U.S. patent application number 09/747142 was filed with the patent office on 2001-07-05 for chemokine inhibition of immunodeficiency virus.
Invention is credited to Gallo, Robert C., Garzino-Demo, Alfredo, Vico, Anthony De.
Application Number | 20010006681 09/747142 |
Document ID | / |
Family ID | 25245797 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006681 |
Kind Code |
A1 |
Vico, Anthony De ; et
al. |
July 5, 2001 |
Chemokine inhibition of immunodeficiency virus
Abstract
The invention relates to therapeutic compositions and methods
for treating and preventing infection by an immunodeficiency virus,
particularly HIV infection, using chemokine proteins, nucleic acids
and/or derivatives or analogs thereof.
Inventors: |
Vico, Anthony De;
(Alexandria, VA) ; Garzino-Demo, Alfredo;
(Baltimore, MD) ; Gallo, Robert C.; (Bethesda,
MD) |
Correspondence
Address: |
Steven J. Hultquist
Intellectual Property/Technology Law
P.O. Box 14329
Research Triangle Park
NC
27709
US
|
Family ID: |
25245797 |
Appl. No.: |
09/747142 |
Filed: |
December 22, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09747142 |
Dec 22, 2000 |
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08826133 |
Mar 26, 1997 |
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6214540 |
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Current U.S.
Class: |
424/500 ;
424/85.1; 435/5; 514/44R |
Current CPC
Class: |
C12Q 1/703 20130101;
A61K 38/00 20130101; A61P 31/18 20180101; C07K 14/521 20130101;
A61K 48/00 20130101; A61P 37/04 20180101; C07K 2319/00
20130101 |
Class at
Publication: |
424/500 ;
424/85.1; 514/44; 435/5 |
International
Class: |
A61K 031/70; A61K
045/00; A61K 009/50 |
Claims
What is claimed is:
1. A method of formulating a composition comprising one or more
chemokines for use in a pharmaceutical composition having anti-HIV
activity against one or more HIV-1 isolates present in an
individual at a given time, the method comprising: (a) contacting a
first aliquot of HIV.sup.+ cells obtained from said individual with
a chemokine, chemokine derivative and/or chemokine analog; and (b)
comparing the ability to isolate HIV from said cells with the
ability to isolate HIV from a second aliquot of HIV.sup.+ cells
obtained from said individual that are not contacted with said
chemokines, chemokine derivatives and/or chemokine analogs; (c)
formulating the composition to comprise one or more chemokines,
chemokine derivatives and/or chemokine analogs, which produce a
decrease in the ability to isolate virus in the presence of said
chemokines, chemokine derivatives and/or chemokine analogs.
2. The method of claim 1, further comprising the step of combining
in the composition two or more of said chemokines, chemokine
derivatives and/or chemokine analogs demonstrating anti-viral
activity against said HIV-1 isolates.
3. The method of claim 2 wherein at least 3 of said chemokines,
chemokine derivatives and/or chemokine analogs are combined.
4. The method of claim 1 further comprising repeating said
contacting and comparing steps for at least 2 individual
chemokines, chemokine derivatives and/or chemokine analogs.
5. The method of claim 1 further comprising repeating said
contacting and comparing steps for at least 3 individual
chemokines, chemokine derivatives and/or chemokine analogs.
6. The method of claim 4 or 5 wherein the chemokines, derivatives,
or analogs are selected from the group consisting of MCP-1, MCP-2,
MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin,
Exodus, I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta.,
GRO-.gamma., ENA-78, GCP-2, and lymphotactin.
7. The method of claim 1 wherein the HIV.sup.+ cells are
co-cultured with uninfected CD4.sup.+ peripheral blood mononuclear
cells prior to said contacting with the chemokines, chemokine
derivatives and/or chemokine analogs.
8. A method of formulating a pharmaceutical composition for a
particular human subject infected with HIV, the method comprising:
(a) assaying a chemokine, chemokine derivative and/or chemokine
analog for the ability to inhibit: (i) HIV infection; (ii) HIV
replication; or (iii) expression of an RNA or protein of HIV;
wherein said HIV is a primary isolate recovered from said subject;
and (b) combining an amount effective for therapy of a disease or
disorder associated with HIV infection of one or more of said
chemokines, chemokine derivatives and/or chemokine analogs
demonstrating said ability with a pharmaceutically acceptable
carrier suitable for use in vivo in humans.
9. The method of claim 9 wherein said assaying of the chemokine,
derivative, or analog is by a method comprising: (a) measuring
HIV-1 levels in primary macrophage cells or primary CD4.sup.+
peripheral blood mononuclear cells incubated with the primary
isolate, which cells have been contacted with the chemokines,
chemokine derivatives and/or chemokine analogs; and (b) comparing
the measured HIV-1 levels in the cells which have been contacted
with the chemokines, chemokine derivatives and/or chemokine analogs
with said levels in cells not so contacted with the chemokines,
chemokine derivatives and/or chemokine analogs, wherein a lower
level in said contacted cells indicates that the chemokines,
chemokine derivatives and/or chemokine analogs have anti-HIV
activity.
10. The method of claim 9 wherein primary CD4.sup.+ peripheral
blood mononuclear cells are incubated with the primary isolate.
11. The method of claim 9 wherein the primary isolate has been
propagated and maintained only in macrophages.
12. The method of claim 9 wherein the primary isolate is syncytia
inducing.
13. The method of claim 9 wherein the primary isolate is
non-syncytia inducing.
14. The method of claim 8 wherein said assaying of the chemokines,
chemokine derivatives and/or chemokine analogs is by a method
comprising: (a) measuring HIV-1 levels in cultures of HIV.sup.+
cells obtained from the patient which have been contacted with the
chemokines, chemokine derivatives and/or chemokine analogs; and (b)
comparing said measured HIV-1 levels with said levels in said cells
not so contacted with the chemokines, chemokine derivatives and/or
chemokine analogs, wherein a lower HIV-1 level in cultures of said
contacted cells indicates that the chemokines, chemokine
derivatives and/or chemokine analogs has anti-HIV activity.
15. The method of claim 14 further comprising repeating steps (a)
and (b) for at least 2 individual chemokines, or derivatives or
analogs.
16. The method of claim 14 further comprising repeating steps (a)
and (b) for at least 3 individual chemokines, or derivatives or
analogs.
17. The method of claim 15 or 16 wherein the chemokines,
derivatives, or analogs are selected from the group consisting of
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha.,
MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10, PF4, NAP-2,
GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2, and
lymphotactin.
18. A method of treating or preventing HIV infection or replication
in a human subject in need of such treatment, the method comprising
administering to the subject a pharmaceutical composition
comprising: (a) a chemokine selected from the group consisting of
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha.,
MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10, PF4, NAP-2,
GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2, and
lymphotactin in an amount effective to inhibit HIV infection or
replication; and (b) a pharmaceutically acceptable carrier.
19. The method of claim 18 wherein the only chemokines in said
composition are those demonstrated to have activity against a
primary HIV isolate from said subject.
20. The method of claim 18 wherein said pharmaceutical composition
comprises at least 2 of said chemokines.
21. The method of claim 20 wherein the chemokines are selected from
the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
and lymphotactin.
22. A method of treating or preventing HIV infection or replication
in a human subject in need of such treatment, the method comprising
administering to the subject a pharmaceutical composition
comprising: (a) a nucleic acid encoding a chemokine selected from
the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
and lymphotactin, in an amount effective to inhibit HIV infection
or replication; and (b) a pharmaceutically acceptable carrier.
23. The method of claim 22 wherein said composition comprises
nucleic acids encoding at least 2 of said chemokines.
24. The method of claim 23 wherein the nucleic acids encode
chemokines selected from the group consisting of MCP-1, MCP-2,
MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin,
Exodus, I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta.,
GRO-.gamma., ENA-78, GCP-2, and lymphotactin.
