interacting with an amino-terminal-modified
chemokine which comprise:
[0073] (a) combining a composition comprising an
amino-terminal-modified chemokine with an indicator molecule and
with a composition comprising molecules to be tested for
interaction; and
[0074] (b) detecting the presence of altered indicator
molecules.
[0075] Methods are also provided for altering receptor function
which comprise causing a receptor to bind at least one
amino-terminal-modified chemokine.
[0076] The present invention also provides methods for inhibiting
the interaction between a chemokine receptor and a ligand of the
receptor which comprise causing the receptor to bind at least one
amino-terminal-modified chemokine.
[0077] Methods are also provided for decreasing receptor function
which comprise causing a receptor to bind at least one
amino-terminal-modified chemokine, resulting in a decrease in the
number of functional receptor molecules.
[0078] The present invention also provides methods for preventing,
treating, or ameliorating HIV infection which comprise
administering therapeutically effective amounts of at least one
composition comprising an amino-terminal-modified chemokine.
[0079] Preferably, the compositions administered comprise:
[0080] (a) an amino-terminal-modified chemokine comprising a
chemokine selected from the group consisting of SDF-1.alpha. and
SDF-1.beta.; and
[0081] (b) an amino-terminal-modified chemokine comprising a
chemokine selected from the group consisting of MIP-1.alpha. and
MIP-1.beta..
[0082] Methods are additionally provided for identifying
amino-terminal-modified chemokines capable of inhibiting the
interaction of HIV with an HIV receptor which comprise:
[0083] (a) combining a composition comprising an
amino-terminal-modified chemokine with a composition comprising HIV
receptor molecules, forming a first mixture;
[0084] (b) combining the first mixture with a composition
comprising HIV molecules, forming a second mixture;
[0085] (c) combining a composition comprising HIV receptor
molecules with a composition comprising HIV molecules, forming a
control mixture;
[0086] (d) determining the amount of interaction between the HIV
molecules and the HIV receptor molecules in the second mixture and
in the control mixture; and
[0087] (e) comparing the amount of interaction between the HIV
molecules and the HIV receptor molecules in the second mixture with
the amount of interaction between the HIV molecules and the HIV
receptor molecules in the control mixture, wherein the
amino-terminal-modified chemokine inhibits the interaction of HIV
with the HIV receptor when the amount of interaction between the
HIV molecules and the HIV receptor molecules is less in the second
mixture than in the control mixture.
[0088] The present invention also provides methods for identifying
amino-terminal-modified chemokines capable of inhibiting the
infection by HIV of cells susceptible to HIV infection which
comprise:
[0089] (a) combining a composition comprising an
amino-terminal-modified chemokine with a composition comprising
cells susceptible to HIV infection, forming a first mixture;
[0090] (b) combining the first mixture with a composition
comprising HIV particles, forming a second mixture;
[0091] (c) combining a composition comprising cells susceptible to
HIV infection with a composition comprising HIV particles, forming
a control mixture;
[0092] (d) determining the amount of infection of the susceptible
cells by HIV in the second mixture and in the control mixture;
and
[0093] (e) comparing the amount of infection of the susceptible
cells by HIV in the second mixture with the amount of infection of
the susceptible cells by HIV in the control mixture, wherein the
amino-terminal-modified chemokine inhibits the infection of the
susceptible cells by HIV when the amount of infection of the
susceptible cells by HIV is less in the second mixture than in the
control mixture.
[0094] The present invention also provides methods for attracting
migratory cells to a region of an organism which comprise
administering therapeutically effective amounts of at least one
composition comprising an amino-terminal-modified chemokine.
[0095] Methods are also provided for stimulating angiogenesis which
comprise administering therapeutically effective amounts of at
least one composition comprising an amino-terminal-modified
chemokine.
[0096] The present invention additionally provides methods for
inhibiting angiogenesis which comprise administering
therapeutically effective amounts of at least one composition
comprising an amino-terminal-modified chemokine.
[0097] Methods are also provided for preventing, treating, or
ameliorating an inflammatory condition which comprise administering
therapeutically effective amounts of at least one composition of
comprising an amino-terminal-modified chemokine.
[0098] Additionally, the present invention provides methods for
preventing, treating, or ameliorating an autoimmune condition which
comprise administering therapeutically effective amounts of at
least one composition comprising an amino-terminal-modified
chemokine.
[0099] Methods are also provided for inducing an immune response
which comprise administering a vaccine and therapeutically
effective amounts of at least one composition comprising an
amino-terminal-modified chemokine.
[0100] The present invention also provides a composition comprising
an amino-terminal-modified chemokine, wherein the chemokine binds
the fusin/CXCR4 chemokine receptor.
[0101] In a further embodiment, the present invention provides a
composition comprising an amino-terminal-modified chemokine,
wherein the amino-terminal-modified chemokine is a more effective
inhibitor of HIV infection than the corresponding unmodified
chemokine.
[0102] Additionally, methods are provided for preventing, treating,
or ameliorating HIV infection of a host which comprises:
[0103] (a) isolating stem cells from the host;
[0104] (b) transforming the stem cells with at least one
composition comprising a polynucleotide of the present invention;
and
[0105] (c) reintroducing the transformed stem cells into the host,
wherein the transformed stem cells will express at least one
amino-terminal-modified chemokine.
[0106] Preferably, the transformed stem cells express an
amino-terminal-modified chemokine comprising a chemokine selected
from the group consisting of SDF-1.alpha. and SDF-1.beta.; and an
amino-terminal-modified chemokine comprising a chemokine selected
from the group consisting of MIP-1.alpha. and MIP-1.beta..
