U.S. patent application number 10/392355 was filed with the patent office on 2003-12-11 for chemokine variants and methods of use.
Invention is credited to Norcross, Michael A., Oravecz, Tamas.
Application Number | 20030229203 10/392355 |
Document ID | / |
Family ID | 22073299 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030229203 |
Kind Code |
A1 |
Oravecz, Tamas ; et
al. |
December 11, 2003 |
Chemokine variants and methods of use
Abstract
The present invention provides the nucleotide and amino acid
sequence of truncated RANTES (3-68), which has the same amino acid
sequence as the wild-type RANTES, but with a Serine/Proline
truncation at positions 1 and 2 from the N-terminus, respectively.
CD26 is a leukocyte activation marker that possesses dipeptidyl
peptidase IV (DPPIV) activity but whose natural substrates and
immunological functions had not been previously defined. Several
chemokines, including RANTES (regulated on activation, normal T
expressed and secreted) are provided, which are substrates for
human CD26. The truncated RANTES (3-68) lacked the ability of
native RANTES (1-68) to increase the cytosolic calcium
concentration in human monocytes, but it still induces this
response in macrophages activated with macrophage
colony-stimulating factor (M-CSF). RANTES (3-68) retains the
ability to stimulate CCR5 receptors and to inhibit the cytopathic
effects of HIV-1. The invention provides methods for identifying
compounds that affect DPPIV-medicated chemokine cleavage, methods
for inhibiting HIV infection and treating individuals having or at
risk of having HIV infection, methods for diagnosis and/or
prognosis of individuals having a chemokine-associated disorder and
methods for accelerating wound healing and angiogenesis, all based
on the discovery of DPPIV-mediated cleavage of chemokines.
Inventors: |
Oravecz, Tamas; (Palo Alto,
CA) ; Norcross, Michael A.; (Bethesda, MD) |
Correspondence
Address: |
NATIONAL INSTITUTE OF HEALTH
C/O NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30303
US
|
Family ID: |
22073299 |
Appl. No.: |
10/392355 |
Filed: |
March 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10392355 |
Mar 18, 2003 |
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09555663 |
Sep 14, 2000 |
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6534626 |
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09555663 |
Sep 14, 2000 |
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PCT/US98/25492 |
Dec 1, 1998 |
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60067033 |
Dec 1, 1997 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 536/23.5 |
Current CPC
Class: |
C07K 14/523 20130101;
A61K 38/00 20130101; C07K 14/522 20130101; C12N 9/48 20130101 |
Class at
Publication: |
530/350 ;
536/23.5; 435/69.1; 435/320.1; 435/325 |
International
Class: |
C07K 014/715; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. A substantially pure polypeptide having an amino acid sequence
as set forth in SEQ ID NO:2.
2. An isolated polynucleotide which encodes an amino acid sequence
as set forth in SEQ ID NO:2.
3. An isolated polynucleotide selected from the group consisting
of: a) SEQ ID NO:1; b) SEQ ID NO:1, wherein T can also be U; c)
nucleic sequences complementary to SEQ ID NO:1; d) fragments of a),
b), or c) that are at least 15 bases in length and that will
hybridize to DNA which encodes SEQ ID NO:2.
4. An expression vector containing in operable linkage the
polynucleotide as in claim 2.
5. A host cell containing the vector of claim 4.
6. The host cell of claim 5, wherein the cell is a eukaryotic
cell.
7. A method for identifying a compound which modulates Dipeptidyl
peptidase IV (DPPIV)-mediated chemokine processing comprising: a)
incubating components comprising the compound, DPPIV and a
chemokine under conditions sufficient to allow the components to
interact; and b) determining the N-terminal amino acid sequence of
the chemokine before and after incubating in the presence of the
compound.
8. The method of claim 7, wherein the modulating is inhibition of
DPPIV-mediated chemokine processing.
9. The method of claim 7, whererin the modulating is stimulation of
DPPIV-mediated chemokine processing.
10. The method of claim 7, wherein the compound is a peptide.
11. The method of claim 7, wherein the compound is a
peptidomimetic.
12. The method of claim 7, wherein the DPPIV is expressed in a
cell.
13. The method of claim 7, wherein the chemokine contains a proline
or an alanine at position 2 from the N-terminus.
14. A method of inhibiting membrane fusion between HIV and a target
cell or between an HIV-infected cell and a CD4 positive uninfected
cell comprising contacting the target or CD4 positive cell with a
fusion-inhibiting effective amount of the polypeptide of SEQ ID
NO:2.
15. The method of claim 14, wherein the contacting is by in vivo
administration to a subject.
16. The method of claim 14, wherein the polypeptide is administered
by intravenous, intramuscular or subcutaneous injection.
17. The method of claim 14, wherein the polypeptide is formulated
in a pharmaceutically acceptable carrier.
18. A method of treating a subject having or at risk of having an
HIV infection or disorder, comprising administering to the subject,
a therapeutically effective amount of a polypeptide of SEQ ID NO:2,
wherein the polypeptide inhibits cell-cell fusion in cells infected
with HIV.
19. The method of claim 18, wherein the subject is suffering from
AIDS or ARC.
20. The method of claim 18, wherein the polypeptide is formulated
in a pharmaceutically acceptable carrier.
21. A method of treating a subject having an HIV-related disorder
associated with expression of CCR5 comprising administering to an
HIV infected or susceptible cell of the subject, a polypeptide of
SEQ ID NO:2 or a nucleic acid sequence encoding the polypeptide of
SEQ ID NO:2 or other variant chemokine.
22. The method of claim 21, wherein the polypeptide or nucleic acid
is introduced into the cell using a carrier.
23. The method of claim 22, wherein the carrier is a vector.
24. The method of claim 21, wherein the administering is ex
vivo.
25. The method of claim 21, wherein the administering is in
vivo.
26. A pharmaceutical composition comprising the polypeptide of SEQ
ID NO:2 in a pharmaceutically acceptable carrier.
27. A pharmaceutical composition comprising CD26 polypeptide in a
pharmaceutically acceptable carrier.
28. A method for producing a variant chemokine having an activity
different from the activity of the wild-type chemokine, comprising
contacting the wild-type chemokine with an N-terminal processing
effective amount of Dipeptidyl peptidase IV (DPPIV), thereby
truncating the chemokine and producing a variant chemokine.
29. The method of claim 28, wherein the chemokine contains a
proline or an alanine at position 2 from the N-terminus.
30. The method of claim 29, wherein the chemokine is selected from
the group consisting of RANTES, MIP-1, IP-10, cotaxin, MDC and
MCP-2.
31. The method of claim 29, wherein the chemokine is RANTES.
32. A method for inhibiting HIV-1 replication in a host cell
susceptible to HIV-1 infection, comprising contacting the cell or
the host with an effective amount of Dipeptidyl peptidase IV
(DPPIV) enzyme such that macrophage-derived chemokine (MDC) is
cleaved to produce truncated MDC, thereby providing antiviral
activity and inhibiting HIV-1 replication.
33. A method for inhibiting Dipeptidyl peptidase IV
(DPPIV)-mediated chemokine processing comprising contacting DPPIV
with an inhibiting effective amount of a compound which inhibits
DPPIV expression or activity.
34. A method for inhibiting an allergic or inflammatory reaction in
a subject, comprising administering to the subject an effective
amount of Dipeptidyl peptidase IV (DPPIV) enzyme such that a
chemokine is cleaved to produce a truncated chemokine, thereby
inhibiting an allergic or inflammatory reaction.
35. The method of claim 34, wherein the chemokine is eotaxin.
36. The method of claim 34, wherein the subject is a human.
37. A method for accelerating angiogenesis or wound healing in a
subject, comprising administering to the subject an effective
amount of an inhibitor of Dipeptidyl peptidase IV (DPPIV) enzyme
activity or gene expression or a DPPIV-insensitive chemokine, such
that chemokine processing is inhibited, thereby accelerating
angiogenesis or wound healing.
38. The method of claim 37, wherein the chemokine is IP-10.
39. The method of claim 37, wherein the DPPIV-insensitive chemokine
is a wild-type chemokine with the proviso that alanine or proline
at position 2 is replaced with any amino acid other than alanine or
proline.
40. A method for inhibiting HIV-1 replication in a host cell
susceptible to HIV-1 infection, comprising contacting the cell or
the host with an effective amount of Dipeptidyl peptidase IV
(DPPIV) enzyme such that RANTES is cleaved to produce truncated
RANTES, thereby providing antiviral activity and inhibiting HIV-1
replication.
41. The method as in any of claims 7, 28, 32, 33, 34, 37, or 39,
wherein the DPPIV enzyme is CD26.
42. A method of diagnosis of a subject having a
chemokine-associated disorder comprising: identifying the presence
of a chemokine of interest from a specimen isolated from the
subject; determining the amino-terminal sequence of the chemokine,
wherein a full-length amino acid sequence is indicative of the
presence of a wild-type chemokine polypeptide and a truncated
amino-terminal sequence is indicative of the presence of a variant
chemokine; and determining the concentration of wild-type chemokine
as compared to variant chemokine, thereby providing a diagnosis of
the subject.
43. The method of claim 42, wherein the determining of the
amino-terminal sequence of the chemokine is by contacting the
chemokine with an antibody which distinguishes wild-type from
variant chemokine polypeptide.
44. The method of claim 42, wherein the specimen is selected from
the group consisting of blood, sputum, urine, saliva, cerebrospinal
fluid, and serum.
45. Antibodies which bind to wild-type chemokine but not to
DPPIV-truncated chemokine.
46. Antibodies which bind to DPPIV-truncated chemokine but not to
wild-type chemokine.
47. The antibodies as in claims 45 or 46, wherein the chemokine is
RANTES.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to chemoattractant
cytokines, called chemokines, and more specifically to truncated or
variant forms of chemokines which have functions different from
their wild-type counterparts, methods of use and methods of
producing such variant chemokines.
BACKGROUND OF THE INVENTION
[0002] Immunomodulatory proteins include chemotactic cytokines,
called "chemokines". Chemokines are small molecular weight immune
ligands which are chemoattractants for leukocytes, such as
especially neutrophils, basophils, monocytes and T cells. There are
two major classes of chemokines which both contain four conserved
cysteine residues which form disulfide bonds in the tertiary
structure of the proteins. The .alpha. class is designated C--X--C
(where X is any amino acid), which includes IL-8, CTAP-III,
gro/MGSA and ENA-78; and the .beta. class, designated C--C, which
includes MCP-1, MIP-1.alpha. and .beta., and regulated on
activation, normal T expressed and secreted protein (RANTES). The
designations of the classes are according to whether an intervening
residue spaces the first two cysteines in the motif. In general,
most C--X--C chemokines are chemoattractants for neutrophils but
not monocytes, whereas C--C chemokines appear to attract monocytes
but not neutrophils. Recently, a third group of chemokines, the "C"
group, was designated by the discovery of a new protein called
lymphotactin (Kelner, et al., Science, 266:1395-1933, 1994). The
chemokine family is believed to be critically important in the
infiltration of lymphocytes and monocytes into sites of
inflammation.