25. A method of treating or preventing HIV infection or replication
in a human subject in need of such treatment, the method comprising
administering to the subject an amount of a purified protein
effective to treat or prevent HIV infection, wherein the protein
comprises a fragment or derivative of a chemokine selected from the
group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.gamma., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
and lymphotactin.
26. The method of claim 25 wherein the only chemokine fragments or
derivatives in said composition are those demonstrated to have
activity against a primary HIV isolate from said subject.
27. The method of claim 25 wherein fragments or derivatives of at
least 2 different chemokines are administered to the subject.
28. The method of claim 25 further comprising administering to the
subject an anti-viral drug other than a chemokine, in an amount
effective to inhibit HIV infection or replication.
29. The method of claim 28 wherein the other anti-viral drug is
selected from one or more of the group consisting of AZT, ddI, ddC,
3TC, and sequinavir.
30. The method of claim 28 wherein the protein is administered
intramuscularly.
31. A method of treating or preventing HIV infection or replication
in a human subject, the method comprising administering to the
subject wherein such treatment or prevention is desired an amount
of a nucleic acid effective to treat or prevent HIV infection,
wherein the nucleic acid encodes a fragment or derivative of a
chemokine selected from the group consisting of MCP-1, MCP-2,
MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin,
Exodus, I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta.,
GRO-.gamma., ENA-78, GCP-2, and lymphotactin.
32. A method of treating or preventing HIV infection or replication
in a human subjec, the method comprising administering to the
subject wherein such treatment or prevention is desired a
composition comprising: (a) a first chemokine selected from the
group consisting of RANTES, MIP- 1.alpha., MIP-1.beta., or IL-8;
(b) a second chemokine selected from the group consisting of MCP-1,
MCP-2, MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta.,
eotaxin, Exodus, I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha.,
GRO-.beta., GRO-.gamma., ENA-78, GCP-2, lymphotactin, and SDF-1;
together in an amount effective to inhibit HIV infection or
replication.
33. The method of claim 32 wherein the total of the chemokines
selected from (a) and (b) is at least 3.
34. The method of claim 32 further comprising administering to the
subject an anti-viral drug other than a chemokine, in an amount
effective to inhibit HIV infection or replication.
35. The method of claim 34 wherein the anti-viral drug is selected
from one or more of the group consisting of AZT, ddI, ddC, 3TC, and
sequinavir.
36. The method of claim 32 wherein the composition is administered
intramuscularly.
37. A method of treating or preventing HIV infection or replication
in a human subject in need of such treatment, the method comprising
administering to the subject a composition comprising: (a) a first
nucleic acid encoding RANTES, MIP-1.alpha., MIP-1.beta., or IL-8,
and (b) a second nucleic acid encoding a chemokine selected from
the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
lymphotactin and SDF-1; together in an amount effective to inhibit
HIV infection or replication.
38. A pharmaceutical composition comprising: (a) a chemokine
selected from the group consisting of MCP-2, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
and lymphotactin, in an amount effective to inhibit HIV infection
or replication; and (b) a pharmaceutically acceptable carrier.
39. The pharmaceutical composition of claim 38 wherein the
chemokine is purified.
40. The pharmaceutical composition of claim 38 further comprising
at least 1, 2, 3, 4, 5, 6, 8, or 9 chemokines in an amount
effective to inhibit HIV infection or replication.
41. The pharmaceutical composition of claim 38 further comprising
RANTES, MIP-1.alpha., MIP-1.beta., MCP-1, MCP-3, IL-8 or SDF-1
together in an amount effective to inhibit HIV infection or
replication.
42. The pharmaceutical composition of claim 41 wherein the
chemokines are purified.
43. A pharmaceutical composition comprising: (a) a derivative or
analog of a chemokine selected from the group consisting of MCP-2,
MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin, Exodus,
I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta.,
GRO-.gamma., ENA-78, GCP-2, and lymphotactin, in an amount
effective to inhibit HIV infection or replication; and (b) a
pharmaceutically acceptable carrier.
44. The pharmaceutical composition of claim 43 wherein the
chemokine derivative or analog is purified.
45. The pharmaceutical composition of claim 43 further comprising
derivatives or analogs of at least 3 of said chemokines, in an
amount effective to inhibit HIV infection or replication.
46. The pharmaceutical composition of claim 43 further comprising
RANTES, MIP-1.alpha., MIP-1.beta., MCP-1, MCP-3 or IL-8 in an
amount effective to inhibit HIV infection or replication.
47. The pharmaceutical composition of claim 43 further comprising a
derivative of RANTES, MIP-1.alpha., MIP-1.beta., MCP-1, MCP-3 and
IL-8 in an amount effective to inhibit HIV infection or
replication.
48. A pharmaceutical composition comprising: (a) one or more
pharmaceutically active components selected from the group
consisting of: (i) a nucleic acid encoding a chemokine selected
from the group consisting of MCP-2, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
lymphotactin and SDF-1, in an amount effective to inhibit HIV
infection or replication; and (ii) an analog of a chemokine of (i);
(iii) a fragment of a chemokine of (i); (iv) a derivative of a
chemokine, analog or fragment of (i), (ii), or (iii); and (v) a
nucleic acid encoding a chemokine chemokine, analog or fragment of
(i), (ii), or (iii); and (b) a pharmaceutically acceptable
carrier.
49. A pharmaceutical composition comprising: (a) two or more
chemokines, each of which binds to at least one chemokine receptor
selected from the group consisting of CC CKR-1, CC CKR-2A, CC
CKR-2B, CC CKR-3, CC CKR-4, CC CKR-5, C.times.C CKR4, IL-8RA,
IL-8RB, Mig receptor, .gamma.IP-10 receptor and Duffy antigen, in
an amount effective to inhibit HIV infection or replication; and
(b) a pharmaceutically acceptable carrier.
50. A method of formulating a pharmaceutical composition having
anti-HIV activity against one or more HIV-1 isolates present in an
individual at a given time, the method comprising: (a) contacting a
first aliquot of CD4.sup.+ cells, one or more virus isolates
obtained from said individual, and a chemokine, chemokine
derivative and/or chemokine analog; and (b) comparing the ability
to isolate HIV from said cells with the ability to isolate HIV from
a second aliquot of CD4.sup.+ cells contacted with said virus
isolates that are not contacted with said chemokines, chemokine
derivatives and/or chemokine analogs, wherein a decrease in the
ability to isolate virus in the presence of said chemokines,
chemokine derivatives and/or chemokine analogs is indicative that
the chemokines, chemokine derivatives and/or chemokine analogs has
anti-viral activity against said HIV-1 isolates.
51. A pharmaceutical composition comprising MDC and I-309.
52. A method for treating HIV infection, the method comprising
administering to a subject in need of such treatment a
therapeutically effective amount of MDC and I-309.
53. The method of claim 52 wherein the MDC and I-309 are
administered together as components of a pharmaceutical
composition, along with a pharmaceutically acceptable carrier.
54. The method of claim 52 wherein the MDC and I-309 are
administered in a synergistically effective and therapeutically
effective amount.
Description
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/826,133, filed Mar. 26, 1997.
2. FIELD OF THE INVENTION
[0002] The present invention relates to therapeutic compositions
and methods for treating and preventing infection by an
immunodeficiency virus, particularly HIV infection, using chemokine
proteins, nucleic acids and/or derivatives or analogs thereof.
3. BACKGROUND OF THE INVENTION
[0003] Human immunodeficiency virus (HIV) induces a persistent and
progressive infection leading, in the vast majority of cases, to
the development of the acquired immunodeficiency syndrome (AIDS)
(Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al.,
1984, Science 224:500-503). There are at least two distinct types
of HIV:HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870;
Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al.,
1986, Science 233:343-346; Guyader et al., 1987, Nature
326:662-669). In humans, HIV replication occurs predominantly in
CD4+ T lymphocyte populations, and HIV infection leads to depletion
of this cell type and eventually to immune incompetence,
opportunistic infections, neurological dysfunctions, neoplastic
growth, and ultimately death.