[0107] 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 DRAWINGS
[0108] FIG. 1 is a graphical representation of the influx of
calcium into cells produced by the binding of N-terminal-modified
or unmodified chemokines to chemokine receptors, as described in
Example 2.
[0109] FIG. 2 is a graphical representation of the binding of a
chemokine-Fc protein to chemokine receptor after incubation with
either N-terminal-modified chemokines or unmodified chemokines, as
described in Example 4.
DETAILED DESCRIPTION OF THE INVENTION
[0110] The present inventors have for the first time constructed
polynucleotides expressing novel amino-terminal-modified
chemokines. These N-terminal modified chemokines interact with
chemokine receptors and have novel and unexpected properties.
[0111] As used herein, "chemokine" includes all protein molecules
with chemotactic activity. An amino-terminal-modified chemokine is
"derived from a chemokine" when the chemokine that has been
modified at its amino terminus has itself been derived from a
chemokine by any kind of alteration, addition, insertion, deletion,
mutation, substitution, replacement, or other 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 and/or 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.
[0112] 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.
[0113] As used herein, "amino-terminal-modified chemokine" includes
the result of covalently attaching any chemical moiety to the
N-terminus of a chemokine polypeptide, wherein the chemical moiety
may include any amino acid(s) or chemically modified amino acid(s);
fragments of or entire chemokines, cytokines, immunoglobulins,
antigens, kinases, proteases (including without limitation CD26,
HIV proteases, granzymes, or cathepsin G), other enzymes, or
structural proteins; polypeptides derived from the foregoing by any
form of alteration, addition, insertion, deletion, mutation,
substitution, replacement, or other modification, including without
limitation alterations to the Leu-25 residue of the mature IL-8
polypeptide (Wells et al., 1996, J. Leukoc. Biol. 59: 53-60),
alterations to the corresponding leucine residue of SDF-1.alpha.
and SDF-1.beta. (e.g. residue 47 of SEQ ID NO:s 1 and 2, residue 27
of SEQ ID NO:s 10 and 11, residue 48 of SEQ ID NO:s 12 and 13, and
residue 26 of SEQ ID NO:s 14 and 15), and alterations to the
tyrosine-28 residue of mature MIP-1.alpha. and MIP-1.beta. (Wells
et al., 1996, J. Leukoc. Biol. 59: 53-60); antibody-binding tags
such as His, Flag, or myc; lectin-binding domains; toxins; etc.
Preferably, the chemical moiety attached to the N-terminus of the
chemokine polypeptide does not interfere with binding of the
chemokine polypeptide to its receptor(s). More preferably, the
amino-terminal-modified chemokine comprises a methionine residue
covalently attached to the amino-terminus of the naturally-occuring
mature (or secreted) form(s) of the chemokine. In another more
preferred embodiment, a serine or threonine residue is attached to
the N-terminus of the chemokine (if its N-terminal residue is not
already serine or threonine), and the chemokine is then subjected
to a mild periodate oxidation to convert the serine or threonine
into an aldehyde, followed by reaction with aminooxypentane (AOP)
to form the desired AOP-chemokine oxime (see Simmons et al., 1997,
Science 276: 276-279, incorporated herein by reference). Other
methods for preparing amino-terminal-modified chemokines are
described in U.S. Pat. No. 5,656,456, incorporated herein by
reference. In another preferred embodiment, the chemical moiety
attached to the N-terminus of the chemokine polypeptide comprises a
enzymatic or chemical cleavage site so that the
amino-terminal-modified chemokine may be cleaved to produce a
molecule or molecule(s) having a desired activity. More preferably,
a GroHEK peptide (SEQ ID NO:5) comprising an enterokinase target
amino acid sequence is attached to the N-terminus of a chemokine,
optionally with additional amino acids(s) linking the GroHek
peptide to the chemokine. The GroHEK peptide can be left attached
to the chemokine as an N-terminal modification, or it can be
cleaved off by enterokinase so that the additional linking amino
acid(s) are now the N-terminal additions to the chemokine. Also
more preferably, a peptide comprising an HIV protease target amino
add sequence is attached to the N-terminus of a chemokine to form
an HIV protease cleavage site, optionally with additional amino
acids(s) linking the HIV protease recognition peptide to the
chemokine. The HIV protease recognition peptide can be left
attached to the chemokine as an N-terminal modification, or it can
be cleaved off by the HIV protease so that the additional linking
amino acid(s), if any, are now the N-terminal additions to the
chemokine. Examples of amino acid sequences cleaved by HIV
proteases are described in Tomasselli and Heinrikson, Methods in
Enzymology 241: 279-301, 1994, incorporated herein by reference. In
another preferred embodiment, the chemical moiety attached to the
N-terminus of the chemokine polypeptide comprises a molecule with a
desired activity, so that the N-terminal-modified chemokine also
possesses this desired activity. More preferably, the chemical
moiety attached to the N-terminus of the chemokine polypeptide
comprises a protease.
[0114] Fragments of amino-terminal-modified chemokines are also
encompassed by the present invention. Preferably, such fragments
retain the desired activity of the amino-terminal-modified
chemokine or modify it to create a desired activity. Fragments of
amino-terminal-modified chemokines 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
amino-terminal-modified chemokines provided herein also include
polypeptides characterized by amino acid sequences similar to those
of purified proteins but into which modifications 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 other
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 add to alter the
conformation of the molecule. 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 other 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 other modification retains
the desired activity of the amino-terminal-modified chemokine or
modifies it to create a desired activity.