[0003] Monocytes differentiate into macrophages as they migrate
from the blood to tissues during immune surveillance. At sites of
inflammation, monocyte infiltration and macrophage accumulation are
coordinated, in part, by chemokines (1). The mechanisms that
control the recruitment of monocytes and macrophages by
chemoattractants have not been clearly defined, but they may
include regulation of the expression of chemokines and their
receptors (2) as well as the modification of chemokine activity by
posttranslational processing (3-5). Several chemokines share a
conserved NH2-X-Pro sequence (X, any amino acid) at the
NH2-terminus (6), which conforms to the substrate specificity of
dipeptidyl exopeptidase IV (DPPIV) (7). DPPIV cleaves the first two
amino acids from peptides with penultimate proline or alanine
residues, although no natural substrate with immune function has
been identified. This enzyme is also a leukocyte differentiation
antigen, known as CD26 (8-10), that is expressed on the cell
surface mostly by T lymphocytes and macrophages. Expression of CD26
has been associated with T cell activation (8-10) and with
susceptibility of a T cell line to infection with macrophage-tropic
(M-tropic) HIV-1 (11).
SUMMARY OF THE INVENTION
[0004] The present invention is based on the discovery that
chemokines having a particular N-terminal motif are natural
substrates for a dipeptidyl dipeptidase (DPPIV). Prior to the
present invention, it was known that CD26 is a leukocyte activation
marker that possesses dipeptidyl peptidase IV (DPPIV) activity but
natural substrates had not been identified. The present invention
shows that several chemokines, including RANTES (regulated on
activation, normal T expressed and secreted) are substrates for
recombinant soluble human CD26 (sCD26). The present invention shows
that DPPIV, e.g., CD26-mediated processing, together with cell
activation induces changes in receptor expression and provides a
mechanism for differential cell recruitment and for the regulation
of target cell specificity of chemokines.
[0005] Abbreviations: [Ca2+]i, cytosolic free Ca2+ concentration;
DPPIV, dipeptidyl peptidase IV; ES-MS, electrospray mass
spectrometry; M-tropic, macrophage-tropic; pNA, p-nitroanilide; rh,
recombinant human; sCD26, soluble CD26.
[0006] In a first embodiment, the invention provides the nucleotide
and amino acid sequence of truncated RANTES (3-68), which is the
same as the wild-type RANTES with a Serine/Proline truncation at
positions 1 and 2 from the N-terminus, respectively
[0007] In another embodiment, the invention provides a method for
identifying a compound which modulates dipeptidyl peptidase IV
(DPPIV)-mediated chemokine processing. The method includes a)
incubating components comprising the compound, DPPIV and a
chemokine under conditions sufficient to allow the components to
interact; and b) determining the N-terminal amino acid sequence of
the chemokine before and after incubating in the presence of the
compound. Modulation of DPPIV-mediated chemokine processing may be
inhibition or stimulation of processing, for example. Compounds
which modulate such processing include peptides, peptidomimetics,
and other small molecule compounds.
[0008] In another embodiment, the invention provides a method of
inhibiting membrane fusion between HIV and a target cell or between
an HIV-infected cell and a CD4 positive uninfected cell by
contacting the target or CD4 positive cell with a fusion-inhibiting
effective amount of the polypeptide of SEQ ID NO:2 (RANTES
3-68).
[0009] The invention also provides a method of treating a subject
having or at risk of having an HIV infection or disorder, including
administering to the subject, a therapeutically effective amount of
a polypeptide of SEQ ID NO:2, wherein the polypeptide inhibits
cell-cell fusion in cells infected with HIV The invention also
provides a method of treating a subject having an HIV-related
disorder associated with expression of CCR5 comprising
administering to an HIV infected or susceptible cell of the
subject, a polypeptide of SEQ ID NO:2 or a nucleic acid sequence
encoding the polypeptide of SEQ ID NO:2 or other variant chemokine.
Preferably, the subject is a human.
[0010] Also included are pharmaceutical compositions including the
polypeptide of SEQ ID NO:2 or CD26, in pharmaceutically acceptable
carriers.
[0011] In yet another embodiment, the invention provides a method
for producing a variant chemokine having an activity different from
the activity of the wild-type chemokine, including contacting the
wild-type chemokine with an N-terminal processing effective amount
of dipeptidyl peptidase IV (DPPIV), thereby truncating the
chemokine and producing a variant chemokine. Chemokines may
include, but are not limited to, RANTES, MIP-1, IP-10, eotaxin,
MDC, and MCP-2.
[0012] The invention also provides a method for inhibiting HIV-1
replication in a host cell susceptible to HIV-1 infection,
comprising contacting the cell or the host with an effective amount
of dipeptidyl peptidase IV (DPPIV) enzyme such that
macrophage-derived chemokine (MDC) is cleaved to produce truncated
MDC, thereby providing antiviral activity and inhibiting HIV-1
replication and a A method for inhibiting HIV-1 replication in a
host cell susceptible to HIV-1 infection, comprising contacting the
cell or the host with an effective amount of dipeptidyl peptidase
IV (DPPIV) enzyme such that RANTES is cleaved to produce truncated
RANTES, thereby providing antiviral activity and inhibiting HIV-1
replication.
[0013] In another embodiment, the invention provides a method for
inhibiting dipeptidyl peptidase IV (DPPIV)-mediated chemokine
processing comprising contacting DPPIV with an inhibiting effective
amount of a compound which inhibits DPPIV expression or
activity.
[0014] In another embodiment, the invention provides a method for
inhibiting an allergic or inflammatory reaction in a subject,
comprising administering to the subject an effective amount of
Dipeptidyl peptidase IV (DPPIV) enzyme such that a chemokine is
cleaved to produce a truncated chemokine, thereby inhibiting an
allergic or inflammatory reaction. Preferably, the chemokine is
eotaxin.
[0015] In another embodiment, the invention provides a method for
accelerating angiogenesis or wound healing in a subject, comprising
administering to the subject an effective amount of an inhibitor of
dipeptidyl peptidase IV (DPPIV) enzyme activity or gene expression
or a DPPIV-insensitive chemokine, such that chemokine processing is
inhibited, thereby accelerating angiogenesis or wound healing. One
exemplary chemokine useful in the method for accelerating
angiogenesis is IP-10.
[0016] In all of the above methods, the exemplary DPPIV shown in
the present invention is CD26.
[0017] In yet another embodiment, the invention provides a method
for diagnosis or prognosis of a subject having a
chemokine-associated disorder. The method includes identifying the
presence of a chemokine of interest from a specimen isolated from
the subject; determining the amino-terminal sequence of the
chemokine, wherein a full-length amino acid sequence is indicative
of the presence of a wild-type chemokine polypeptide and a
truncated amino-terminal sequence is indicative of the presence of
a variant chemokine; and determining the concentration of wild-type
chemokine as compared to variant chemokine, thereby providing a
diagnosis of the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. RANTES cleavage products after digestion with sCD26.
RANTES was incubated overnight with the indicated amounts of
enzymatically active (E+) or enzymatically deficient (E( ) sCD26
and samples were subjected to ES-MS analysis. The peaks in the
spectrum at masses of 7905 to 7906 and 7887 to 7890 are tentatively
identified as [M+K+]+ of RANTES with (7904 daltons) and without
(7886 daltons) a molecule of H2O, respectively; the labeled peaks
at the left of the spectrum correspond to each of these molecular
ions minus a Ser-Pro dipeptide (184 daltons).
[0019] FIG. 2. Competitive inhibition of DPPIV by RANTES(1-68).
Colorimetric DPPIV enzyme assay was performed using human placental
DPPIV and the Gly-Pro-pNA substrate, in the presence or absence of
the test competitors Ile-Pro-Ile, RANTES(1-68), or RANTES(3-68);
the competitor concentration is indicated on the horizontal axis.
Data are means.+-.SEM (n=3), except for the highest concentration
of RANTES(1-68) and RANTES(3-68), for which only one sample was
assayed in order to conserve material. Similar results were
obtained in a repeat experiment.
[0020] FIG. 3. RT-PCR analysis of chemokine receptor transcripts in
monocytes cultured in the absence (M) or presence (M+M-CSF) of
M-CSF. Total cellular RNA was subjected to RT-PCR analysis as
described in Materials and Methods. Control reactions performed
without reverse transcriptase were negative for each PCR
product.
[0021] FIG. 4. Effects of chemokines on [Ca2+]i in monocytes
cultured in the absence (M) or presence (M+M-CSF) of M-CSF Fura-2
labeled cells were exposed (at the times indicated by arrowheads)
to chemically synthesized RANTES variants (100 nM) or other
indicated rh chemokines (30 nM) (R & D Systems), and Ca2+
responses were measured. The final concentrations of chemokines in
this and subsequent experiments were sufficient to induce a maximal
increase in [Ca2+]i in the responding cells, and further challenge
with the same dose produced little or no detectable change in
[Ca2+]i. The duration (.about.100 s) and amplitude (.about.20 to
30% of Fura-2 saturation) of Ca2+ responses were similar to those
obtained for chemokines with human monocytes (36). Similar results
were obtained in two additional experiments.
[0022] FIG. 5. Desensitization of chemokine-induced Ca2+ responses
by full-length or truncated RANTES. Fura-2 labeled cells were
stimulated first with 100 nM RANTES(1-68) or RANTES(3-68), or were
left unstimulated. After .about.150 s, the cells were challenged
with the RANTES variants (100 nM) or other chemokines (30 nM) as
indicated, and Ca2+ responses were measured.
[0023] FIG. 6. Activity of full-length and truncated RANTES in
cells expressing recombinant CCR5 or CCR1 receptors. The [Ca2+]i
was measured in HEK-293 cells expressing CCR5 (A and C) and HOS-CD4
cells expressing CCR1 (B and D). (A and B) Cells were stimulated
with various concentrations of the two RANTES variants as indicated
and maximal fluorescence values were calculated from the peaks of
the Ca2+ response curves. (C and D) Homologous and heterologous
desensitization of the responses induced by RANTES(1-68) and
RANTES(3-68) was measured in transfectants as described in FIG.
5.