[0004] HIV is a member of the lentivirus family of retroviruses
(Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds.,
CSH-press, pp. 949-956). Retroviruses are small enveloped viruses
that contain a single-stranded RNA genome, and replicate via a DNA
intermediate produced by a virally-encoded reverse transcriptase,
an RNA-dependent DNA polymerase (Varmus, H., 1988, Science
240:1427-1439). Other retroviruses include, for example, oncogenic
viruses such as human T-cell leukemia viruses (HTLV-1,-II,-III),
and feline leukemia virus.
[0005] The HIV viral particle consists of a viral core, composed in
part of capsid proteins designated p24 and p18, together with the
viral RNA genome and those enzymes required for early replicative
events. Myristylated gag protein forms an outer viral shell around
the viral core, which is, in turn, surrounded by a lipid membrane
envelope derived from the infected cell membrane. The HIV envelope
surface glycoproteins are synthesized as a single 160 kilodalton
precursor protein, which is cleaved by a cellular protease during
viral budding into two glycoproteins, gp41 and gp120. gp41 is a
transmembrane glycoprotein and gp120 is an extracellular
glycoprotein which remains non-covalently associated with gp41,
possibly in a trimeric or multimeric form (Hammerskjold, M. and
Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
[0006] HIV, like other enveloped viruses, introduces viral genetic
material into the host cell through a viral-envelope mediated
fusion of viral and target membranes. HIV is targeted to CD4.sup.+
cells because a CD4 cell surface protein (CD4) acts as the cellular
receptor for the HIV-1 virus (Dalgleish et al., 1984, Nature
312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et
al., 1986, Cell 47:333-348). Viral entry into cells is dependent
upon gp120 binding the cellular CD4 receptor molecules (Pal et al.,
1993, Virology 194:833-837; McDougal et al., 1986, Science
231:382-385; Maddon et al., 1986, Cell 47:333-348), explaining
HIV's tropism for CD4.sup.- cells, while gp41 anchors the envelope
glycoprotein complex in the viral membrane. The binding of gp120 to
CD4 induces conformational changes in the viral glycoproteins, but
this binding alone is insufficient to lead to infection (reviewed
by Sattentau and Moore, 1993, Philos. Trans. R. Soc. London (Biol.)
342:59-66).
[0007] Studies of HIV-1 isolates have revealed a heterogeneity in
their ability to infect different human cell types (reviewed by
Miedema et al., 1994, Immunol. Rev. 140:35-72). The majority of
extensively passaged laboratory strains of HIV-1 readily infect
cultured T cell lines and primary T lymphocytes, but not primary
monocytes or macrophages. These strains are termed T-tropic.
T-tropic HIV-1 strains are more likely to be found in HIV-1
infected individuals during the late stages of aids (Weiss et al.,
1996, Science 272:1885-1886). The majority of primary HIV-1
isolates (i.e., viruses not extensively passaged in culture)
replicate efficiently in primary lymphocytes, monocytes and
macrophages, but grow poorly in established T cell lines. These
isolates have been termed M-tropic. The viral determinant of T- and
M-tropism maps to alterations in the third variable region of gp120
(the V3 loop)(Choe et al., 1996, Cell 85:1135-1148; Cheng-Mayer et
al., 1991, J. Virol. 65:6931-6941; Hwang et al., 1991, Science
253:71-74; Kim et al., 1995, J. Virol., 69:1755-1761; and O'Brien
et al., 1990, Nature 348:69-73). The characterization of HIV
isolates with distinct tropisms taken together with the observation
that binding to the CD4 cell surface protein alone is insufficient
to lead to infection, suggest a requirement for cell-type specific
cofactors, in addition to CD4, for HIV-1 entry into the host
cell.
[0008] Recently, certain chemokines produced by CD8.sup.+ T cells
have been implicated in suppression of HIV infection. The
chemokines RANTES (regulated on activation normal T cell expressed
and secreted), macrophage-inflammatory protein-1.alpha. and
-1.beta. (MIP-1.alpha. and MIP-1.beta., respectively), which are
secreted by CD8.sup.+ T cells, were shown to suppress HIV-1 p24
antigen production in cells infected with HIV-1 or HIV-2 isolates
in vitro (Cocchi et al., 1995, Science 270:1811-1815).
Additionally, high levels of these chemokines have been found to be
secreted by CD4.sup.+ T lymphocytes in individuals that have been
exposed to HIV-1 on multiple occasions but, remain uninfected
(Paxton et al., 1996, Nature Med. 2:412-417). While RANTES,
MIP-1.alpha. and MIP-1.beta. alone or in combination, potently
suppress a variety of primary HIV-1 isolates and macrophage tropic
isolates, such as HIV-1.sub.BaL, some established laboratory
strains, such as HIV-1.sub.IIIB, are refractory to inhibition of
infection or replication by these chemokines (Cocchi et al., 1995,
Science 270:1811-1815).
[0009] Chemokines, or chemoattractant cytokines, are a subgroup of
immune factors that mediate chemotactic and other pro-inflammatory
phenomena (See, Schall, 1991, Cytokine 3:165-183). Chemokines are
small molecules of approximately 70-80 residues in length and can
generally be divided into two subgroups, .alpha. which have two
N-terminal cysteines separated by a single amino acid (C.times.C)
and .beta. which have two adjacent cysteines at the N terminus
(CC). RANTES, MIP-1.alpha. and MIP-1.beta. are members of the
.beta. subgroup (reviewed by Horuk, R., 1994, Trends Pharmacol.
Sci, 15:159-165; Murphy, P. M., 1994, Annu. Rev. Immunol.,
12:593-633). The amino terminus of the .beta. chemokines RANTES,
MCP-1, and MCP-3 have been implicated in the mediation of cell
migration and inflammation induced by these chemokines. This
involvement is suggested by the observation that the deletion of
the amino terminal 8 residues of MCP-1, amino terminal 9 residues
of MCP-3, and amino terminal 8 residues of RANTES and the addition
of a methionine to the amino terminus of RANTES, antagonize the
chemotaxis, calcium mobilization and/or enzyme release stimulated
by their native counterparts (Gong et al., 1996 J. Biol. Chem.
271:10521-10527; Proudfoot et al., 1996 J. Biol. Chem.
271:2599-2603). Additionally, .alpha. chemokine-like chemotactic
activity has been introduced into MCP-1 via a double mutation of
Tyr 28 and Arg 30 to leucine and valine, respectively, indicating
that internal regions of this protein also play a role in
regulating chemotactic activity (Beall et al., 1992, J. Biol. Chem.
267:3455-3459).
[0010] The monomeric forms of all chemokines characterized thus far
share significant structural homology, although the quaternary
structures of .alpha. and .beta. groups are distinct. While the
monomeric structures of the .beta. and .alpha. chemokines are very
similar, the dimeric structures of the two groups are completely
different. An additional chemokine, lymphotactin, which has only
one N terminal cysteine has also been identified and may represent
an additional subgroup (.gamma.) of chemokines (Yoshida et al.,
1995, FEBS Lett. 360:155-159; and Kelner et al., 1994, Science
266:1395-1399).
[0011] Receptors for chemokines belong to the large family of
G-protein coupled, 7 transmembrane domain receptors (GCR's) (See,
reviews by Horuk, R., 1994, Trends Pharmacol. Sci. 15:159-165; and
Murphy, P. M., 1994, Annu. Rev. Immunol. 12:593-633). Competition
binding and cross-desensitization studies have shown that chemokine
receptors exhibit considerable promiscuity in ligand binding.
Examples demonstrating the promiscuity among .beta. chemokine
receptors include: CC CKR-1, which binds RANTES and MIP-1.alpha.