[0115] Other fragments and derivatives of the sequences of
amino-terminal-modified chemokines which would be expected to
retain biological 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. For
example, amino-terminal-modified chemokines can be attached through
"linker" sequences to the Fc portion of an immunoglobulin. For a
bivalent form of the amino-terminal-modified 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, an amino-terminal-modified chemokine-IgM fusion would
generate a decavalent form of the chemokine. In addition, it is
possible to create a multivalent form of an amino-terminal-modified
chemokine by connecting the amino-terminal-modified chemokine
through a P.sub.i linkage to the phosphatidyl inositol present in
micellular preparations.
[0116] The present invention also provides both
amino-terminal-modified chemokines and forms of
amino-terminal-modified chemokines that futher include secretory
leader sequences. When an amino-terminal-modified chemokine to
which a secretory leader sequence has been attached is expressed in
host cells, the secretory leader sequence is cleaved off as the
amino-terminal-modified chemokine is translated, producing a
secreted amino-terminal-modified chemokine that has the desired
amino-terminal modification, or has a precursor molecule attached
to the N-terminus of the chemokine that may be converted to the
desired N-terminal-modification by a chemical or biological
process. The secretory leader sequence may be the same as that
found on the naturally-occurring full-length form of the chemokine,
or it may be a "synthetic" secretory leader sequence specifically
chosen for expression of the amino-terminal modified chemokine in a
particular host cell.
[0117] Amino-terminal-modified chemokines including those
comprising chemokines that are species homologs of disclosed
chemokines 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
chemokines or chemokine-encoding polynucleotides; that is,
naturally-occurring alternative forms of the disclosed
polynucleotides which also encode polypeptides which are identical,
homologous, or related to that encoded by the polynucleotides.
[0118] 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.
[0119] Expression and Purification of Amino-Terminal-Modified
Chemokines
[0120] 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.
19, 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.
[0121] 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.
[0122] 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.
[0123] 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."
[0124] 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
protein.
[0125] 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-S-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 (San
Diego, Calif.), 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.).
[0126] 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.
[0127] 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."
[0128] 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.
[0129] Uses of Amino-Terminal-Modified Chemokines
[0130] Amino-terminal-modified chemokines can be used as tools for
identifying cells expressing receptor for the chemokine, or for
studying binding of chemokine to isolated receptor molecules. The
amino-terminal-modified chemokine when incubated with cells
expressing a receptor for the chemokine will bind to these cells
and can be indicated using an indicator molecule, preferably a
commercially available fluorescently tagged antibody or other
protein, able to bind to and be localized by the
amino-terminal-modified chemokine. This will indicate cells having
a surface receptor for a given chemokine as well as the density of
this receptor on the cell surface.
[0131] Interactions between amino-terminal-modified chemokines and
chemokine receptors or other molecules can also be detected
directly by measuring changes in surface plasmon resonance using a
BIAcore.TM. sensor (Pharmacia). The chemokine receptor or the
amino-terminal-modified chemokine can be covalently immobilized to
different flow cells on the BIAcore.TM. 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. In this example the molecules of
the flow cells are acting as indicator molecules, as their state is
altered when the molecules being tested interact with the chemokine
receptor or the amino-terminal-modified chemokine that is
covalently immobilized to the flow cells.
[0132] Interactions between amino-terminal-modified chemokines and
chemokine receptors or other molecules can also be detected using a
two-hybrid or "interaction trap" system such as that developed in
yeast. (See Bai and Elledge, 1996, Methods in Enzymology 273:
331-347; Allen et al., 1995, Trends in Biochem. Sci. 20: 511-516;
and White, 1996, Proc. Natl. Acad. Sci. USA 93: 10001-10003; all of
which are incorporated herein by reference.) For example, the
amino-terminal-modified chemokine is fused or covalently linked to
a protein having a DNA binding domain, and the indicator molecule
comprises the molecule to be tested fused or covalently linked to a
protein having a transcription activation domain. Interaction
between the amino-terminal-modified chemokine and the
tested-molecule portion of the indicator molecule allows the
transcription activation portion of the indicator molecule to
activate transcription of a reporter gene.
[0133] Other suitable assays for receptor-chemokine binding
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. 168:11451156, 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; Daugherty et al., J. Exp. Med. 183: 2349-2354,
1996; Wu et al., Nature 384: 179-183, 1996; and Trkola et al.,
Nature 384: 184-187, 1996; all of which are incorporated herein by
reference.
[0134] Amino-terminal-modified chemokines 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 an
amino-terminal-modified chemokine the infiltration of the necessary
APCs and lymphocytes can be induced by the chemoattractive presence
of the chemokine. Potential advantages of using an
amino-terminal-modified chemokine is that the
amino-terminal-modified chemokine can have an enhanced activity
relative to the unmodified chemokine, or have a longer biological
half life than the chemokine alone would have.
[0135] Amino-terminal-modified chemokines can also be used to
enhance the activity of antigen-presenting cells (APCs). The
presence of the chemokine domain of the amino-terminal-modified
chemokine would chemotactically attract APCs. An antigenic molecule
could be attached to the N-terminus of the chemokine for delivery
to the APC. When such an antigen-containing amino-terminal-modified
chemokine binds to the surface of an APC and is internalized, and
the amino-terminal-modified chemokine is degraded within the APC,
the antigenic portion of the amino-terminal-modified chemokine
would be freed for interaction with MHC proteins and presentation
on the surface of the APC.
[0136] Amino-terminal-modified chemokines can also be used to
affect the chemotactic recruitment of migratory cells.