[0024] FIG. 7. Effects of full-length and truncated RANTES on
HIV-1-induced cytopathicity. (A) HOS-CD4.CCR5 cells were incubated
with uninfected PM1 cells or PM1 cells chronically infected with
MV3-HXB2 virus in the presence or absence of the indicated
concentrations of RANTES variants. After 3 days, cell viability was
measured by the XTT method. Data are means of triplicate samples
(SEM, <20% of mean). (B) Representative photomicrographs of
HOS-CD4.CCR5 cells cultured with HIV-1-infected PM1 cells in the
absence or presence of RANTES (1-68) or RANTES(3-68) as
indicated.
[0025] FIG. 8. The nucleotide and deduced amino acid sequences for
RANTES 3-68 (SEQ ID NO:1 and 2, respectively) are shown.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] The present invention is based on the discovery of variant
forms of chemokines which have different functions than their
wild-type counterparts. These variant chemokines are produced by
cleavage with a dipeptidyl peptidase IV (DPPIV) which cleaves at
the N-terminus of a polypeptide when there is a proline or an
alanine at position 2.
[0027] Overview
[0028] CD26 is a leukocyte activation marker that possesses
dipeptidyl peptidase IV (DPPIV) activity but whose natural
substrates and immunological functions have not been clearly
defined. Several chemokines, including RANTES (regulated on
activation, normal T expressed and secreted) have now been shown to
be substrates for recombinant soluble human CD26 (sCD26). The
truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to
increase the cytosolic calcium concentration in human monocytes,
but it still induced this response in macrophages activated with
macrophage colony-stimulating factor (M-CSF). Analysis of chemokine
receptor messenger RNAs and patterns of desensitization of
chemokine responses showed that the differential activity of the
truncated molecule results from an altered receptor specificity.
RANTES(3-68) showed a reduced activity, relative to that of
RANTES(1-68), with cells expressing the recombinant CCR1 chemokine
receptor, but it retained the ability to stimulate CCR5 receptors
and to inhibit the cytopathic effects of HIV-1. Our results
indicate that CD26-mediated processing together with cell
activation induced changes in receptor expression provide an
integrated mechanism for differential cell recruitment and for the
regulation of target cell specificity of RANTES, and possibly other
chemokines.
[0029] Nucleotide and Amino Acid Sequences of RANTES Variant (3-68)
or other Chemokine Variants
[0030] In a first embodiment, the invention provides a
substantially purified RANTES variant polypeptide exemplified by
the amino acid sequence of SEQ ID NO:2. The term "polypeptide"
means any chain of amino acids, regardless of length or
post-translational modification (e.g., glycosylation or
phosphorylation), and includes natural proteins as well as
synthetic or recombinant polypeptides and peptides.
[0031] The term "substantially pure" as used herein refers to
RANTES (3-68) or other variant chemoline which is substantially
free of other proteins, lipids, carbohydrates or other materials
with which it is naturally associated. One skilled in the art can
purify RANTES (3-68) or other variant chemokine using standard
techniques for protein purification. The substantially pure
polypeptide will yield a single major band on a non-reducing
polyacryl-amide gel. The purity of the RANTES (3-68) or other
variant chemokine polypeptide can also be determined by
amino-terminal amino acid sequence analysis. RANTES (3-68) or other
variant chemokine polypeptide includes functional fragments of the
polypeptide, as long as the activity of RANTES (3-68) or other
variant chemokine remains. Such functional variants would include
the N-terminus which is truncated as compared to the wild-type
RANTES or other chemokine. The term "variant" as used herein refers
to a polypeptide having substantially the same polypeptide sequence
as the corresponding wild-type polypeptide, with minor amino acid
variations. These amino acid variations result in a polypeptide
having various additional and/or different functions from the
wild-type polypeptide, and possibly having altered receptor
specificity as compared to the wild-type polypeptide. Smaller
peptides containing the biological activity of RANTES (3-68) or
other variant chemokine are included in the invention. The term
"substantially pure," when referring to an chemokine polypeptide,
means a polypeptide that is at least 60%, by weight, free from the
proteins and naturally-occurring organic molecules with which it is
naturally associated. A substantially pure RANTES (3-68) or other
variant chemokine polypeptide is at least 75%, more preferably at
least 90%, and most preferably at least 99%, by weight, RANTES
(3-68) or other variant chemokine polypeptide. A substantially pure
RANTES (3-68) or other variant chemokine can be obtained, for
example, by extraction from a natural source; by expression of a
recombinant nucleic acid encoding a RANTES (3-68) or other variant
chemokine polypeptide, or by chemically synthesizing the protein.
Purity can be measured by any appropriate method, e.g., column
chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
[0032] Minor modifications of the recombinant RANTES (3-68) or
other variant chemokine primary amino acid sequence may result in
proteins which have substantially equivalent activity as compared
to the RANTES (3-68) or other variant chemokine polypeptide
described herein. Such modifications may be deliberate, as by
site-directed mutagenesis, or may be spontaneous. All of the
polypeptides produced by these modifications are included herein as
long as the biological activity of RANTES (3-68) or other variant
chemokine still exists. Further, deletion of one or more amino
acids can also result in a modification of the structure of the
resultant molecule without significantly altering its biological
activity. This can lead to the development of a smaller active
molecule which would have broader utility.
[0033] The polynucleotide sequence encoding the RANTES (3-68) or
other variant chemokine polypeptide of the invention includes the
disclosed sequence and conservative variations thereof The term
"conservative variation" as used herein denotes the replacement of
an amino acid residue by another, biologically similar residue.
Examples of conservative variations include the substitution of one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine, and the
like. The term "conservative variation" also includes the use of a
substituted amino acid in place of an unsubstituted parent amino
acid provided that antibodies raised to the substituted polypeptide
also immunoreact with the unsubstituted polypeptide.
[0034] The invention provides isolated polynucleotides encoding the
RANTES (3-68) or other variant chemokine polypeptide. In one
embodiment, the polynucleotide is the nucleotide sequence of SEQ ID
NO: 1. These polynucleotides include DNA, cDNA and RNA sequences
which encode RANTES (3-68) or other variant chemokine. It is
understood that all polynucleotides encoding all or a portion of
RANTES (3-68) or other variant chemokine are also included herein,
as long as they encode a polypeptide with RANTES (3-68) or other
variant chemokine activity (e.g., does not bind to CCR1 but binds
to CCR5). Such polynucleotides include naturally occurring,
synthetic, and intentionally manipulated polynucleotides. For
example, RANTES (3-68) or other variant chemokine polynucleotide
may be subjected to site-directed mutagenesis. The polynucleotide
sequence for RANTES (3-68) or other variant chemokine also includes
antisense sequences. The polynucleotides of the invention include
sequences that are degenerate as a result of the genetic code.
There are 20 natural amino acids, most of which are specified by
more than one codon. Therefore, all degenerate nucleotide sequences
are included in the invention as long as the amino acid sequence of
RANTES (3-68) or other variant chemokine polypeptide encoded by the
nucleotide sequence is functionally unchanged. Abbreviations for
the amino acid residues are follows: A, Ala; C, Cys; D, Asp: E,
Glu: F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N,
Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y,
Tyr.
[0035] As used herein, "polynucleotide" also refers to a nucleic
acid sequence of deoxyribonucleotides or ribonucleotides in the
form of a separate fragment or a component of a larger construct.
DNA encoding portions or all of the polypeptides of the invention
can be assembled from cDNA fragments or from oligonucleotides that
provide a synthetic gene which can be expressed in a recombinant
transcriptional unit.
[0036] An isolated polynucleotide as described herein is a nucleic
acid molecule that is separated in some way from sequences in the
naturally occurring genome of an organism. Thus, the term "isolated
polynucleotide" includes any nucleic acid molecules that are not
naturally occurring. The term therefore includes, for example, a
recombinant polynucleotide which is incorporated into a vector,
into an autonomously replicating plasmid or virus, or into the
genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule independent of other sequences. It also includes
a recombinant DNA which is part of a hybrid gene encoding
additional polypeptide sequences.
[0037] Specifically disclosed herein is a DNA sequence containing
the RANTES polypeptide gene encoding RANTES truncated at positions
1 and 2. The polynucleotide encoding RANTES (3-68) includes FIG. 8
(SEQ ID NO:1), as well as nucleic acid sequences complementary to
SEQ ID NO:1. A complementary sequence may include an antisense
nucleotide. When the sequence is RNA, the deoxynucleotides A, G, C,
and T of SEQ ID NO:1 are replaced by ribonucleotides A, G, C, and
U, respectively. Also included in the invention are fragments of
the above-described nucleic acid sequences that are at least 15
bases in length, which is sufficient to permit the fragment to
selectively hybridize to DNA that encodes the protein of SEQ ID NO:
2 under physiological conditions or a close family member of
RANTES. The term "selectively hybridize" refers to hybridization
under moderately or highly stringent conditions which excludes
non-related nucleotide sequences.
[0038] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0039] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0040] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
techniques which are well known in the art. These include, but are
not limited to: 1) hybridization of genomic or cDNA libraries with
probes to detect homologous nucleotide sequences, 2) polymerase
chain reaction (PCR) on genomic DNA or cDNA using primers capable
of annealing to the DNA sequence of interest, and 3) antibody
screening of expression libraries to detect cloned DNA fragments
with shared structural features.
[0041] Preferably the RANTES (3-68) or other variant chemokine
polynucleotide of the invention is derived from a mammalian
organism, and most preferably from a mouse, rat, or human.
Screening procedures which rely on nucleic acid hybridization make
it possible to isolate any gene sequence from any organism,
provided the appropriate probe is available. Oligonucleotide
probes, which correspond to a part of the sequence encoding the
protein in question, can be synthesized chemically. This requires
that short, oligopeptide stretches of amino acid sequence must be
known. The DNA sequence encoding the protein can be deduced from
the genetic code, however, the degeneracy of the code must be taken
into account. It is possible to perform a mixed addition reaction
when the sequence is degenerate. This includes a heterogeneous
mixture of denatured double-stranded DNA. For such screening,
hybridization is preferably performed on either single-stranded DNA
or denatured double-stranded DNA. Hybridization is particularly
useful in the detection of cDNA clones derived from sources where
an extremely low amount of MRNA sequences relating to the
polypeptide of interest are present. In other words, by using
stringent hybridization conditions directed to avoid non-specific
binding, it is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res., 9:879, 1981;
Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y. 1989).
[0042] The development of specific DNA sequences encoding RANTES
(3-68) or other variant chemokine can also be obtained by: 1)
isolation of double-stranded DNA sequences from the genomic DNA; 2)
chemical manufacture of a DNA sequence to provide the necessary
codons for the polypeptide of interest; and 3) in vitro synthesis
of a double-stranded DNA sequence by reverse transcription of MRNA
isolated from a eukaryotic donor cell. In the latter case, a
double-stranded DNA complement of mRNA is eventually formed which
is generally referred to as cDNA.