(Neote et al., 1993, Cell 72:415-425), CC CKR-4, which binds
RANTES, MIP-1.alpha., and MCP-1 (Power et al., 1995, J. Biol. Chem.
270:19495-19500), and CC CKR-5, which binds RANTES, MIP-1.alpha.,
and MIP-1.beta. (Alkhatib et al., 1996, Science, in press and
Dragic et al., 1996, Nature 381:667-674). Erythrocytes possess a
receptor (known as the Duffy antigen) which binds both .alpha. and
.beta. chemokines (Horuk et al., 1994, J. Biol. Chem.
269:17730-17733; Neote et al., 1994, Blood 84:44-52; and Neote et
al., 1993, J. Biol. Chem. 268:12247-12249). Thus the sequence and
structural homologies evident among chemokines and their receptors
allows some overlap in receptor-ligand interactions.
[0012] CC CKR-5 is the major coreceptor for macrophage-tropic
strains of HIV-1 (Alkhatib et al., 1996, Science, in press; Choe et
al., 1996, Cell 85:1135-1148; Deng et al., 1996, Nature
381:661-666; Doranz et al., 1996, Cell 85:1149-1158; Dragic et al.,
1996, Nature 381:667-674). RANTES, MIP-1.alpha., or MIP-1.beta.,
the chemokine ligands for this receptor block HIV Env-mediated cell
fusion directed by CC CKR-5 (Alkhatib et al., 1996, Science, in
press; and Dragic et al., 1996, Nature 381:667-674). Additional
support for the role of CC CKR-5 as an M-tropic HIV-1 cofactor
comes from the finding that a 32-base pair deletion in the CC CKR-5
gene found in three multiply exposed but uninfected individuals,
prevents HIV from infecting macrophages (Liu et al., 1996, Cell
86:367-377). However, only three of the 25 uninfected individuals
studied had this mutation.
[0013] The V3 loop of gp120 is the major determinant of sensitivity
to chemokine inhibition of infection or replication (Cocchi et al.,
1996, Nature Medicine 2:1244-1247; and Oravecz et al., 1996, J.
Immunol. 157:1329-1332). Signal transduction through .beta.
chemokine receptors is not required for inhibition of HIV infection
or replication, since RANTES inhibits HIV-1 infection in the
presence of pertussis toxin, an inhibitor of G-protein-mediated
signaling pathways (P. M. Murphy 1994, Ann. Rev. Immunol.
12:593-633; Bischoff et al., 1993, Eur. J. Immunol. 23:761-767; and
Simon et al., 1991, Science 252:802-807). C.times.C CKR4, a
C.times.C (.alpha.) chemokine receptor, has been shown to be a
coreceptor involved in infection by laboratory-adapted HIV-1
strains (Fong et al., 1996, Science 272:872-877). The .alpha.
chemokine SDF-1, the ligand for this receptor, has been
demonstrated to block infection by T-tropic HIV-1 isolates.
C.times.C CKR4 does not bind the beta chemokines RANTES,
MIP-1.alpha., or MIP-1.beta..
[0014] Recently, it has been shown that certain primary,
syncytium-inducing/T-tropic isolates use both CC CKR5 and C.times.C
CKR4 as coreceptors and are able to switch between the two. Thus,
in the presence of RANTES, MIP-1.alpha. and MIP-1.beta., the
chemokine ligands for CC CKR5, T-tropic strains are still able to
infect cells via the C.times.C CKR4 coreceptor (Zhang et al., 1996,
Nature 383:768).
[0015] HIV infection is pandemic and HIV-associated diseases
represent a major world health problem. Although considerable
effort is being put into the design of effective therapeutics,
currently no curative anti-retroviral drugs against AIDS exist. In
attempts to develop such drugs, several stages of the HIV life
cycle have been considered as targets for therapeutic intervention
(Mitsuya et al., 1991, FASEB J. 5:2369-2381). Many viral targets
for intervention with the HIV life cycle have been suggested, as
the prevailing view is that interference with a host cell protein
would have deleterious side effects. For example, virally encoded
reverse transcriptase has been one focus of drug development. A
number of reverse-transcriptase-targeted drugs, including
2N,3N-dideoxynucleoside analogs such as AZT, ddI, ddc, and d4T have
been developed which have been shown to been active against HIV
(Mitsuya et al., 1991, Science 249:1533-1544).
[0016] The new treatment regimens for HIV-1 show that a combination
of anti-HIV compounds, which target reverse transcriptase (RT),
such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine
(ddi), dideoxycytidine (ddc) used in combination with an HIV-1
protease inhibitor have a far greater effect (2 to 3 logs
reduction) on viral load compared to AZT alone (about 1 log
reduction). For example, impressive results have recently been
obtained with a combination of AZT, ddI, 3TC and ritonavir
(Perelson et al., 1996, Science 15:1582-1586). However, it is
likely that long-term use of combinations of these chemicals will
lead to toxicity, especially to the bone marrow. Long-term
cytotoxic therapy may also lead to suppression of CD8.sup.+ T
cells, which are essential to the control of HIV, via killer cell
activity (Blazevic et al., 1995, AIDS Res. Hum. Retroviruses
11:1335-1342) and by the release of factors which inhibit HIV
infection or replication, notably the chemokines Rantes,
MIP-1.alpha. and MIP-1.beta. (Cocchi et al., 1995, Science
270:1811-1815). Another major concern in long-term chemical
anti-retroviral therapy is the development of HIV mutations with
partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum.
Retroviruses 10:S77-82). It is thought that such mutations may be
an inevitable consequence of anti-viral therapy. The pattern of
disappearance of wild-type virus and appearance of mutant virus due
to treatment, combined with coincidental decline in CD4.sup.+ T
cell numbers strongly suggests that, at least with some compounds,
the appearance of viral mutants is a major underlying factor in the
failure of AIDS therapy.
[0017] Attempts are also being made to develop drugs which can
inhibit viral entry into the cell, the earliest stage of HIV
infection, by focusing on CD4, the cell surface receptor for HIV.
Recombinant soluble CD4, for example, has been shown to inhibit
infection of CD4.sup.+ T cells by some HIV-1 strains (Smith et al.,
1987, Science 238:1704-1707). Certain primary HIV-1 isolates,
however, are relatively less sensitive to inhibition by recombinant
CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579).
In addition, recombinant soluble CD4 clinical trials have produced
inconclusive results (Schooley et al., 1990, Ann. Int. Med.
112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261;
Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP
137).
[0018] The late stages of HIV replication, which involve crucial
virus-specific processing of certain viral encoded proteins, have
also been suggested as possible anti-HIV drug targets. Late stage
processing is dependent on the activity of a viral protease, and
drugs are being developed which inhibit this protease (Erickson,
J., 1990, Science 249:527-533). The clinical outcome of these
candidate drugs is still in question.
[0019] Attention is also being given to the development of vaccines
for the treatment of HIV infection. The HIV-1 envelope proteins
(gp160, gp120, gp41) have been shown to be the major antigens for
anti-HIV antibodies present in AIDS patients (Barin et al., 1985,
Science 228:1094-1096). Thus far, therefore, these proteins seem to
be the most promising candidates to act as antigens for anti-HIV
vaccine development. Several groups have begun to use various
portions of gp160, gp120, and/or gp41 as immunogenic targets for
the host immune system. See for example, Ivanoff et al., U.S. Pat.
No. 5,141,867; Saith et al., WO 92/22654; Shafferman, A., WO
91/09872; Formoso et al., WO 90/07119. To this end, vaccines
directed against HIV proteins are problematic in that the virus
mutates rapidly rendering many of these vaccines ineffective.
Clinical results concerning these candidate vaccines, however,
still remain far in the future.
[0020] Thus, although a great deal of effort is being directed to
the design and testing of anti-retroviral drugs, effective,
non-toxic treatments are still needed.