Amino-terminal-modified 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 attaching a large or particularly
stable heterologous polypeptide to the amino-terminus of the
chemokine, the amino-terminal-modified chemokine 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, an N-terminal modification may be
selected that, by binding to particular molecules or cells, will
target the amino-terminal-modified chemokine to a particular site
in order to establish a chemoattractive gradient at that site. By
altering chemoattractive gradients, amino-terminal-modified
chemokines 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,
amino-terminal-modified chemokines can 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
amino-terminal-modified chemokine within the bone marrow of a bone
marrow transplant recipient, the amino-terminal-modified chemokine
can be used to recruit the transplanted bone marrow cells to the
bone marrow where they are needed. Similarly, other cellular
processes can be affected by amino-terminal-modified chemokines, by
using them to attract particular classes of migratory cells
secreting determined factors. As another example, bone resorption
is controlled by the production within the marrow of soluble
regulatory molecules such as IL-1.beta., IL-6, and TNF-.alpha. that
mediate osteoclast recruitment, differentiation, and activation.
IL-6 influences bone resorption by stimulating the development of
osteoclasts from precursor cells and has a mitogenic effect on
osteoblasts. An amino-terminal-modified chemokine can be used to
attract cells secreting factors that stimulate osteoclasts, or by
obscuring an existing chemoattractive gradient can be used to
inhibit the recruitment of such cells to a site within bone.
[0137] Amino-terminal-modified chemokines can also be used to
affect the nature of chemokine-receptor interactions, and may block
the binding of endogenous molecules to their receptors. "Receptor
functions" that may be affected by N-terminal-modified chemokines
include, without limitation, the ability to bind ligand molecules,
the ability to interact with other proteins, the ability to
generate a "signal" affecting the properties or behaviors of the
cell expressing the receptor, or the ability to interact with or
affect other cells. By binding to a receptor,
amino-terminal-modified chemokines may deliver a signal similar to
that received via the normal ligand. The signal delivered by
binding the amino-terminal-modified chemokine may have some
properties different from that of the normal ligand because of the
structure of the amino-terminal-modified chemokine. This could
include prolonged triggering/activation or decreased activation.
The amino-terminal-modified chemokines, because of their larger
size or the nature of the structure of the N-terminal modification,
can have a longer half life in vivo compared to unmodified ligand,
possibly leading to prolonged signaling/activation. Also the larger
size of the amino-terminal-modified chemokine will cause some
stearic hindrance which may block the binding of the unmodified
ligand. An amino-terminal-modified chemokine can 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
amino-terminal-modified chemokine can inhibit one form of signaling
while enhancing or altering another. Also, an
amino-terminal-modified chemokine can bind to a receptor and cause
down regulation and/or internalization of the receptor.
Additionally, an amino-terminal-modified chemokine can 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 an
amino-terminal-modified chemokine can cause another receptor or
membrane protein to become desensitized or unable to carry out its
normal function.
[0138] HIV-1 infection of cells expressing CD4 and the fusin/CXCR4
receptor is greatly decreased by the addition of purified SDF-1
chemokine, which is bound by fusin/CXCR4. Preincubation of cells in
the presence of purified SDF-1 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. Amino-terminal-modified
chemokines can also be used to prevent infection of cells by HIV or
other viruses by blocking the binding of virus to chemokine
receptors. "HIV molecule" refers to any part of the HIV virus,
including isolated polypeptides and fragments thereof, which may or
may not be capable of infecting cells susceptible to HIV viral
isolates. "HIV particles" refers to HIV virions or derivatives
thereof which are capable of infecting certain cell types. As used
herein, "susceptible cells" are cell types capable of being
infected by certain HIV viral isolates, preferably T1 cells which
can be infected by HIV-1.sub.IIIB. An amino-terminal-modified
chemokine is a more effective inhibitor of HIV infection than the
corresponding unmodified chemokine when incubation of susceptible
cells with the amino-terminal-modified chemokine results in lower
incidence of HIV infection, as assayed by the presence of
HIV-specific proteins in the cell culture supernatant, than
incubation with the unmodified chemokine. For example, Tables 2 and
3 in Example 6 demonstrate that the amino-terminal-modified
chemokine met-hSDF-1.alpha., mature human SDF-1.alpha. with an
additional amino-terminal methionine, is a more effective inhibitor
of HIV infection than the corresponding unmodified chemokine
lys-hSDF-1.alpha. lysine being the amino-terminal amino acid of the
unmodified mature protein).
[0139] The amino-terminal-modified chemokine met-hSDF-1.alpha. has
been shown to bind to cells expressing the fusin/CXCR4 receptor.
This binding can 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 other
amino-terminal-modified chemokines such as met-MIP-1.alpha. or
met-MIP-1.beta. can 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. Further modifications of
the amino-terminal-modified chemokine, such as changes in
glycosylation or additions of chemical moieties to other parts of
the amino-terminal-modified chemokine, may result in enhanced
binding with loss of signaling, resulting in strong antagonism. By
making amino-terminal-modified chemokines 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, for example, by changing the leucine in
met-hSDF-1 (at position 27 of SEQ ID NO:s 10 and 11) to a tryosine
to change its binding specificity from CXCR receptors to CCR
receptors; thus, one modified chemokine could bind to CCR5 as well
as other CCR receptors, another modified chemokine could bind to
CXCR4 as well as a variety of other CXCR receptors, and yet another
could bind to both CCR and CXCR receptors. By simultaneously
administering a combination of amino-terminal-modified chemokines,
the greatest number of chemokine receptor types could be protected
from binding by HIV or other viral isolates.