[0043] Of the three above-noted methods for developing specific DNA
sequences for use in recombinant procedures, the isolation of
genomic DNA isolates is the least common. This is especially true
when it is desirable to obtain the microbial expression of
mammalian polypeptides due to the presence of introns.
[0044] The synthesis of DNA sequences is frequently the method of
choice when the entire sequence of amino acid residues of the
desired polypeptide product is known. When the entire sequence of
amino acid residues of the desired polypeptide is not known, the
direct synthesis of DNA sequences is not possible and the method of
choice is the synthesis of cDNA sequences. Among the standard
procedures for isolating cDNA sequences of interest is the
formation of plasmid- or phage-carrying cDNA libraries which are
derived from reverse transcription of mRNA which is abundant in
donor cells that have a high level of genetic expression. When used
in combination with polymerase chain reaction technology, even rare
expression products can be cloned. In those cases where significant
portions of the amino acid sequence of the polypeptide are known,
the production of labeled single or double-stranded DNA or RNA
probe sequences duplicating a sequence putatively present in the
target cDNA may be employed in DNA/DNA hybridization procedures
which are carried out on cloned copies of the cDNA which have been
denatured into a single-stranded form (Jay, et al., Nucl. Acid
Res., 11:2325, 1983).
[0045] A cDNA expression library, such as lambda gt11, can be
screened indirectly for RANTES (3-68) or other variant chemokine
peptides having at least one epitope, using antibodies specific for
RANTES (3-68) or other variant chemokine. Such antibodies can be
either polyclonally or monoclonally derived and used to detect
expression product indicative of the presence of RANTES (3-68) or
other variant chemokine cDNA.
[0046] The isolated polynucleotide sequences of the invention also
include sequences complementary to the polynucleotides encoding
RANTES (3-68) or other variant chemokine (antisense sequences).
Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub et al., Scientific American 262:40, 1990). The invention
includes all antisense polynucleotides that inhibit production of
RANTES (3-68) or other variant chemokine polypeptides. In the cell,
the antisense nucleic acids hybridize to the corresponding MRNA,
forming a double-stranded molecule. Antisense oligomers of about 15
nucleotides are preferred, since they are easily synthesized and
introduced into a target RANTES (3-68) or other variant
chemokine-producing cell. The use of antisense methods to inhibit
the translation of genes is known in the art, and is described,
e.g., in Marcus-Sakura (Anal. Biochem., 172:289, 1988).
[0047] In addition, ribozyme nucleotide sequences for RANTES (3-68)
or other variant chemokine are included in the invention. Ribozymes
are RNA molecules possessing the ability to specifically cleave
other single-stranded RNA in a manner analogous to DNA restriction
endonucleases. Through the modification of nucleotide sequences
encoding these RNAs, molecules can be engineered to recognize
specific nucleotide sequences in an RNA molecule and cleave it
(Cech (1988) J. Amer. Med. Assn. 260:3030). A major advantage of
this approach is that, because they are sequence-specific, only
mRNAs with particular sequences are inactivated.
[0048] There are two basic types of ribozymes, tetrahymena-type
(Hasselhoff (1988) Nature 334:585) and "hammerhead"-type.
Tetrahymena-type ribozymes recognize sequences which are four bases
in length, while "hammerhead"-type ribozymes recognize base
sequences 11-18 bases in length. The longer the sequence, the
greater the likelihood that the sequence will occur exclusively in
the target mRNA species. Consequently, hammerhead-type ribozymes
are preferable to tetrahymena-type ribozymes for inactivating a
specific mRNA species, and 18-base recognition sequences are
preferable to shorter recognition sequences.
[0049] DNA sequences encoding RANTES (3-68) or other variant
chemokine can be expressed in vitro by DNA transfer into a suitable
host cell. "Host cells" are cells in which a vector can be
propagated and its DNA expressed. The term also includes any
progeny of the subject host cell. It is understood that all progeny
may not be identical to the parental cell since there may be
mutations that occur during replication. However, such progeny are
included when the term "host cell" is used. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art.
[0050] In the present invention, the RANTES (3-68) or other variant
chemokine polynucleotide sequences may be inserted into a
recombinant expression vector. The term "recombinant expression
vector" refers to a plasmid, virus or other vehicle known in the
art that has been manipulated by insertion or incorporation of the
RANTES (3-68) or other variant chemokine genetic sequences. Such
expression vectors contain a promoter sequence which facilitates
the efficient transcription of the inserted genetic sequence of the
host. The expression vector typically contains an origin of
replication, a promoter, as well as specific genes which allow
phenotypic selection of the transformed cells. Vectors suitable for
use in the present invention include, but are not limited to the
T7-based expression vector for expression in bacteria (Rosenberg,
et al., Gene, 56:125, 1987), the pMSXND expression vector for
expression in mammalian cells (Lee and Nathans, J. Biol. Chem.,
263:3521, 1988) and baculovirus-derived vectors for expression in
insect cells. The DNA segment can be present in the vector operably
linked to regulatory elements, for example, a promoter (e.g., T7, m
etallothionein I, or polyhedrin promoters).
[0051] Polynucleotide sequences encoding RANTES (3-68) or other
variant chemokine can be expressed in either prokaryote or
eukaryotes. Hosts can include microbial, yeast, insect and
mammalian organisms. Methods of expressing DNA sequences having
eukaryotic or viral sequences in prokaryote are well known in the
art. Biologically functional viral and plasmid DNA vectors capable
of expression and replication in a host are known in the art. Such
vectors are used to incorporate DNA sequences of the invention.
[0052] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgC.sub.2l or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired.
[0053] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransformed with DNA
sequences encoding the RANTES (3-68) or other variant chemokine of
the invention, and a second foreign DNA molecule encoding a
selectable phenotype, such as the herpes simplex thymidine kinase
gene. Another method is to use a eukaryotic viral vector, such as
simian virus 40 (SV40) or bovine papilloma virus, to transiently
infect or transform eukaryotic cells and express the protein. (see
for example, Eukaryotic Viral Vectors, Cold Spring Harbor
Laboratory, Gluzman ed., 1982).
[0054] Isolation and purification of microbial expressed
polypeptide, or fragments thereof, provided by the invention, may
be carried out by conventional means including preparative
chromatography and immunological separations involving monoclonal
or polyclonal antibodies.
[0055] Antibodies that Distiguish Wild-type Chemokine from
Truncated Chemokine
[0056] The present invention also provides antibodies useful for
distinguishing between wild-type and DPPIV-truncated chemokine
polypeptides. Preferably, the antibodies are produced by using
N-terminal peptides having about 8 or more amino acids. Therefore,
antibodies produced will distinguish between a chemokine, such as
RANTES, that contains N-terminal amino acids, and a chemokine that
has been cleaved, for example by CD26. The preparation of
polyclonal antibodies is well-known to those skilled in the art.
See, for example, Green et al., Production of Polyclonal Antisera,
in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press
1992); Coligan et al., Production of Polyclonal Antisera in
Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN
IMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated by
reference.
[0057] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature
256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor
Pub. 1988), which are hereby incorporated by reference. Briefly,
monoclonal antibodies can be obtained by injecting mice with a
composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the B lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures. Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections
2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG),
in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana
Press 1992). Methods of in vitro and in vivo multiplication of
monoclonal antibodies is well-known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g., osyngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane (tetramethylpentadecane) prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0058] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention may also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg et
al., International Patent Publication WO 91/11465 (1991) and Losman
et al., Int. J. Cancer 46:310 (1990), which are hereby incorporated
by reference.
[0059] Alternatively, a therapeutically useful anti-RANTES (3-68)
or other variant chemokine antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementarity determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain, and then substituting
human residues in the framework regions of the murine counterparts.
The use of antibody components derived from humanized monoclonal
antibodies obviates potential problems associated with the
immunogenicity of murine constant regions. General techniques for
cloning murine immunoglobulin variable domains are described, for
example, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833
(1989), which is hereby incorporated in its entirety by reference.
Techniques for producing humanized monoclonal antibodies are
described, for example, by Jones et al., Nature 321: 522 (1986);
Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science
239: 1534 (1988); Carteret al., Proc. Nat'l Acad. Sci. USA 89: 4285
(1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et
al., J. Immunol. 150: 2844 (1993), which are hereby incorporated by
reference.
[0060] Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., METHODS: A COMPANION TO
METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12: 433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
[0061] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet. 7:13 (1994); Lonberg et
al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579
(1994), which are hereby incorporated by reference.
[0062] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of the antibody or by expression in E.
coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, by Goldenberg, U.S. Pat. Nos.
4,036,945 and 4,331,647, and references contained therein. These
patents are hereby incorporated in their entireties by reference.
See also Nisonhoff et al., Arch. Biochem. Biophys. 89:230 (1960);
Porter, Biochem. J. 73:119 (1959); Edelman et al., METHODS IN
ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et
al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0063] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0064] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by whitlow et al.,
METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97
(1991); Bird et al., Science 242:423-426 (1988); Ladner et al.,
U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271-77
(1993); and Sandhu, supra.
[0065] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., METHODS: A COMPANION TO
METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).
[0066] Screen for Compounds Which Modulate DDPPIV
[0067] In another embodiment, the invention provides a method for
identifying a compound which modulates dipeptidyl peptidase IV
(DPPIV)-mediated chemokine processing. The method includes: a)
incubating components comprising the compound, DPPIV and a
chemokine under conditions sufficient to allow the components to
interact; and b) determining the N-terminal amino acid sequence of
the chemokine before and after incubating in the presence of the
compound. Compounds that inhibit DPPIV include peptides,
peptidomimetics, polypeptides, chemical compounds and biologic
agents. Preferably the DPPIV is CD26. If a compound inhibits the
DPPIV or CD26 enzymatic activity, the chemokine will have an
N-terminal amino acid sequence which corresponds to the wild-type
polypeptide. Alternatively, if the compound stimulates DPPIV or
CD26 enzymatic activity, the chemokine will have a truncated
amino-terminal amino acid sequence. The amino acid sequence can be
determined by standard N-terminal sequencing methods or by
contacting the chemokine with a monoclonal antibody which
distinguishes between wild-type and truncated or variant chemokine,
for example.
[0068] Incubating includes conditions which allow contact between
the test compound and the chemokine and a DPPIV. Contacting
includes in solution and in solid phase, or in a cell. The test
compound may optionally be a combinatorial library for screening a
plurality of compounds. Compounds identified in the method of the
invention can be further evaluated, detected, cloned, sequenced,
and the like, either in solution or after binding to a solid
support, by any method usually applied to the detection of a
specific DNA sequence such as PCR, oligomer restriction (Saiki, et
al., Bio/Technology, 3:1008-1012, 1985), allele-specific
oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl.
Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays
(OLAs) (Landegren, et al., Science, 241:1077, 1988), and the like.
Molecular techniques for DNA analysis have been reviewed
(Landegren, et al., Science, 242:229-237, 1988).
[0069] Methods for Producing Variant Chemokines
[0070] In another embodiment, the invention provides a method for
producing a variant chemokine having an activity different from the
activity of the wild-type chemokine, including contacting the
wild-type chemokine with an N-terminal processing effective amount
of dipeptidyl peptidase IV (DPPIV), thereby truncating the
chemokine and producing a variant chemokine. The term "N-terminal
processing effective amount" refers to that amount of a DPPIV that
cleaves the amino terminus of a wild-type chemokine polypeptide to
produce a chemokine lacking the first two amino terminal amino
acids. For example, incubation of RANTES with an "N-terminal
processing effective amount" of CD26 results in RANTES (3-68) which
has different activity than wild type RANTES. Chemokines that
contain amino acid motifs at the N-terminus include but are not
limited to RANTES, MP-1, IP-10, eotaxin, macrophage-derived
chemokine (MDC) and MCP-2. Other chemokines known in the art can be
assessed for sensitivity to cleavage by DPPIVs as described herein
by determining the first two amino terminal amino acids.
[0071] Contacting the chemokine can be in vitro or in vivo. For
example, a specimen isolated from a subject, such as a human, or a
mixture or pure sample of chemokine, can be contacted with DPPIV in
vitro. The contacting of the DPPIV and chemokine is deemed
sufficient when cleavage of the chemokine has occurred. It may be
desirable to only cleave a fraction of the total chemokine
population, therefore, samples can be analyzed at various time of
incubation to determine the optimal conditions for the desired
concentration of wild-type versus truncated variant chemokine
achieved.
[0072] The preferred chemokine illustrated herein is RANTES and the
preferred DPPIV is CD26. Other chemokines and DPPIVs are also
included in the method of the invention.
[0073] Inhibition of DPPIV
[0074] In another embodiment, the invention provides a method for
inhibiting dipeptidyl peptidase IV (DPPIV)-mediated chemokine
processing comprising contacting DPPIV with an inhibiting effective
amount of a compound which inhibits DPPIV expression or activity.
For example, the method includes inhibiting CD26 expression or
activity. To determine whether the DPPIV activity or expression is
inhibited, an assay to detect cleavage of a chemokine having an
alanine or proline at position 2, or a Northern blot analysis, can
be performed, respectively. Other standard methods can be used to
detect inhition of gene expression or enzymatic activity. For
example, incubation of CD26, RANTES and a compound suspected of
inhibiting CD26 activity, would result in wild-type RANTES, but
little or no cleaved RANTES (or RANTES "variant").
[0075] Methods of use for Inhibiting HIV-1 Replication, Allergic or
Inflammatory Reactions, and Angiogenesis
[0076] In another embodiment, the invention provides a method for
inhibiting HIV-1 replication in a host cell susceptible to HIV-1
infection, comprising contacting the cell or the host with an
effective amount of dipeptidyl peptidase IV (DPPIV) enzyme such
that macrophage-derived chemokine (MDC) or RANTES is cleaved to
produce truncated MDC or RANTES, respectively, thereby providing
antiviral activity and inhibiting HIV-1 replication. The present
invention provides data demonstrating that cleaved RANTES blocks
HIV-1 infection (EXAMPLE 7). While not wanting to be bound to a
particular theory, it is believed that the activity of MDC is
increased upon cleavage. MDC suppresses HIV-1 replication, thus, it
is desirable for AIDS patients, or individuals at risk of HIV-1
infection to have increased levels of cleaved MDC. Other chemokines
may also be useful in the method of the invention fro inhibiting
HIV-1 replication.
[0077] In yet another embodiment, the invention provides a method
for inhibiting an allergic or inflammatory reaction in a subject,
comprising administering to the subject an effective amount of
dipeptidyl peptidase IV (DPPIV) enzyme such that a chemokine is
cleaved to produce a truncated chemokine, thereby inhibiting an
allergic or inflammatory reaction. Preferably, a chenokine useful
for inhibition allergic or inflammatory reactions is a truncated
eotaxin.
[0078] The use of a truncated chemokine in the method of the
invention may inhibit or depress an immune or inflammatory response
where desirable, such as in graft rejection responses after organ
and tissue transplantations, or autoimmune disease. Some of the
commonly performed transplantation surgery today includes organs
and tissues such as kidneys, hearts, livers, skin, pancreatic
islets and bone marrow. However, in situations where the donors and
recipients are not genetically identical, graft rejections can
still occur. Autoimmune disorders refer to a group of diseases that
are caused by reactions of the immune system to self antigens
leading to tissue destruction. These responses may be mediated by
antibodies, auto-reactive T cells or both. Some important
autoimmune diseases include diabetes, autoimmune thyroiditis,
multiple sclerosis, rheumatoid arthritis, systemic lupus
erythematosis, and myasthenia gravis. Other allergic or
inflammatory responses are included in the method of the
invention.
[0079] In another embodiment, the invention provides a method for
accelerating angiogenesis or wound healing in a subject, comprising
administering to the subject an effective amount of an inhibitor of
dipeptidyl peptidase IV (DPPIV) enzyme activity or gene expression
or a DPPIV-insensitive chemokine, such that chemokine processing is
inhibited, thereby accelerating angiogenesis or wound healing. For
example, new blood vessels are required for tissue repair and
enhanced blood vessel growth may aid in improving circulation to
ischernic limbs and heart tissue suffering from atherosclerotic
disease, healing skin ulcers or other wounds, and establishing
tissue grafts. Preferably, a chemokine useful for accelerating
angiogenesis is a wild-type IP-10. Cleavage of IP-10 appears to
inactivate the activity of IP-10, therefore it is desirable to
inhibit cleavage of IP-10. Alternatively, it may be desirable to
provide a variant IP-10 polypeptide which contains an amino acid
substitution at position 2, such that neither proline nor alanine
is present, which would result in a DPPIV-insensitive chemokine.
However, such a variant must retain the activity of wild-type
IP-10, e.g., a chemoattractant for NK cells.
[0080] Methods of Diagnosis of Chemokine-associated Disorders
[0081] In another embodiment, the invention provides a method for
diagnosis and prognosis of chemokine-associated disorders. The
method includes identifying the presence of a chemokine of interest
from a specimen isolated from the subject; determining the
amino-terminal sequence of the chemokine, wherein a full-length
amino acid sequence is indicative of the presence of a wild-type
chemokine polypeptide and a truncated amino-terminal sequence is
indicative of the presence of a variant chemokine; and determining
the concentration of wild-type chemokine as compared to variant
chemokine, thereby providing a diagnosis of the subject. This
method is also useful for prognosis of a subject, for example, a
subject having AIDS and being treated with a particular therapeutic
regimen. The amino-terminal sequence of the chemokine is
determined, for example, by standard N-terminal sequencing, or by
contacting the chemokine with an antibody which distinguishes
wild-type from variant chemokine polypeptide, as described above.
Use of monoclonal antibodies, for example, allows simple detection
by ELISA or other methods. Specimens useful for such diagnosis
include but are not limited to blood, sputum, urine, saliva,
cerebrospinal fluid, and serum.
[0082] Pharmaceutical Compositions
[0083] The invention also includes various pharmaceutical
compositions that are useful for therapeutic applications as
described herein. The pharmaceutical compositions according to the
invention are prepared by bringing a polypeptide such as SEQ ID
NO:2 (RANTES (3-68)) or a DPPIV, such as CD26, into a form suitable
for administration to a subject using carriers, excipients and
additives or auxiliaries. Frequently used carriers or auxiliaries
include magnesium carbonate, titanium dioxide, lactose, mannitol
and other sugars, talc, milk protein, gelatin, starch, vitamins,
cellulose and its derivatives, animal and vegetable oils,
polyethylene glycols and solvents, such as sterile water, alcohols,
glycerol and polyhydric alcohols. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial, anti-oxidants, chelating agents and inert gases.
Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co.,
1405-1412, 1461-1487 (1975) and The National Formulary XIV., 14th
ed. Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's The Pharmacological Basis for Therapeutics
(7th ed.).
[0084] In another embodiment, the invention relates to a method of
treating a subject having an HIV-related disorder associated with
expression of CCR5 including administering to an HIV-infected or
susceptible cell of a subject a therapeutically effective dose of a
pharmaceutical composition containing the compounds of the present
invention and a pharmaceutically acceptable carrier.
"Administering" the pharmaceutical composition of the present
invention may be accomplished by any means known to the skilled
artisan. By "subject" is meant any mammal, preferably a human. Such
a method can be performed in vivo or ex vivo for example. For
example, a vector containing a nucleic acid sequence encoding SEQ
ID NO:2 or another truncated chemokine can be utilized for
introducing the composition into a cell of the subject.
[0085] In another embodiment, the invention provides a method of
treating a subject having or at risk of having an HIV infection or
disorder, comprising administering to the subject, a
therapeutically effective amount of a polypeptide of SEQ ID NO:2,
wherein the polypeptide inhibits cell-cell fusion in cells infected
with HIV This method is performed as discussed above.
[0086] In another embodiment, the invention provides a method of
inhibiting membrane fusion between HIV and a target cell or between
an HIV-infected cell and a CD4 positive uninfected cell comprising
contacting the target or CD4 positive cell with a fusion-inhibiting
effective amount of the polypeptide of SEQ ID NO:2.
[0087] The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units are tablets, capsules
and suppositories. For treatment of a patient, depending on
activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the patient,
different daily doses are necessary. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or else
several smaller dose units and also by multiple administration of
subdivided doses at specific intervals.
[0088] The pharmaceutical compositions according to the invention
are in general administered topically, intravenously, orally or
parenterally or as implants, but even rectal use is possible in
principle. Suitable solid or liquid pharmaceutical preparation
forms are, for example, granules powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, aerosols, drops or injectable solution in ampule form and
also preparations with protracted release of active compounds, in
whose preparation excipients and additives and/or auxiliaries such
as disintegrants, binders, coating agents, swelling agents,
lubricants, flavorings, sweeteners or solubilizers are customarily
used as described above. The pharmaceutical compositions are
suitable for use in a variety of drug delivery systems. For a brief
review of present methods for drug delivery, see Langer, Science,
249: 1527-1533 (1990), which is incorporated herein by
reference.