[0021] Citation of a reference hereinabove shall not be construed
as an admission that such reference is prior art to the present
invention.
4. SUMMARY OF THE INVENTION
[0022] The present invention relates to prophylactic and
therapeutic methods and compositions based on chemokine proteins,
nucleic acids, derivatives or analogs thereof that inhibit
replication and/or infection of an immunodeficiency virus in vitro
or in vivo, decrease viral load, and/or treating or preventing
diseases and disorders associated with infection of an
immunodeficiency virus. In specific embodiments, the
immunodeficiency virus inhibited by the methods and compositions of
the invention is HIV.
[0023] According to the present invention, different chemokine
receptors are involved in immunodeficiency virus infection,
depending on the particular isolate. The present invention provides
methods of identifying the particular chemokine(s) capable of
inhibiting the infection or replication of a viral isolate of a
particular patient and of treating such patient. Pharmaceutical
compositions comprising chemokines heretofore unknown to be active
against HIV are also provided, as well as related methods of
treatment or prophylaxis.
[0024] The invention also relates to chemokine derivatives or
analog(s) that bind to a plurality of chemokine receptors and that
are effective at preventing diseases or disorders associated with
infection of an immunodeficiency virus, particularly HIV infection.
The invention also relates to pharmaceutical compositions
containing such therapeutically and prophylactically effective
chemokine derivatives or analogs, or the nucleic acids encoding
such. In one embodiment, the chemokine derivative or analog binds
to one or more .beta. chemokine receptors selected from a group
consisting of CC CKR-1, CC CKR-2A, CC CKR-2B, CC CKR-3, CC CKR-4
and CC CKR-5. In a preferred embodiment, the derivative or analog
binds to the chemokine receptor CC CKR-5. In another embodiment,
the chemokine derivative or analog binds to one or more .alpha.
chemokine receptors selected from the group consisting of C.times.C
CKR4, IL-8RA, IL-8RB, Mig receptor, .gamma.IP-10 receptor, and
Duffy antigen. In a preferred embodiment, the derivative or analog
binds to both an .alpha. chemokine receptor and a .beta. chemokine
receptor. In a most preferred embodiment, the derivative or analog
binds to both C.times.C CKR4 and CC CKR-5. In another embodiment,
the chemokine derivative or analog binds to 3, 4, 5, 6, 7, 8, 9,
10, 11 or 12 chemokine receptors.
[0025] The present invention also relates to pharmaceutical
compositions comprising one or more .alpha., .beta., or .gamma.
chemokines, or nucleic acids encoding the foregoing, in an amount
effective to inhibit HIV infection or replication. In one
embodiment, the pharmaceutical compositions of the invention
comprise RANTES, MIP-1.alpha., MIP-1.beta., MCP-1, MCP-3 or IL-8
nucleic acid encoding RANTES, MIP-1.alpha., MIP-1.beta., MCP-1,
MCP-3 or IL-8 or a therapeutically and prophylactically effective
derivative or analog thereof or nucleic acid encoding the same, in
combination with another chemokine, nucleic acid encoding another
chemokine, or derivative or analog thereof, in an amount effective
to treat or prevent diseases or disorders associated with
immunodeficiency virus infection, particularly HIV infection, e.g.,
ARC, AIDS. In another embodiment, the pharmaceutical composition
comprises a .beta. chemokine, or nucleic acid encoding a .beta.
chemokine, selected from the group consisting of MCP-2, MCP-4,
MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, and
I-309, MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, or a
therapeutically or prophylactically effective derivative or analog
thereof. In an additional embodiment, the pharmaceutical
composition comprises an .alpha. chemokine, nucleic acid encoding
an .alpha. chemokine, or therapeutically or prophylactically
effective derivative or analog thereof. In a further embodiment,
the pharmaceutical composition comprises the .gamma. chemokine
lymphotactin, nucleic acid encoding lymphotactin, or a
therapeutically or prophylactically effective derivative or analog
thereof. In one embodiment, the pharmaceutical composition of the
invention comprises an .alpha. chemokine, or nucleic acid encoding
an .alpha. chemokine, selected from the group consisting of
.gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma.,
ENA-78, GCP-2, or a therapeutically effective derivative or analog
thereof. In yet another embodiment, the pharmaceutical composition
of the invention contains a combination of .alpha., .beta. and/or
.gamma. chemokines, nucleic acids encoding .alpha., .beta. and/or
.gamma. chemokines, or therapeutically or prophylactically
effective derivatives or analogs thereof.
[0026] The present invention also relates to therapeutic
compositions based on chemokines and nucleic acids encoding
chemokines. Therapeutic compounds of the invention include but are
not limited to chemokines, nucleic acids encoding chemokines, and
derivatives (including fragments and chimerics) and analogs
thereof, that are effective at inhibiting replication or infection
by an immunodeficiency virus.
[0027] The invention further relates to therapeutic methods for
treatment and prevention of diseases and disorders associated with
infection with an immunodeficiency virus, in particular HIV
infection, by administering a therapeutic composition of the
invention. More specifically, the invention provides methods for
formulating and administering pharmaceutical compositions of the
invention that inhibit infection or replication of one or more
known isolates of an immunodeficiency virus, preferably of HIV.
[0028] The invention further provides methods for inhibiting the
infection or replication of an immunodeficiency virus isolate, in
particular, an HIV isolate. In a preferred embodiment, the
invention provides methods for formulating, on a patient-to-patient
basis, a therapeutic composition of the invention for treating
diseases and disorders associated with the immunodeficiency virus
isolate(s) present in an individual at a given time. Methods for
administering the prophylactic or therapeutic compositions of the
invention are also provided.
[0029] The invention further provides methods for treating or
preventing diseases and disorders associated with infections by
immunodeficiency viruses, particularly HIV infections, comprising
administering a pharmaceutical composition of the invention
containing one or more therapeutically and/or prophylactically
effective chemokine derivative(s) and/or analog(s) that bind to a
plurality of chemokine receptors. Methods for identifying such
derivatives or analogs and formulating the prophylactic or
therapeutic compositions are also provided.
[0030] In a preferred embodiment, the invention relates to a
pharmaceutical composition comprising MDC and I-309. In a related
aspect, the invention relates to a method for treating HIV
infection, the method comprising administering to a subject in need
of such treatment a therapeutically effective amount of MDC (and/or
analogs and/or derivatives thereof) and I-309 (and/or analogs
and/or derivatives thereof). The MDC (and/or analogs and/or
derivatives thereof) and I-309 (and/or analogs and/or derivatives
thereof) may be administered simultaneously or sequentially.
Moreover, the MDC (and/or analogs and/or derivatives thereof) and
I-309 (and/or analogs and/or derivatives thereof) are suitably
administered together as components of a pharmaceutical
composition, along with a pharmaceutically acceptable carrier. The
components are preferably administered in a synergistic amount and
in a therapeutically effective amount.
5. DEFINITIONS
[0031] A "therapeutically effective" amount or dose is an amount or
dose which prevents or delays the onset or progression of an
indicated disease or other adverse medical condition. The term also
includes an amount sufficient to arrest or reduce the severity of
an ongoing disease or other adverse medical condition, and also
includes an amount necessary to enhance normal physiological
functioning.
[0032] As used herein, "treatment" of a disease or other adverse
medical condition, should be broadly interpreted based on the
therapeutic effects described herein as variously including
palliative, active, causal, conservative, medical, palliative,
prophylactic, and/or symptomatic treatment, treatment designed to
delay the onset or progression of the disease or other adverse
medical condition, as well as treatment designed to arrest or
reducing the severity of an ongoing disease or other adverse
medical condition.