[0140] Amino-terminal-modified chemokines could also interact with
the T cell protein CD26 in such a way as to alter the role that
CD26 plays in HIV infection.
[0141] Administration and Dosing
[0142] An amino-terminal-modified chemokine 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.alpha., IFN.beta., 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, sapomin,
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. Amino-terminal-modified
chemokines 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
amino-terminal-modified chemokine 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
amino-terminal-modified chemokine 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 mg to about 100
mg (preferably about 0.1 pg to about 10 mg, more preferably about
0.1 pg 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 amino-terminal-modified chemokine. Such
antibodies may be obtained using either the entire
amino-terminal-modified chemokine or fragments thereof as an
immunogen, the fragments preferably comprising portions of both the
chemokine and the N-terminal modification. 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 amino-terminal-modified chemokine may
also be useful therapeutics for both conditions associated with the
chemokine portion of the amino-terminal-modified chemokine 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
amino-terminal-modified chemokine 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 portion
of the amino-terminal-modified chemokine.
[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 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.
[0156] 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 amino-terminal-modified chemokine compositions from
disassociating from the matrix.
[0157] 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 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorbtion of the amino-terminal-modified
chemokine 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.
[0158] 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).
[0159] 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.
[0160] 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 amino-terminal-modified
chemokines, 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
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
amino-terminal-modified chemokines 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. For example, stem cells, preferably stem
cells that are progenitors of cells susceptible to infection by
HIV, can be obtained from an organism to be treated, preferably a
human, and transformed ex vivo with polynucleotides of the present
invention. When reintroduced into the body, the stem cells will
differentiate into particular cell types, or will produce daughter
cells that will differentiate into particular cell types, these
cell types preferably being cells susceptible to infection by HIV.
If the stem cells were transformed with a polynucleotide encoding
an amino-terminal-modified chemokine attached to a secretory leader
sequence, the differentiated cells can secrete the
amino-terminal-modified chemokine which can then bind to chemokine
receptors expressed by those differentiated cells or by other
cells, protecting the cells from HIV infection.
[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
Expression and Purification of N-Terminal-Modified Chemokines
[0165] The amino acid sequences of the full-length human chemokines
SDF-1.alpha. and SDF-1.beta. (hSDF-1.alpha. and hSDF-1.beta.,
GeneSeq accession numbers R75419 and R75420) are provided as SEQ ID
NO:s 1 and 2, respectively, and SEQ ID NO:s 3 and 4 are the
nucleotide sequences of cDNA molecules encoding hSDF-1.alpha. and
hSDF-1.beta. (GeneSeq accession numbers Q74089 and Q74091). The
amino acid sequences of the mature hSDF-1.alpha. and hSDF-1.beta.
proteins begin at amino acid 22 (lysine) in both SEQ ID NO:1 and
SEQ ID NO:2. Polymerase chain reaction (PCR) with hSDF-1.alpha. or
hSDF-1.beta. cDNA as a template was used to make expression
constructs encoding mature hSDF-1.alpha. and hSDF-1.beta. proteins,
or mature hSDF-1.alpha. and hSDF-1.beta. proteins fused to the
C-terminus of an expression/purification accessory sequence such as
GroHEK (SEQ ID NO:5, AAKDVKHHHHHHGSGSDDDDK). Cloning
NdeI/XbaI-restricted hSDF-1.alpha., hSDF-1.beta.,
GroHEK/hSDF-1.alpha., and GroHEK/hSDF-1.beta. PCR products
(generally referred to as the hSDF-1 PCR products) into the E. coli
expression vector pAL781 (LaVallie et al., 1993, Biotechnology (NY)
11: 187-193) fused the hSDF-1 PCR products in-frame to an ATG codon
which serves as the translation initiation codon, producing the
four coding sequences shown as SEQ ID NO:6-SEQ ID NO:9. When
hSDF-1.alpha. and hSDF-1.beta. are expressed from these vectors,
the resulting proteins have a methionine residue attached to the
N-terminus of the mature hSDF-1.alpha. or hSDF-1.beta. protein;
these proteins are referred to as met-hSDF-1.alpha. and
met-hSDF-1.beta. and have the amino acid sequences shown in SEQ ID
NO:10 and SEQ ID NO:11, respectively. Similarly, when
GroHEK/hSDF-1.alpha. and GroHEK/hSDF-1.beta. are expressed from
these vectors, the resulting proteins have the GroHEK peptide
attached to the N-terminus of the mature hSDF-1.alpha. or
hSDF-1.beta. protein; these proteins are referred to as
GroHEK/hSDF-1.alpha. and GroHEK/hSDF-1.beta. and have the amino
acid sequences shown in SEQ ID NO:12 and SEQ ID NO:13,
respectively. The expression vectors containing the hSDF-1 PCR
products were sequenced and used to transform the E. coli strain
GI934 (Lu et al., 1996, J. Biol. Chem. 271: 5059-5065). The
resulting transformed strains hSDF-1.alpha., hSDF-1.beta.,
GroHEK/hSDF-1.alpha., and GroHEK/hSDF-1.beta. were deposited with
the American Type Culture Collection on Aug. 15, 1997 and were
given the accession numbers ATCC 98506, ATCC 98507, ATCC 98508, and
ATCC 98509, respectively.