[0089] The pharmaceutical compositions according to the invention
may be administered locally or systemically. By "therapeutically
effective dose" is meant the quantity of a compound according to
the invention necessary to prevent, to cure or at least partially
arrest the symptoms of the disease and its complications. Amounts
effective for this use will, of course, depend on the severity of
the disease and the weight and general state of the patient.
Typically, dosages used in vitro may provide useful guidance in the
amounts useful for in situ administration of the pharmaceutical
composition, and animal models may be used to determine effective
dosages for treatment of particular disorders. Various
considerations are described, e.g., in Gilman et al. (eds.) (1990)
GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS,
8th ed., Pergamon Press; and REMINGTON'S PHARMACEUTICAL SCIENCES,
17th ed. (1990), Mack Publishing Co., Easton, Pa., each of which is
herein incorporated by reference.
[0090] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLE 1
[0091] Materials and Methods
[0092] Cell cultures and transfections. Monocytes were isolated
from human PBMCs of healthy donors by counter-current centrifugal
elutriation. Monocyte-derived macrophages were prepared by
culturing monocytes for 6 days at a density of 106 cells/ml in
serum-free macrophage medium (Gibco BRL, Grand Island, N.Y.)
supplemented with recombinant human (rh) M-CSF (10 ng/ml) (R&D
Systems, Minneapolis, Minn.).
[0093] Human embryonic kidney (HEK)-293 cells grown to confluence
in DMEM supplemented with 10% heat-inactivated FCS, penicillin,
streptomycin, 2 mM glutamine, and 10 mM Hepes (pH 7.4) were
transfected with plasmid DNA encoding CCR5 (12). CD4-positive human
osteosarcoma (HOS-CD4) cell lines transfected with individual
chemokine receptor cDNAs were obtained from N. Landau, and were
grown in the above culture medium supplemented with puromycin.
[0094] The derivative of the PM1 cell line chronically infected
with the recombinant HIV-1 clone MV3-HXB2 has been described
previously (11). sCD26 cleavage and electrospray mass spectrometry
(ES-MS). To create the recombinant soluble human CD26 (sCD26)
construct, a signal peptidase cleavage consensus sequence was
introduced in the pTZ-CD26.11 cDNA (13) by a Leu to Ala
substitution at residue 28. To obtain enzyme negative construct,
the Ser at residue 630 was further replaced by Ala. The two
constructs were cloned into the pEE14.HCMV expression vector and
transfected into CHO-K1 cells (14). The enzymatically active (E+)
and enzymatically deficient (E-) sCD26 proteins were purified from
cell culture supernatants of stable transfectants, and were tested
in Western blotting and DPPIV enzyme assays (15). Both proteins had
a relative molecular weight of 110 kDa, bound equally well to
several CD26 mAbs, but only the E+ sCD26 showed detectable DPPIV
activity. rhRANTES, MCP-1, MCP-2, eotaxin, and IP-10 (100 nM)
(Peprotech, Rocky Hill, N.J.) were incubated overnight at
37.degree. C. with different amounts of E+ or E(sCD26 in 50 (1 of
PBS. Samples were desalted and concentrated by using a peptide trap
(Michrom BioResources, Inc., Auburn, Calif.), or a reversed-phase
(RP) HPLC interface. ES-MS analysis of samples was performed in 50%
acetonitrile, supplemented with 0.1% (v/v) glacial acetic acid,
using a Finnigan (San Jose, Calif.) TSQ 7000 triple-stage
quadrupole mass spectrometer. Several scans were summed to obtain
the final spectrum. Peptide synthesis. Full-length and truncated
RANTES were synthesized with an Applied Biosystems (Foster City,
Calif.) peptide synthesizer according to fluorenyl methoxycarbonyl
(FMOC) chemistry. FMOC-protected amino acids were added stepwise
with ninhydrin monitoring at each cycle. The peptides were folded
by air oxidation and purified by RP HPLC. Peptide sequences were
confirmed by amino acid analysis and Edman sequence analysis, and
the molecular masses were confirmed by ES-MS analysis. There was no
substantial difference in the activities of chemically synthesized
full-length RANTES and rhRANTES(1-68) as judged by the Ca2+ influx
and anti-HIV-1 assays used in this study.
[0095] Colorimetric DPPIV Enzyme Assay.
[0096] The p-nitroanilide (pNA)-conjugated Gly-Pro dipeptide
substrate and test competitors were mixed and added to human
placental DPPIV (Enzyme System Products), and the resulting mixture
was incubated at room temperature in a final volume of 150 (1
containing 50 mM tris-HCl (pH 8.0) and 0.15 M NaCl. The final
concentrations of DPPIV and Gly-Pro-pNA were 1.25 mU/ml and 400 (M,
respectively. The kinetics of the enzyme reaction were monitored by
measuring absorbance at 405 nm with a Vmax kinetic microplate
reader (Molecular Devices, Menlo Park Calif.). The percentage
inhibition of enzyme activity was calculated from the maximal
velocity for each sample and from that apparent in the absence of
competitor (100% activity).
[0097] RT-PCR analysis. Isolated total cellular RNA of monocytes
was subjected to first-strand cDNA synthesis. PCR amplification of
cDNA was performed for 30 cycles (92.degree. C. for 1 min,
40.degree. C. for 1 min, 72.degree. C. for 1 min) with primers
specific for CCR1, CCR2b, CCR3, CCR5, CXCR4, and GAPDH. Separated
products were stained with SYBR Green I (Molecular Probes, Eugene,
Oreg.).
[0098] Cytosolic Calcium Measurements.
[0099] Cells (107/ml) were washed and incubated in the dark at
37.degree. C. for 45 min in Ca2+ buffer [136 mM NaCl, 4.8 mM KCl, 5
mM glucose, 1 mM CaCl2, 20 mM Hepes (pH 7.4)] supplemented with 5
(M Fura-2 acetoxymethyl ester that had been premixed with 10%
Pluronic<<F-127 (Molecular Probes). The cells were then
washed and resuspended at 2 (106 cells/ml in Ca2+ buffer containing
BSA (1 mg/ml), and portions (2 ml) of the cell suspension were
exposed at different time points in a stirred cuvette at 37.degree.
C. to chemokines. Fluorescence was monitored with a Photon
Technology International d scan (South Brunswick, N.J.), and data
were recorded as the relative ratio of fluorescence at excitation
wavelengths of 340 and 380 nm, with emission measured at 510 nm.
After each measurement, maximal and minimal fluorescence were
assessed by addition of 20 (M ionomycin followed by 5 mM MnCl2.
[0100] Assay for HIV-1-induced cytopathicity. HOS-CD4.CCR5 cells
(2(104) were incubated for 1 hour at 37.degree. C. with RANTES
variants in 150 (1 of culture medium containing 20% FCS, and were
then mixed with 50 (1 (2 (105 cells/ml) of uninfected PM1 cells or
PM1 cells chronically infected with MV3-HXB2 virus. After 3 days,
photormicrographs of cultures were taken and cell viability was
measured by adding of 50 (1 of 1 mg/ml
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
solution containing 20 (M phenazine methosulfate and recording the
OD at 450 nm. Data are expressed as the percentage inhibition of
cytopathicity [calculated as 100% ((R (V)/(U (V), where U, V, and R
represent OD values obtained for HOS-CD4.CCR5 cells cultured with
uninfected PM1 cells, or with HIV-1-infected cells in the absence
or presence of chemokine, respectively].
EXAMPLE 2
[0101] RANTES, MCP-2, Eotaxin, and IP-10 are Substrates of CD26
[0102] ES-MS analysis revealed that 100 nM rhRANTES underwent
partial to complete hydrolysis when incubated overnight at
37.degree. C. with increasing amounts (25 to 250 (U) of sCD26 (FIG.
1). Taking into account cationization (K+) of the multiply charged
ions, the measured molecular masses of the native and degraded
polypeptides corresponded to the theoretical masses of full-length
(residues 1 to 68) and truncated (residues 3 to 68) forms of
RANTES, respectively. The calculated difference between the
molecular masses of the native and the truncated forms ranged from
183 to 185 daltons, which is consistent with the expected mass (184
daltons) of a released Ser-Pro dipeptide, the predicted
NH2-terminus of RANTES (16). In contrast to the effect of
enzymatically active sCD26, shortened RANTES was not generated by
incubation of the chemokine with a mutant sCD26 deficient in enzyme
activity (FIG. 1). RANTES also inhibited, possibly in a competitive
manner, the rapid hydrolysis of a pNA-conjugated Gly-Pro dipeptide
by human placental DPPIV, as measured in a colorimetric enzyme
assay (FIG. 2). The efficacy of inhibition by chemically
synthesized RANTES(1-68) was similar to that observed with the
DPPIV substrate and competitive inhibitor Ile-Pro-Ile (Diprotin A)
(17), whereas RANTES (3-68) did not inhibit the reaction.
EXAMPLE 3
[0103] Sensitivity to CD26-mediated Cleavage
[0104] Sensitivity to CD26-mediated cleavage was not a unique
property of RANTES (Table 1.). Cleavage products with the predicted
molecular masses were also evident in samples of MCP-2, eotaxin and
IP-10 after incubation with sCD26. In contrast, MCP-1, which has a
62% sequence similarity with MCP-2 including the NH2-terminal QP
dipeptides, was not cleaved by the enzyme under the same
experimental conditions.
EXAMPLE 4
[0105] CD26-specific Truncation of RANTES Modifies Its Target Cell
Specificity
[0106] To investigate the functional significance of DPPIV-mediated
truncation of RANTES, we compared the effects of chemically
synthesized RANTES(1-68) and RANTES(3-68) on monocytes and
monocyte-derived macrophages. Both resting cells and cells
activated with M-CSF were analyzed because RT-PCR revealed marked
changes in the abundance of chemokine receptor transcripts in
response to M-CSF activation (FIG. 3). In resting cells,
transcripts encoding the chemokine receptors CCR1, CCR2, or CXCR4,
as well as control glyceraldehyde phosphate dehydrogenase (GAPDH)
MRNA, were readily detectable, whereas CCR5 receptor transcripts
were virtually absent. After differentiation to macrophages, the
intensity of the CXCR4 and GAPDH signals remained virtually
unchanged, whereas the abundance of CCR1 and CCR5 mRNAs increased
substantially and the CCR2b transcript virtually disappeared. CCR3
MRNA was not detected in either cell type.