[0033] As used herein, a "pharmaceutically acceptable" component
(such as a salt, carrier, excipient or diluent) of a formulation
according to the present invention is a component which (1) is
compatible with the other ingredients of the formulation in that it
can be combined with the therapeutics of the invention without
eliminating the biological activity of the therapeutics; and (2) is
suitable for use in non-human animals or humans without undue
adverse side effects (e.g., toxicity, irritation, and allergic
response). Side effects are "undue" when their risk outweighs the
benefit provided by the pharmaceutical composition.
[0034] As used herein, a "pharmaceutically acceptable" with
reference to the degree of purity of a polypeptide (e.g., a
chemokine or chemokine analog or chemokine fragment) or nucleic
acid indicates that the polypeptide or nucleic acid (1) is free of
contaminating materials that would eliminate the biological
activity of the polypeptide or nucleic acid; and (2) is free of
contaminating materials that would render the therapeutic (e.g.,
polypeptide or nucleic acid) unsuitable for administration to
humans (for pharmaceutical use) or other animals (for veterinary
use) by causing undue adverse side effects (e.g., toxicity,
irritation, and allergic response). Side effects are "undue" when
their risk outweighs the benefit provided by the therapeutic (e.g.,
polypeptide or nucleic acid).
[0035] The term "substantially pure" when used in reference to a
polypeptide or nucleic acid is defined herein to mean a therapeutic
(e.g., polypeptide or nucleic acid) that is substantially free from
other contaminating proteins, nucleic acids, and other biologicals
derived from an original source organism, recombinant DNA
expression system, or from a synthetic procedure employed in the
synthesis or purification of the therapeutic (e.g., chromatography
reagents and polymers, such as acrylamide or agarose). Purity may
be assayed by standard methods. Purity evaluation may be made on a
mass or molar basis.
6. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 demonstrates how mixtures of chemokines at the low
concentrations released by primary activated CD8+ T cells (as
determined by ELISA) block both R5 and X4 HIV infection. The figure
also shows that even at much higher concentrations, either I-309 or
MDC alone have much less antiviral effect, so they must cooperate
or synergize to mediate potent antiviral activity in the mix. Also
presented for comparison are tests (right panel) with four randomly
selected supernatants from activated CD8+ T cells. Two test
dilutions are shown. The "50%" sups contain the levels of the
chemokines used in the mix.
[0037] FIG. 2 shows the contribution of MDC to soluble HIV.sub.IIIB
suppressive activity produced by primary CD8+ T cells. The figure
demonstrates that the more MDC the cells make, the more it
contributes to the antiviral effect.
[0038] FIG. 3 shows the same form of analyses on I-309, using an
anti-I-309 antibody.
[0039] FIG. 4 shows the same analyses using a mixture of antibodies
to I-309 and MDC. This figure shows that the two chemokines
contribute very significantly to the natural activity produced by
primary CD8- T cells.
7. DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention relates to therapeutic compositions
comprising chemokines, nucleic acids encoding chemokines, or
derivatives and/or analogs thereof or nucleic acids encoding the
same, that are effective at inhibiting replication and/or infection
of an immunodeficiency virus in vitro or in vivo, decreasing viral
load, and/or treating or preventing diseases and disorders
associated with human infection with an immunodeficiency virus. The
immunodeficiency virus can be but is not limited to HIV, simian
immunodeficiency virus, and feline immunodeficiency virus, and is
most preferably HIV.
[0041] The invention also relates to therapeutic methods and
compositions for the treatment and prevention of diseases and
disorders associated with infection by immunodeficiency viruses,
preferably HIV infections, by administration of chemokine
preparations. The invention provides for treatment of HIV infection
by administration of one or more therapeutic compositions of the
invention. Therapeutic compounds of the invention include
chemokines, nucleic acids encoding chemokines, and related
therapeutically and prophylactically effective derivatives and
analogs thereof and nucleic acids encoding the same.
[0042] Without being bound by any theory, the following theoretical
model for HIV transmission is suggested: the HIV-1 envelope
initiates infection by binding to the CD4 cell surface protein.
This binding induces conformational changes in the envelope protein
that increase the exposure of the gp120 V3 loop. The exposed V3
loop then binds to a chemokine receptor, an event that itself
triggers further conformational changes leading to fusion and entry
of the virus. Depending on their origin (macrophage, CD4.sup.+ PBL,
T cell line) various isolates of HIV-1 display a requirement for a
distinct array of chemokine receptors. The envelope sequence and
structure of a given isolate most likely governs which receptor(s)
or receptor array is required for entry into the target cell. It is
likely that the envelope molecules share a structural homology with
chemokines that allows them to interact with various chemokine
binding domains.
[0043] The Inventors believe that .alpha.-chemokines and
.beta.-chemokines other than RANTES, MIP-1.alpha. and MIP-1.beta.
(previously demonstrated by the Inventors to inhibit the infection
or replication of HIV-1) will also be able to inhibit the infection
or replication of HIV-1, depending upon the GCR requirement of the
isolate and the target cell. Accordingly, the invention provides
methods for formulating pharmaceutical compositions containing one
or more chemokines, nucleic acids encoding chemokines, and/or
therapeutically and prophylactically effective derivatives or
analogs thereof or nucleic acids encoding the same, that will
target and treat diseases and disorders associated with infection
by an immunodeficiency virus isolate of interest, particularly a
primary HIV isolate. Additionally, the invention provides methods
for formulating pharmaceutical compositions which contain
(preferably as the only chemokines) the chemokines, nucleic acids
encoding chemokines, therapeutically and prophylactically effective
derivatives or analogs thereof and/or nucleic acids encoding the
same, that treat diseases and disorders associated with isolates of
immunodeficiency viruses present in an individual at a given time,
by a method comprising testing HIV recovered from the individual
for inhibition by one or more, preferably a panel of at least 2, 3,
4, 5, 6, 7, 8, 9, or 10 chemokines, and identifying the
chemokine(s), derivative(s), or analog(s) effective at inhibiting
HIV infection or replication. Alternatively, the invention provides
methods by which to formulate pharmaceutical compositions
containing numerous of the chemokines, derivatives and analogs that
inhibit the infection or replication of one or more, preferably
numerous of the isolates known for a particular immunodeficiency
virus, preferably a HIV.
[0044] The invention further provides methods by which to formulate
pharmaceutical compositions comprising one or more therapeutically
and prophylactically effective chemokine derivative(s) or
analog(s), or nucleic acids encoding the foregoing, that bind
separately to a plurality of .alpha. and/or .beta. chemokine
receptors. Methods for identifying such derivatives or analogs are
also provided.
[0045] The invention additionally relates to pharmaceutical
compositions comprising one or more .alpha., .beta., or .gamma.
chemokines, or nucleic acid encoding one or more .alpha., .beta.,
or .gamma. chemokines, for treatment or prevention of disorders
associated with HIV infection. In a specific embodiment, such a
composition comprises a therapeutically or prophylactically
effective amount of one or more of MCP-1, MCP-2, MCP-3, MCP-4,
MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309,
.gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma.,
ENA-78, GCP-2, or lymphotactin. In another embodiment, the
pharmaceutical composition further comprises a therapeutically or
prophylactically effective amount of a chemokines, chemokine
derivatives and/or chemokine analogs thereof, selected from the
group consisting of RANTES, MIP-1.alpha., MIP-1.beta., MCP-1,
MCP-3, IL-8, and/or SDF-1. Pharmaceutical compositions comprising
nucleic acids encoding such chemokines, derivatives or analogs are
also provided.
[0046] In another embodiment, the pharmaceutical composition of the
invention comprises a therapeutically or prophylactically effective
amount of a derivative or analog of one or more .alpha., .beta., or
.gamma. chemokines, or nucleic acid encoding a derivative or analog
of one or more .alpha., .beta., or .gamma. chemokines, for
treatment and prevention of disorders associated with HIV
infection. In a specific embodiment, such composition comprises a
therapeutically or prophylactically effective amount of a
derivative or analog of MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2, or
lymphotactin. In another embodiment, such pharmaceutical
composition further comprises a therapeutically or prophylactically
effective amount of a chemokines, chemokine derivatives and/or
chemokine analogs thereof, selected from the group consisting of
RANTES, MIP-1.alpha., MIP-1.beta., IL-8, and/or SDF-1.