[0166] Epression and purification of met-hSDF-1 and GroHEK/hSDF-1
proteins. A fresh overnight culture of GI934 harboring a plasmid
expressing met-hSDF-1.alpha., met-hSDF-1.beta.,
GroHEK/hSDF-1.alpha., or GroHEK/hSDF-1.beta. was used to inoculate
IMC/Amp medium (M9 medium supplemented with 0.2% casamino acids,
0.5% glucose, 1 mM MgSO.sub.4, and 100 .mu.g/ml ampicillin) to an
OD550 of 0.05. The culture was grown at 30.degree. C. until the
OD550 reached 0.5, then L-tryptophan was added to a concentration
of 100 .mu.g/ml and the culture temperature shifted to 37.degree.
C. Four hours following tryptophan addition the cells were
harvested by centrifugation and stored at -80.degree. C. until
use.
[0167] Cells with inclusion bodies containing met-hSDF-1.alpha.,
met-hSDF-1.beta., GroHEK/hSDF-1.alpha., or GroHEK/hSDF-1.beta.
proteins were resuspended in 100 mM Tris solution, pH 8, containing
10 mM EDTA, 1 mM p-aminobenzamidine (PABA), and 1 mM
phenylmethylsulfonyl fluoride (PMSF) and were lysed in a
microfluidizer (Microfluidics, Newton, Mass.) or a French Pressure
cell (SLM Instruments, Inc.). After centrifugation of the cell
lysate in a GSA rotor at 6000 for 30 minutes, the pellet was washed
first with a 100 mM Tris solution, pH 8, containing 1 M NaCl, 1 mM
PABA, and 1 mM PMSF, and then with a 100 mM Tris solution, pH 8,
containing 0.5% Triton X-100, 1 mM PABA, and 1 mM PMSF.
[0168] In order to refold the expressed proteins, washed inclusion
bodies were solubilized in 100 ml of a pH 6.5 (or 5.5) solution
containing 15 mM sodium phosphate, 15 mM sodium acetate, 1 mM PABA,
and 6 M guanidine hydrochloride. After removing the insoluble
material, the supernatant was placed in dialysis tubing with a MW
cut-off of 5000 for dialysis at 4.degree. C. for 16 hours against a
solution containing 15 mM sodium phosphate, 15 mM sodium acetate, 1
mM PABA, and 10 mM EDTA, pH 6.5 (or 5.5). The dialysate containing
the refolded met-hSDF-1 or GroHEK/hSDF-1 proteins was then
clarified by centrifugation.
[0169] The solution containing refolded met-hSDF-1 or GroHEK/hSDF-1
proteins was pH-adjusted to 7.5 and loaded on QAE columns
equilibrated with a buffer of 15 mM sodium phosphate, pH 7.5. The
flow-through of the column was collected and pH-adjusted to 5.5 and
loaded onto an SP-650 column equilibrated with a buffer of 15 mM
sodium phosphate, 15 mM sodium acetate, pH5.5. The bound material
was then eluted with a linear gradient of 1 M NaCl in a buffer of
15 mM sodium phosphate, 15 mM sodium acetate, pH 5.5. The eluate
fractions containing the desired hSDF-1 proteins were identified by
SDS-PAGE.
[0170] Enterokinase cleavage to remove the expression/purification
accessory sequence. Solutions containing purified GroHEK/hSDF-1
proteins are dialyzed against PBS and then cleaved with
enterokinase. The digest is loaded on a Ni-IDA column in order to
separate the mature hSDF-1 proteins from the enterokinase and
GroHEK fragments. Cleavage of the GroHEK peptide from the
N-terminus of these GroHEK/hSDF-1.alpha. or GroHEK/hSDF-1.beta.
proteins produces the mature form of the hSDF-1.alpha. or
hSDF-1.beta. protein having lysine as its N-terminal amino acid;
these proteins are referred to as lys-hSDF-1.alpha. or
lys-hSDF-1.beta. and have the amino acid sequences shown in SEQ ID
NO:14 and SEQ ID NO:15, respectively.
EXAMPLE 2
Stimulation of Calcium Flux by N-Terminal-Modified Chemokines
[0171] When chemokines bind to receptors present within the
membranes of cells, a calcium flux may be induced. When
N-terminal-modified chemokines bind to these receptors, the
duration, intensity, or other properties of the calcium flux may be
altered, or the calcium flux may be inhibited. The calcium fluxes
induced by the binding of met-hSDF-1.beta., lys-hSDF-1.beta., and
lys-hSDF-1.alpha.-Fc were measured using the following protocol,
and the effects of the binding of mature chemokines (lys-) to
chemokine receptors were compared to the effects of binding
displayed by N-terminal-modified chemokines (met-). The
lys-hSDF-1.alpha.-Fc protein (or "chemokine-Fc protein") has the
same chemokine N-terminus as a mature hSDF-1.alpha. or hSDF-1.beta.
protein, but this chemokine domain has been fused to the Fc domain
of a human IgG4 molecule so that when expressed the Fc regions
interact to form a dimer. This protocol can also be used to assay
the calcium flux induced by the interaction of other
N-terminal-modified chemkoines with cells containing appropriate
chemokine receptors.
[0172] U937 cells expressing the appropriate chemokine receptor
(fusin/CXCR4) were harvested, washed twice in phenol-red-free RPMI
1640 buffer (10 mM HEPES, 0.02% BSA), and adjusted to 10.sup.7
cells per ml. A 50 .mu.g vial of FLUO-3 ester (Molecular Probes,
Eugene, Oreg., catalogue no. F-1242) was dissolved in 50 .mu.l DMSO
right before use. 5 .mu.l of this 1 mg/ml FLUO-3 ester solution was
added for each ml of cells. The mixture was incubated for 20-30
minutes at room temperature, then washed twice with phenol-red-free
RPMI 1640 buffer (phenol-red-free RPMI 1640 with 2.5% fetal calf
serum and 10 mM HEPES may also be used). The cells were resuspended
at 10.sup.7 per ml in RPMI 1640 buffer and stored on ice until
ready to use. To test for calcium flux, 50 .mu.l of cells were
diluted to 500 .mu.l with phenol-red-free RPMI 1640 buffer. Using a
FACSCAN (BD) fluorescence-activated cell analyzer, the background
reading for the loaded cells was determined (FL1 channel). Cells
were stimulated appropriately with amino-terminal-modified or
unmodified chemokine 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.