[0107] Transient changes in the cytosolic free Ca2+ concentration
([Ca2+]i) were recorded after stimulation of monocytes or
macrophages with an optimal concentration of RANTES(1-68) or
RANTES(3-68), and the effects were compared with those of other
chemokines (FIG. 4). Addition of 100 nM RANTES(1-68) to cells
loaded with the fluorescent Ca2+ probe Fura-2 induced a rapid
increase in [Ca2+]i in both monocytes and macrophages. In contrast,
the same concentration of RANTES(3-68) increased [Ca2+]i in
macrophages but not in monocytes. Among the other chemokines
tested, macrophage inflammatory protein-1((MIP-1( ), monocyte
chemotactic protein-1 (MCP-1), MCP-3 (1, 6), and stromal-derived
factor-1((SDF-10 (18-20) also increased [Ca2+]i in resting
monocytes, whereas MCP-2 (21) induced a barely detectable response
and MIP-1((1, 6) was inactive. On the basis of the previously
described receptor specificities of these chemokines (1, 6, 19,
20), the obtained activity pattern is consistent with expression of
CCR1, CCR2b, and CXCR4 receptors on monocytes (FIG. 3). Macrophages
showed marked Ca2+ responses to MIP-1(, MIP-1(, MCP-2, MCP-3, and
SDF-1(, but were resistant to MCP-1, consistent with the presence
of transcripts encoding CCR1, CCR5, and CXCR4, and the absence of
those encoding CCR2b, in these cells (FIG. 3).
EXAMPLE 5
[0108] RANTES(3-68) is a Chemokine Agonist, with Altered Receptor
Specificity
[0109] Agonists that act at common chemokine receptors block each
other's activity as a result of receptor desensitization, whereas
responses to chemokines that act at different receptors are
generally not affected (1, 6). We therefore performed comparative
desensitization experiments to define the types of receptors that
mediate the effects of native versus truncated RANTES in
macrophages (FIG. 5). Macrophages that were stimulated first with
100 nM RANTES(1-68) did not exhibit a second Ca2+ response when
challenged with the same dose of either full-length or truncated
RANTES. In contrast, cells stimulated with 100 nM RANTES(3-68)
fully retained their ability to respond to a subsequent challenge
with full-length RANTES, but were desensitized to the effect of the
truncated form. These results suggest that the receptor repertoire
available for truncated RANTES is more restricted than that
available for the native chemokine. To characterize further the
receptor usage of the different forms of RANTES and other
chemokines, we also studied the sensitivity of MIP-1(-, MCP-3-, and
SDF-1(-induced Ca2+ responses to RANTES-mediated receptor
desensitization (FIG. 5). Of the known receptors, RANTES signals
via CCR, CCR4, and CCR5, whereas MIP-I(acts at CCR5 exclusively and
MCP-3 binds only to CCR1 and CCR2b at the concentrations used in
our experiments (1, 6). The only receptor known to bind SDF-1(is
CXCR4 (19, 20). Pretreatment of macrophages with full-length RANTES
blocked the ability of MIP-1(and MCP-3, but not that of SDF-1(, to
increase [Ca2+]i. In contrast, RANTES(3-68) desensitized cells to
the effect of MIP-1(but did not affect the response to MCP-3 or
SDF-1. These results are consistent with previous data on
RANTES-induced receptor desensitization (1) and with our data on
chemokine receptor mRNA abundance (FIG. 3). They suggest that, in
M-CSF-activated macrophages, full-length RANTES shares CCR1 and
CCR5 receptors with MCP-3 and MIP-1(, respectively. Our results
also indicate that, without its two NH2-terminal residues, RANTES
is still able to signal via CCR5 but can no longer act at the CCR1
receptor.
EXAMPLE 6
[0110] CCR1- and CCR5-mediated Signaling of RANTES
[0111] HEK-293 cells expressing CCR5 and HOS-CD4 cells expressing
CCR1 were loaded with Fura-2 and exposed to various concentrations
of RANTES(1-68) or RANTES(3-68) The two RANTES variants showed
similar abilities to increase [Ca2+]i in the CCR5 transfectant
(FIG. 6 A); the responses were dose dependent, with 10 nM of each
variant sufficient to induce a maximal Ca2+ response. In contrast,
in the cells expressing CCR1, the amount of RANTES(3-68) required
to produce a detectable Ca2+ response was .about.100 times that for
RANTES (1-68) (FIG. 6 B); the effect of RANTES(1-68) saturated at
50 nM, whereas that of RANTES(3-68) appeared not to have achieved
saturation at 200 nM. Furthermore, bidirectional
cross-desensitization between the two RANTES variants was evident
only with the cells expressing CCR5 (FIG. 6 C); in the CCR1
transfectant, cross-desensitization was induced by full-length
RANTES but not by the truncated form, which also did not exhibit
self-desensitization (FIG. 6 D). Control cells transfected with
vector alone or with vectors encoding CCR2b, CCR3, or CXCR4 did not
respond to these ligands (data not shown). These results thus
confirm that the native and CD26-truncated RANTES variants exhibit
markedly different activities at the CCR1 receptor.
EXAMPLE 7
[0112] RANTES(3-68) is a Potent Inhibitor of HIV-1
[0113] In addition to their function in chemotaxis, RANTES, MIP-1(,
and MIP-1(each inhibit HIV-1 infection by competitive binding to
CCR5 (22-27), and this inhibition does not require
receptor-mediated cell signaling (27, 28). To examine whether
removal of the two NH2-terminal residues affects the antiviral
activity of RANTES, we mixed HOS-CD4 cells expressing recombinant
CCR5 and PM1 cells chronically infected with the M-tropic
recombinant MV3-HXB2 virus and cocultured them in the absence or
presence of various concentrations of RANTES(1-68) or RANTES
(3-68). Both RANTES variants inhibited HIV-1-induced syncytium
formation and cytopathicity (FIG. 7). Thus, similar to signaling
activity through CCR5, competitive inhibition of HIV-1 infection
does not require the NH2-terminal Ser-Pro residues of RANTES.
[0114] The CD26 cleavage product of RANTES, RANTES(3-68), acts as a
chemokine agonist with altered receptor-specificity. Hydrolysis by
CD26 might explain why RANTES(3-68) has been isolated as a second
component in addition to intact RANTES from culture supernatants of
stimulated human fibroblasts, skin samples, and platelet
preparations (29, 30). The CC-chemokines RANTES, MCP-2, and
eotaxin, and the CXC-chemokine IP-10 are the first immune
modulators and the longest polypeptides identified as natural
substrates for CD26.
[0115] CD26 exists in both soluble and membrane-expressed forms.
Secreted forms of CD26 have been identified in cell cultures and in
human serum (31, 32), although CD26 may be more active when
expressed as an ectoenzyme at high concentrations on endothelial
cells, hepatocytes, kidney brush border membranes, and leukocytes
(10). Up-regulation of CD26 expression on T lymphocytes and
macrophages has been linked to cell activation and development of
immunological memory (10). Thus, activation-induced changes in CD26
expression could affect the course of an inflammatory response by
modifying the target cell specificity of RANTES or other
chemokines, and by regulating the equilibrium between the migrating
cell subsets. We are currently addressing whether cells with
different levels of CD26 expression (e.g. naive versus memory T
cells) secrete truncated forms of RANTES or other chemoattractants,
or are capable of modifying exogenous chemokines.
[0116] The differential effects of CD26-truncated RANTES on
monocytes versus macrophages illustrate a role for cell
differentiation in regulating chemokine sensitivity through altered
receptor expression. Our functional and receptor transcript data
indicate that CCR1 and CCR2b may be the two principal CC chemokine
receptors in resting monocytes, although other unidentified and
functionally overlapping receptors may also contribute to chemokine
function. Cell differentiation markedly changes the pattern of
chemokine sensitivity by reducing CCR2b expression, thereby
rendering the cells resistant to MCP-1, while increasing CCR5
expression, thereby augmenting the responses to CD26-truncated
RANTES and MIP-1(. An increase in CCR5 expression also may render
macrophages more susceptible to infection by M-tropic variants of
HIV-1. We have shown that macrophages also express CXCR4, the
coreceptor for T cell line-tropic HIV-1 variants (33-34), as
assessed by receptor transcript abundance and functional activity
of the CXCR4 ligand SDF-1(. Nevertheless, activated macrophages are
relatively resistant to infection by T cell line-tropic HIV-1
variants (35), which suggests that factors other than CXCR4 may
also be required for efficient infection of macrophages by these
types of viruses.
[0117] Removal of two NH2-terminal residues by CD26 abolishes the
interaction of RANTES with CCR1, but does not affect the anti-HIV-1
activity or the CCR5 signaling properties of the chemokine. Proline
residues also influence the susceptibility of proximal peptide
bonds to proteolytic enzymes (6), and so the removal of such
residues by CD26 may also reduce the half-life of RANTES and other
chemokines during an inflammatory response. It will be important to
determine whether CD26-mediated cleavage is a general mechanism for
changing the receptor specificity and functional activity of other
chemokines, including those examined in this study (MCP-2, eotaxin,
and IP-10).
[0118] Many, but not all CC- and CXC-chemokines contain X-Pro- or
X-Ala-amino-terminal sequence and are potential substrates of DPPIV
We are currently exploring whether the inability of CD26 to cleave
MCP-1 is due to aggregation of this chemokine under these
experimental conditions or to a conformational requirement of the
enzyme that is not fulfilled by MCP-1. Selectivity of CD26 activity
on chemokines may function to reduce redundancy in chemokine target
cell specificity as illustrated by the different activity of
full-length and truncated RANTES on monocytes versus macrophages.
Finally, truncated analogs of chemokines with selective activity on
distinct functional receptors, or analogs that resist CD26
cleavage, may prove therapeutically beneficial in blocking or
inducing the infiltration of specific subsets of effector cells
mediating inflammation, allergy and anti-tumor responses.
1TABLE 1 Chemokine cleavage products after digestion with sCD26.
Molecular masses by mass spectrometry (Da) NH2-terminal CD26 Full
length Truncated Chemokine dipeptide cleavage Theoretical Observed
Theoretical Observed Eotaxin GP Yes 8361 8361 8207 8207 IP-10 VP
Yes 8633 8637/8751* 8437 8440/8555* MCP-1 QP No 8681 8678 8456 ND
MCP-2 QP Yes 8910 8909 8685 8686/8703# *Tentatively identified as
[M + trifluoroacetic acid (TFA)]+; molecular mass of TFA is 114 Da.
#Tentatively identified as [M + H2O]+. ND = not detected.
REFERENCES
[0119] 1. Murphy, P. M. 1996. Chemokine receptors: structure,
function and role in microbial pathogenesis. Cytokine Growth Factor
Rev. 7:47-64.
[0120] 2. Sica, A., A. Saccani, A. Borsatti, C. A. Power, T. N.
Wells, W. Luini, N. Polentarutti, S. Sozzani, and A. Mantovani.
1997. Bacterial lipopolysaccharide rapidly inhibits expression of
C-C chemokine receptors in human monocytes. J. Exp. Med.