Pharmaceutical compositions comprising nucleic acids encoding such
chemokines, derivatives or analogs are also provided.
[0047] In specific embodiments, the pharmaceutical composition
comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chemokines,
derivatives or analogs, or nucleic acids encoding the same.
Embodiments wherein the pharmaceutical composition of the invention
comprises a plurality of chemokines, derivatives and/or analogs
selected from those listed above are further described herein.
[0048] The pharmaceutical composition of the invention optionally
further comprises a therapeutically or prophylactically effective
amount of another anti-HIV agent.
[0049] The invention also provides in vitro and in vivo assays for
assessing the efficacy of therapeutics of the invention for
treatment or prevention of infection with an immunodeficiency
virus, in particular HIV infection.
[0050] The invention further relates to methods for treating or
preventing immunodeficiency virus infection, in particular HIV, in
mammals, including humans, by administering the therapeutic
compositions of the invention. Methods of administration of the
therapeutics of the invention for treatment or prevention of
immunodeficiency virus infection are also provided.
[0051] The invention also provides methods for inhibiting the
infection or replication of any isolate of an immunodeficiency
virus, in particular, an HIV isolate. More specifically, the
invention provides methods for formulating, on a patient-to-patient
basis, a pharmaceutical composition of specific chemokines
effective against immunodeficiency virus isolates present in the
patient at a given time. Methods for administering a pharmaceutical
composition containing chemokines or derivatives or analogs
thereof, which inhibit the infection or replication of one or more
of all known isolates of a immunodeficiency virus are also
provided. In a preferred embodiment the inhibited immunodeficiency
virus is HIV.
[0052] Additionally, the invention provides methods for treating or
preventing immunodeficiency virus infections, by administering an
effective amount of a pharmaceutical composition containing
chemokine derivatives or analog(s) that bind a plurality of
chemokine receptors.
[0053] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections which follow.
[0054] 7.1 Chemokines, Derivatives and Analogs
[0055] The invention provides pharmaceutical compositions
comprising chemokines, nucleic acids encoding chemokines,
derivatives or analogs thereof, or nucleic acids encoding the
derivatives or analogs, that have activity in the treatment and
prevention of disorders associated with immunodeficiency virus
infection, preferably HIV infection. In a specific embodiment, the
compounds of the invention inhibit HIV infection or
replication.
[0056] In various embodiments, the invention relates to
pharmaceutical compositions comprising one or more .alpha., .beta.,
or .gamma. chemokines, or nucleic acid encoding one or more
.alpha., .beta., or .gamma. chemokines, for treatment and
prevention of disorders associated with HIV infection. In a
specific embodiment, such composition comprises a therapeutic or
prophylactically effective amount of an .alpha.(C.times.C)
chemokine or nucleic acid encoding an a chemokine selected from the
group consisting of .gamma. interferon inducible protein-10
(.gamma.IP-10), interleukin-8 (IL-8), platelet factor-4 (PF4),
neutrophil activating protein (NAP-2), GRO-.alpha., GRO-.beta.,
GRO-.gamma., neutrophil-activating peptide (ENA-78), granulocyte
chemoattractant protein-2 (GCP-2), and stromal cell-derived
factor-1 (SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a
.beta.(CC) chemokine or nucleic acid encoding a .beta. chemokine
selected from the group consisting of: RANTES (regulated on
activation, normal T expressed and secreted), macrophage
inflammatory protein-1.alpha. (MIP-1.alpha.), macrophage
inflammatory protein-1.beta. (MIP-1.beta.), monocyte chemotactic
protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2), monocyte
chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4
(MCP-4) macrophage inflammatory protein-1.gamma. (MIP-1.gamma.),
macrophage inflammatory protein-3.alpha. (MIP-3.alpha.), macrophage
inflammatory protein-3.beta. (MIP-3.beta.), eotaxin, Exodus, and
I-309; and/or the .gamma.(C) chemokine, or nucleic acid encoding
the .gamma. chemokine, lymphotactin. In another embodiment, such
compositions comprise a plurality of chemokines, e.g. at least 2,
3, 4, 5, 6, 7, 8, 9, or 10 chemokines, selected from those listed
above.
[0057] In one embodiment, the pharmaceutical compositions of the
invention comprise 1, 2, 3, or 4 chemokines selected from among
RANTES, MIP-1.alpha., MIP-1.beta., or IL-8 or a therapeutically and
prophylactically effective derivative or analog thereof, in
combination with one or more other chemokines, or a therapeutically
and prophylactically effective derivative or analog thereof. Such
other chemokines are selected from the group consisting of MCP-1,
MCP-2, MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta.,
eotaxin, Exodus, I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha.,
GRO-.beta., GRO-.gamma., ENA-78, GCP-2, lymphotactin and SDF-1.
Pharmaceutical compositions comprising nucleic acids encoding such
chemokines are also provided. In further embodiments, the
pharmaceutical composition comprises at least 2, 3, 4, 5, 6, 7, 8,
9, or 10 chemokines, or nucleic acids encoding the same.
[0058] In another embodiment, the pharmaceutical composition
comprises a .beta. chemokine, or nucleic acid encoding a .beta.
chemokine. In specific embodiments, the pharmaceutical composition
comprises a .beta. chemokine, or nucleic acid encoding a .beta.
chemokine, selected from the group consisting of MCP-2, MCP-4,
MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, and
I-309, or a therapeutically and prophylactically effective
derivative or analog thereof.
[0059] In another embodiment, the pharmaceutical composition of the
invention comprises an .alpha. chemokine, or nucleic acid encoding
an .alpha. chemokine. In specific embodiments, the pharmaceutical
composition comprises an .alpha. chemokine, or nucleic acid
encoding a .alpha. chemokine, selected from the group consisting of
.gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma.,
ENA-78, and GCP-2, and optionally, SDF-1, or a therapeutically or
prophylactically effective derivative thereof.
[0060] In another embodiment, the pharmaceutical composition
comprises an .gamma. chemokine, or nucleic acid encoding a .gamma.
chemokine, or a therapeutically or prophylactically effective
derivative or analog thereof. In a specific embodiment, the
pharmaceutical composition comprises the chemokine lymphotactin, or
nucleic acid encoding lymphotactin, or a therapeutically or
prophylactically effective derivative or analog thereof.
[0061] In a further embodiment, the pharmaceutical composition of
the invention comprises a combination of .alpha., .beta. and/or
.gamma. chemokines, nucleic acids encoding .alpha., .beta., or
.gamma. chemokines, or therapeutically and prophylactically
effective derivatives or analogs thereof. For example, in one
embodiment, a pharmaceutical composition comprises a .beta.
chemokine and an .alpha. chemokine, in an amount therapeutically or
prophylactically effective against disease or disorder associated
with HIV infection.
[0062] In a specific embodiment, the pharmaceutical composition
comprises a therapeutically or prophylactically effective amount
(i.e., an amount effective to inhibit immunodeficiency virus
replication or infection) of one or more of MCP-1, MCP-2, MCP-3,
MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin, Exodus,
I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta.,
GRO-.gamma., ENA-78, GCP-2, and lymphotactin. Preferably, the
chemokine has been purified. In another embodiment, the
pharmaceutical composition further comprises a therapeutically or
prophylactically effective amount of one or more chemokines,
chemokine derivatives and/or chemokine analogs thereof, selected
from the group consisting of RANTES, MIP-1.alpha., MIP-1.beta.,
IL-8, and SDF-1. Pharmaceutical compositions comprising nucleic
acids encoding such chemokines, derivatives and analogs are also
provided.