[0173] The results of this experiment are shown in FIG. 1, and
demonstrate that the binding of met-hSDF-1.beta. to its receptor
induces a stronger calcium flux, and at a lower concentration, than
either lys-hSDF-1.beta. or lys-hSDF-1.alpha.-Fc. This result is
surprising in view of the experimental results observed by Wells et
al. (1996, J. Leukoc. Biol. 59: 53-60), who concluded that the
amino-terminal-modified chemokine met-RANTES was unable to induce
chemotaxis or calcium mobilization in the RANTES-responsive THP-1
pro-monocytic cell line.
EXAMPLE 3
Stimulation or Inhibition of Chemotaxis by N-Terminal-Modified
Chemokines
[0174] N-terminal-modified chemokines can be tested for their
ability to stimulate or inhibit 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 4
Binding of Chemokine to Receptor After Incubation
Within-Terminal-Modified or Unmodified Chemokines
[0175] The ability of N-terminal-modified and unmodified chemokines
to compete with a chemokine-Fc protein for binding to chemokine
receptor was tested. Cells were preincubated with chemokine and
then reacted with chemokine-Fc protein and a fluorescently-labeled
anti-Fc antibody to determine if the chemokine-Fc was able to bind
the fusin/CXCR4 receptor. As shown in FIG. 2, both the unmodified
form and the N-terminal-modified methionine form of hSDF-1.beta.
affect the binding of hSDF-1.beta.-Fc with the fusin/CXCR4
receptor.
[0176] The ability of the met-hSDF-1.beta. proteins to affect the
availability of chemokine receptors for binding was compared to
that of mature human SDF-1.beta. proteins having a lysine residue
at the N-terminus (lys-hSDF-1.beta.). U937 cells were preincubated
with either met-hSDF-1.beta. or lys-hSDF-1.beta. for 1 hour at 4
degrees C. in D-PBS containing 0.02% azide, fetal calf serum, and
rabbit serum, followed by incubation with 450 ng/ml
lys-hSDF-1.alpha.-Fc for 20 minutes on ice, a wash of the cells,
and staining with goat anti-human phycoerythrin-conjugated antibody
(GaH). After a brief incubation on ice the stained cells are washed
and analyzed by fluorescent flow cytometry on a FACSscan machine
(BD Instruments, Mountain View, Calif.). Each data point represents
the average of two duplicate samples; the "GaH" control shown in
FIG. 2 is a sample to which the lys-hSDF-1.alpha.-Fc protein was
not added. The results of these experiments are shown in FIG. 2,
and indicate that both met-hSDF-1.beta. and lys-hSDF-1.beta. can
equally block binding of lys-hSDF-1.alpha.-Fc to the
receptor-expressing cells. It is possible to conclude from this
result that the enhanced ability of met-hSDF-1.beta. to inhibit HIV
infection (see Example 6 and Tables 2 and 3) is not due to a
greater ability to bind the fusin/CXCR4 receptor, since both
met-hSDF-1.beta. and lys-hSDF-1.beta. apparently block binding of
lys-hSDF-1.alpha.-Fc to the receptor to the same extent.
[0177] The ability of other N-terminal-modified chemokines to block
binding to chemokine receptors is determined by an assay analogous
to that described above.
EXAMPLE 5
Down-Modulation of Chemokine Receptor by N-Terminal-Modified
Chemokine Binding
[0178] Although chemokines can inhibit HIV infection (see Example
6) it has not been established whether this occurs through
competition with the virus for the co-receptor binding sites, by
making the receptor nonfunctional for HIV binding, or by having
altered signaling properties that affect events downstream to
infection, i.e. viral replication or production of virus particles.
It is possible that binding of the receptor by the chemokine will
either cause desensitization or down-modulation (also called
"down-regulation"). Chemokine receptors and other seven-span
G-protein-coupled receptors can become desensitized: still present
on the surface of the cell but no longer able to bind ligand. To
investigate this question we have incubated cells with chemokine
and then reacted them with a fluorescently-labeled anti-receptor
antibody to determine if they still express the receptor. As shown
in Table 1, both the unmodified form ("lys-") and the
N-terminal-modified ("met-") form of hSDF-1.beta. will
down-modulate the fusin/CXCR4 receptor, with the
N-terminal-modified met-hSDF-1.beta. demonstrating greater
effectiveness in down-modulation.
[0179] U937 cells were incubated overnight (for 16 hours) at 37
degrees C. with 500 ng/ml of either met-hSDF-1.beta.,
lys-hSDF-1.beta., or lys-hSDF-1.alpha.-Fc. Following the incubation
the cells were stained with the fluorescently labeled
anti-fusin/CXCR4 12G5 monoclonal antibody and analyzed by FACS. The
median fluorescence observed using an isotype control, 3.6, was
subtracted from the raw fluorescence data to determine the net
median fluorescence reported in Table 1. The results of these
experiments are shown in Table 1, and indicate that the enhanced
ability of met-hSDF-1.beta. to inhibit HIV infection (see Example 6
and Tables 2 and 3), presumably via binding of HIV to the
fusin/CXCR4 receptor, could be due to increased down-modulation of
the receptor, since met-hSDF-1.beta. causes down-modulation of the
receptor to a greater extent than lys-hSDF-1.beta..