185:969-974
[0121] 3. Weber, M., M. Uguccioni, M. Baggiolini, I. Clark-Lewis,
and C. A. Dahinden. 1996. Deletion of the NH2-terminal residue
converts monocyte chemotactic protein 1 from an activator of
basophil mediator release to an eosinophil chemoattractant. J. Exp.
Med. 183:681-685.
[0122] 4. Gong, J. -H., M. Uguccioni, B. Dewald, M. Baggiolini, and
I. Clark-Lewis. 1996. RANTES and MCP-3 antagonists bind multiple
chemokine receptors. J. Biol. Chem. 271:10521-10527.
[0123] 5. Arenzana-Seisdedos, F., J. -L. Virelizier, D. Rousset, I.
Clark-Lewis, P. Loetscher, B. Moser, and M. Baggiolini. HIV blocked
by chemokine antagonist. 1996. Nature. 383:400.
[0124] 6. Murphy, P. M. 1994. The molecular biology of leukocyte
chemoattractant receptors. Annu. Rev. Immunol. 12:593-633.
[0125] 7. Walter, R., W. H. Simmons, and T. Yoshimoto. 1980.
Proline specific endo- and exopeptidases. Mol. Cell. Biochem.
30:111-127.
[0126] 8. Fox, D. A., R. E. Hussey, K. A. Fitzgerald, O. Acuto, C.
Poole, L. Palley, J. F. Daley, S. F. Schlossman, and E. L.
Reinherz. 1984. Tal , a novel 105 kD human T cell activation
antigen defined by a monoclonal antibody. J. Immunol.
133:1250-1256.
[0127] 9. Hegen, M., G. Niedobitek, C. E. Klein, H. Stein, and B.
Fleischer. 1990. The T cell triggering molecule Tp103 is associated
with dipeptidyl aminopeptidase IV activity. J. Immunol.,
144:2908-2914.
[0128] 10. Fleischer, B. 1994. CD26: a surface protease involved in
T-cell activation. Immunol. Today. 15:180-184.
[0129] 11. Oravecz, T., G. Roderiquez, J. Koffi, J. Wang, M. Ditto,
D. C. Bou-Habib, P. Lusso, and M. A. Norcross. 1995. CD26
expression correlates with entry, replication and cytopathicity of
monocytotropic HIV-1 strains in a T-cell line. Nature Med.
1:919-926.
[0130] 12. Samson, M., O. Labbe, C. Mollereau, G. Vassart, and M.
Parmentier. 1996. Molecular cloning and functional expression of a
new human CC-chemokine receptor gene. Biochemistry.
35:3362-3367.
[0131] 13. Tanaka, T., D. Camerini, B. Seed, Y. Torimoto, N. H.
Dang, J. Karneoka, H. N. Dahlberg, S. F. Schlossman, and C.
Morimoto. 1992. Cloning and functional expression of the T cell
activation antigen CD26. J Immunol. 149:481-486.
[0132] 14. Davis, S. J., H. A. Ward, M. J. Puklavec, A. C. Willis,
A. F. Williams, and A. N. Barclay. 1990. High level expression in
Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte
glycoprotein including glycosylation variants. J. Biol. Chem.
265:10410-10418.
[0133] 15. McCaughan, G. W., J. E. Wickson, P. F. Creswick, and M.
D. Gorrell. 1990. Identification of the bile canalicular cell
surface molecule GP110 as the ectopeptidase dipeptidyl peptidase
IV: an analysis by tissue distribution, purification and N-terminal
amino acid sequence. Hepatology. 11:534-544.
[0134] 16. Schall, T. J., J. Jongstra, B. J. Dyer, J. Jorgensen, C.
Clayberger, M. M. Davis, and A. M. Krensky. 1988. A human T
cell-specific molecule is a member of a new gene family. J.
Immunol. 141:1018-1025.
[0135] 17. Rahfeld, J., M. Schierhorn, B. Hartrodt, K. Neubert, and
J. Heins. 1991. Are diprotin A (Ile-Pro-Ile) and diprotin B
(Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?
Biochim. Biophys. Acta. 1076:314-316.
[0136] 18. Nagasawa, T., H. Kikutani, and T. Kishimoto. 1994.
Molecular cloning and structure of a pre-B-cell growth-stimulating
factor. Proc. Natl. Acad. Sci. U.S.A. 91:2305-2309.
[0137] 19. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I.
Clark-Lewis, J. Sodroski, and T. A. Springer. 1996. The lymphocyte
chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1
entry. Nature. 382:829-833.
[0138] 20. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. -L.
Virelizier, F. Arenzana-Seisdedos, O. Schwartz, J. -M. Heard, I.
Clark-Lewis, D. F. Legler, M. Loetscher, M. Baggiolini, and B.
Moser. 1996. The CXC chemokine SDF-1 is the ligand for LESTR/fusin
and prevents infection by T-cell-line-adapted HIV-1. Nature.
382:833-835.
[0139] 21. Van Damme, J., P. Proost, J. -P. Lenaerts, and G.
Opdenakker. 1992. Structural and functional identification of two
human, tumor-derived monocyte chemotactic proteins (MCP-2 and
MCP-3) belonging to the chemokine family. J. Exp. Med.
176:59-65.
[0140] 22. Alkathib, G., C. Combadiere, C. C. Broder, Y. Feng, P.
E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: A
RANTES, MIP-1(, MIP-1(, receptor as a fusion cofactor for
macrophage-tropic HIV-1. Science. 272:1955-1958.
[0141] 23. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P.
D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C.
Gerard, and J. Sodroski. 1996. The (-chemokine receptors CCR3 and
CCR5 facilitate infection by primary HIV-1 isolates. Cell.
85:1135-1148.
[0142] 24. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson,
S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A
dual-tropic primary HIV-1 isolate that uses fusin and the
(-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.
Cell. 85:1149-1158.
[0143] 25. Deng, H., R. Liu, W Ellmeier, S. Choe, D. Unutmaz, M.
Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B.
Davis, S. C. Peiper, T. J. Schall, D. R. Littman, and N. R. Landau.
1996. Identification of a major co-receptor for primary isolates of
HIV-1. Nature. 381:661-673.
[0144] 26. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y.
Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P.
Moore, and W. A. Paxton. 1996. HIV-1 entry into CD4+ cells is
mediated by the chemokine receptor CC-CKR-5. Nature
381:667-673.
[0145] 27. Oravecz, T., M. Pall, and M. A. Norcross. 1996.
(-chemokine inhibition of monocytotropic HIV-1 infection:
Interference with a postbinding fusion step. J. Immunol.
157:1329-1332.
[0146] 28. Farzan, M., H. Choe, K. A. Martin, Y. Sun, M. Sidelko,
C. R. Mackay, N. P. Gerard, J. Sodroski, and C. Gerard. 1997. HIV-1
entry and macrophage inflammatory protein-1 beta-mediated signaling
are independent functions of the chemokine receptor CCR5. J. Biol.
Chem. 272:6854-6857.
[0147] 29. Mallet, A. I., and I. Kay. 1995. Characterization of
chemokine proinflammatory proteins by combined liquid
chromatography-mass spectrometry. Biochem. Soc. Trans.
23:911-913.
[0148] 30. Noso, N. M., Sticherling, J. Bartels, A. I. Mallet, E.
Christophers, and J. -M. Schr.div.der. 1996. Identification of an
N-terminally truncated form of the chemokine RANTES and
granulocyte-macrophage colony-stimulating factor as major
eosinophil attractants released by cytokine-stimulated dermal
fibroblasts. J. Immunol. 156:1946-1953.
[0149] 31 . Tanaka, T., J. S. Duke-Cohan, J. Kameoka, A. Yaron, I.
Lee, F. F. Schlossman, and C. Morimoto. 1994. Enhancement of
antigen-induced T-cell proliferation by soluble CD26/dipeptidyl
peptidase IV Proc. Natl. Acad. Sci. U.S.A. 91:3082-3086.
[0150] 32. Duke-Cohan, J. S., C. Morimoto, J. A. Rocker, and S.
Schlossman. 1996. Serum high molecular weight dipeptidyl peptidase
IV (CD26) is similar to a novel antigen DPPT-L released from
activated T cells. J. Immunol. 156:1714-1721.
[0151] 33. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger.
1996. HIV-1 entry cofactor: Functional cDNA cloning of a
seven-transmembrane, G protein-coupled receptor. Science.
272:872-876.
[0152] 34. Berson, J. F., D. Long, B. J. Doranz, J. Rucker, F. R.
Jirik, and R. W. Doms. 1996. A seven-transmembrane domain receptor
involved in fusion and entry of T-cell-tropic human
immunodeficiency virus type 1 strains. J. Virol. 70:6288-6295.
[0153] 35. Cheng-Mayer, C., M. Quiroga, J. W. Tung, D. Dina, and J.
A. Levy. 1990. Viral determinants of human immunodeficiency virus
type 1 T-cell or macrophage tropism, cytopathogenicity, and CD4
antigen modulation. J. Virol. 64:4390-4398.
[0154] 36. Wang, J. M., D. W. McVicar, J. J. Oppenheim, and D. J.
Kelvin. 1993. Identification of RANTES receptors on human monocytic
cells: competition for binding and desenzitization by homologous
chemotactic cytokines. J. Exp. Med. 177:699-705.
[0155] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof,
that the foregoing description is intended to illustrate and not
limit the scope of the invention, which is defined by the scope of
the appended claims. Other aspects, advantages, and modifications
are within the scope of the following claims.
Sequence CWU 1
1
2 1 198 DNA Homo sapiens CDS (1)..(198) 1 tat tcc tcg gac acc aca
ccc tgc tgc ttt gcc tac att gcc cgc cca 48 Tyr Ser Ser Asp Thr Thr
Pro Cys Cys Phe Ala Tyr Ile Ala Arg Pro 1 5 10 15 ctg ccc cgt gcc
cac atc aag gag tat ttc tac acc agt ggc aag tgc 96 Leu Pro Arg Ala
His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys Cys 20 25 30 tcc aac
cca gca gtc gtc ttt gtc acc cga aag aac cgc caa gtg tgt 144 Ser Asn
Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln Val Cys 35 40 45
gcc aac cca gag aag aaa tgg gtt cgg gag tac atc aac tct ttg gag 192
Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser Leu Glu 50
55 60 atg agc 198 Met Ser 65 2 66 PRT Homo sapiens 2 Tyr Ser Ser
Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala Arg Pro 1 5 10 15 Leu
Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly Lys Cys 20 25
30 Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln Val Cys
35 40 45 Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser
Leu Glu 50 55 60 Met Ser 65
* * * * *