1TABLE 1 Down-modulation of fusin/CXCR4 receptors after incubation
with N-terminal-modified chemokine (met-hSDF-1.beta.) or chemokines
not modified at the N-terminus (lys-hSDF-1.beta., lys-hSDF-1-Fc).
Median Sample: Fluorescence: % of Control: Control 23.2 100%
met-hSDF-1.beta. 0.5 2.3% lys-hSDF-1.beta. 2.0 9.3% lys-hSDF-1-Fc
2.2 9.6%
[0180] Down-regulation of other chemokine receptors by binding of
N-terminal-modified chemokines to cells is determined by an assay
for receptor down-regulation analogous to that described above.
EXAMPLE 6
Use of N-Terminal-Modified Chemokines to Inhibit HIV Infection of T
Cells
[0181] The T cell line T1 expresses CD4 and the chemokine receptor
fusin/CXCR4, and are readily infected with the T-tropic virus
HIV-1.sub.IIIB. The ability of different forms of hSDF-1.beta. to
inhibit HIV binding to the chemokine receptor was tested as
follows. T1 cells were preincubated at 37 degrees C. for two hours
with a chemokine added at an approximate concentration of 115 nM.
The T1 cells were then infected with HIV-1.sub.IIIB added at a
multiplicity of infection (MOI) of 10.sup.-2. After a four-hour
incubation at 37 degrees C., the cells were washed twice and
5.times.10.sup.5 cells per well were added to 24-well plates in 2
ml of medium. Every three days thereafter, half the medium (1 ml)
was removed and replaced with 1 ml fresh medium containing
approximately 115 nM of the chemokine. Starting on day 4, samples
were taken every three days for analysis of HIV-p24 by ELISA. As a
control, virus-infected T1 cells were cultured without
preincubation or incubation with exogenous chemokine. The
lys-hSDF-1.alpha. was obtained from PeproTech (Rocky Hill, N.J.).
As indicated in Table 2, preincubation with met-hSDF-1.beta. and
the readdition of this chemokine to the medium every three days
results in near complete inhibition of HIV-1 infection of the T1
CD4.sup.+ T cell line. In contrast to the inhibition seen with the
N-terminal-modified methionine form of hSDF1-.beta., the
preincubation and addition every three days of approximately 115 nM
of the unmodified lys-hSDF-1.alpha. or lys-hSDF-1.beta. gives a
much lower level of inhibition, about 60% at day 10 of culture.
Thus the unmodified hSDF-1.alpha. and hSDF-1.beta. having an
amino-terminal sequence of KPV . . . (SEQ ID NO:14 and SEQ ID
NO:15) appear to give roughly equivalent levels of inhibition of
HIV infection, but hSDF-1.beta. with a modified amino-terminal
sequence of MKPV . . . (SEQ ID NO:11) gives a level of inhibition
of HIV infection (99+% at day 10 of culture) that is three logs
greater than that seen with the unmodified chemokines.
2TABLE 2 Inhibition of HIV infection of T1 T cells by unmodified
(lys-hSDF-1.alpha. or .beta.) and N-terminal-modified
(met-hSDF-1.beta.) chemokines. HIV-1 p24 (pg/ml) % Inhibition %
Inhibition Chemokine: Day 7: vs. Control: Day 10: vs. Control:
Control 632 -- 646,000 -- (no chemokine) lys-hSDF-1.alpha. 107 83%
266,000 59% lys-hSDF-1.beta. 160 75% 280,000 57% met-hSDF-1.beta. 5
99% 308 99+%
[0182] When T1 cells were cultured for two hours with
met-hSDF-1.beta. before infection with HIV-1.sub.IIIB and
met-hSDF-1.beta. was not added again, the level of inhibition of
infection was 81% at day 5 and 72% at day 10 (Table 3). In this
same experiment, culture with met-hSDF-1.beta. followed by addition
to culture of this N-terminal-modified chemokine at three-day
intervals resulted in complete inhibition of HIV infection. Even
when the T cells were not pretreated with N-terminal-modified
chemokine before infection with virus, but the met-hSDF-1.beta. was
added at 115 nM every three days after infection, the inhibition of
infection was 93% at day 5 and 98% at day 10. In contrast, the
addition of unmodified chemokine lys-hSDF-1.alpha. after infection
produced much weaker inhibition of infection (data not shown).
3TABLE 3 Inhibition of HIV infection of T1 T cells by pre-treatment
and post-treatment with N-terminal-modified (met-hSDF-1.beta.)
chemokines. Treatment with N-terminal Modified % Inhibition vs.
Control Chemokine met-SDF-1.beta.: Day 5: Day 10: Control (no
chemokine) -- -- Pretreatment only 81% 72% Pretreatment and
addition every 3rd day 99.9% 99.9% No pretreatment, addition every
3rd day 93% 98%
[0183] The results shown in Tables 2 and 3 are surprising in view
of the experimental results observed by Simmons et al. in testing
the ability of the amino-terminal-modified chemokine met-RANTES to
inhibit HIV-1 infection of peripheral blood mononuclear cells or
primary macrophage cultures (1997, Science 276: 276279). Simmons et
al. found that met-RANTES was either about as effective or less
effective in inhibiting HIV-1 infection of these cells than the
unmodified RANTES chemokine.
Sequence CWU 1
1
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