[0063] In another specific embodiment, the pharmaceutical
composition comprises a therapeutically or prophylactically
effective amount of a derivative or analog of one or more of MCP-1,
MCP-2, MCP-3, MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta.,
eotaxin, Exodus, I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha. ,
GRO-.beta., GRO-.gamma., ENA-78, GCP-2, and lymphotactin.
Preferably, the chemokine derivative or analog has been purified.
In another embodiment, the pharmaceutical composition further
comprises a therapeutically or prophylactically effective amount of
one or more chemokines, chemokine derivatives and/or chemokine
analogs thereof, selected from the group consisting of RANTES,
MIP-1.alpha., MIP-1.beta., IL-8, and SDF-1. Pharmaceutical
compositions comprising nucleic acids encoding such chemokines,
derivatives and analogs listed above are also provided.
[0064] In another specific embodiment, the pharmaceutical
composition comprises a therapeutically or prophylactically
effective amount of one or more of MCP-2, MCP-4, MIP-1.gamma.,
MIP-3.alpha., MIP-3.beta., eotaxin, Exodus, I-309, .gamma.IP-10,
PF4, NAP-2, GRO-.alpha., GRO-.beta., GRO-.gamma., ENA-78, GCP-2,
and lymphotactin. Preferably, the chemokine has been purified. In
another embodiment, such pharmaceutical composition further
comprises a therapeutically or prophylactically effective amount of
one or more chemokines, chemokine derivatives and/or chemokine
analogs selected from the group consisting of RANTES, MIP-1.alpha.,
MIP-1.beta., MCP-1, MCP-3, IL-8, and SDF-1. Pharmaceutical
compositions comprising nucleic acids encoding such chemokines,
derivatives or analogs are also provided.
[0065] In another specific embodiment, the pharmaceutical
composition comprises a therapeutically or prophylactically
effective amount of a derivative or analog of one or more of MCP-2,
MCP-4, MIP-1.gamma., MIP-3.alpha., MIP-3.beta., eotaxin, Exodus,
I-309, .gamma.IP-10, PF4, NAP-2, GRO-.alpha., GRO-.beta.,
GRO-.gamma., ENA-78, GCP-2, lymphotactin, and SDF-1. Preferably,
the derivative or analog has been purified. In another embodiment,
such pharmaceutical composition further comprises a therapeutically
or prophylactically effective amount of one or more chemokines,
chemokine derivatives and/or chemokine analogs selected from the
group consisting of RANTES, MIP-1.alpha., MIP-1.beta., MCP-1,
MCP-3, and IL-8. Pharmaceutical compositions comprising nucleic
acids encoding such chemokines, derivatives or analogs are also
provided.
[0066] In specific embodiments, the pharmaceutical composition
comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chemokines,
derivatives (including fragments) or analogs, or nucleic acids
encoding the same. In further embodiments, the pharmaceutical
composition of the invention comprises only chemokines,
derivatives, and/or analogs that have been demonstrated using
assays. Examples include those described herein to have activity
against a primary HIV isolate.
[0067] The pharmaceutical composition of the invention optionally
further comprises a therapeutically or prophylactically effective
amount of another anti-HIV agent. Examples of other anti-HIV
compounds that can be used in such multi-drug regimens include:
protease inhibitors (e.g., CRIXIVAN.TM. (indinavir); FORTOVASE.TM.
and INVIRASE.TM. (saquinavir); NORVIR.TM. (ritonavir); and
VIRACEPT.TM. (nelfinavir)); non-nucleoside reverse transcriptase
inhibitors (e.g., RESCRIPTOR.TM. (delavirdine); SUSTIVA.TM.
(efavirenz); and VIRAMUNE.TM. (nevirapine)); and nucleoside reverse
transcriptase inhibitors (e.g., VIDEX.TM. (didanosine, also known
as DDI); EPIVIR.TM. (lamivudine, also known as 3TC); ZERIT.TM.
(stavudine, also known as d4T); HIVID.TM. (Zalcitabine, also known
as DDC); RETROVIR.TM. (zidovudine, also known as AZT or ZDV); and
COMBIVIR.TM. (lamivudine and zidovudine)).
[0068] In one embodiment, the pharmaceutical composition of the
invention comprises a chemokine, or nucleic acid encoding a
chemokine, or derivative or analog thereof, that binds to a .beta.
chemokine receptor, including, but not limited to, CC CKR-1, CC
CKR-2A, CC CKR-2B, CC CKR-3, CC CKR-4 or CC CKR-5. In a preferred
embodiment, such chemokines, chemokine derivatives and/or chemokine
analogs, binds the chemokine receptor CC CKR-5.
[0069] In another embodiment, the pharmaceutical composition
comprises a chemokine, or nucleic acid encoding a chemokine, or
derivative or analog thereof that binds to an .alpha. chemokine
receptor, including but not limited to, C.times.C CKR4, IL-8RA,
IL-8RB, Mig receptor, .gamma.IP-10 receptor, and Duffy antigen.
Preferably, the pharmaceutical composition comprises a chemokine,
nucleic acid encoding a chemokine, or derivative or analog thereof
that binds separately to an .alpha. and .beta. chemokine receptor.
In another preferred embodiment, the pharmaceutical composition
comprises a chemokines, chemokine derivatives and/or chemokine
analogs that binds separately to a plurality of .alpha. and/or
.beta. chemokine receptors. In a most preferred embodiment, the
pharmaceutical composition comprises a chemokine, or a derivative
or analog thereof, that binds separately to both C.times.C CKR4 and
CC CKR-5. In further embodiments, the chemokine, derivative and/or
analog binds separately to 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
chemokine receptors. In another preferred embodiment, the
derivative or analog of the invention is capable of binding
separately to at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 chemokine
receptors selected from the group consisting of CC CKR-1, CC
CKR-2a, CC CKR-2b, CC CKR-3, CC CKR-4, CC CKR-5, C.times.C CKR4,
IL-8RA, IL-8RB, Mig receptor, .gamma.IP-10 receptor, and Duffy
antigen. Pharmaceutical compositions containing nucleic acids
encoding such chemokines, derivatives and/or analogs are also
provided.
[0070] In a specific embodiment, the invention relates to RANTES
and SDF-1 derivatives and analogs, or nucleic acids encoding RANTES
and SDF-1 derivatives and analogs, that comprise, or alternatively
consist of an amino acid sequence capable of binding to a chemokine
receptor. In a preferred embodiment, the chemokine derivative or
analog is a molecule that comprises the amino acid sequence
Lys-Asn-Asn-Asn-Arg-Gln-Val (SEQ ID NO:1)(amino acids 45-51 of
mature SDF-1 (SWISS-PROT:P48061, Jun. 1, 1996)), which is believed
by the Inventors to bind the C.times.C CKR4 receptor, with the
proviso that the molecule is less than 61, 60, 55, 50, 45, 40, 35,
30, 20, 15, 14, 13, 12 ,11, 10, 9, 8, or 7 amino acids. In a
particular embodiment, the chemokine derivative or analog is a
molecule that comprises the amino acid sequence
Cys-Ala-Leu-Gln-Ile-Val-Ala-Arg-Le-
u-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys (SEQ ID NO:2, amino acids 36-52
of mature SDF-1 (SWISS-PROT:P48061, Jun. 1, 1996)). In a particular
embodiment, a fusion protein is provided wherein an SDF-1 sequence
is found fused via a peptide bond to a different sequence, and
wherein the entire SDF-1 sequence contained in the fusion protein
consists of amino acids 45-51 or 36-52 of mature SDF-1, or
comprises amino acids 45-51 or 36-52 of mature SDF-1 but has less
than 61, 60, 55, 50, 45, 40, 35, 30, 20, 15, 14, 13, 12 ,11, 10, 9,
8, or 7 contiguous amino acids of the SDF-1 sequence where
applicable, or with conservative substitutions therein. In another
embodiment, a peptide is provided whose amino acid sequence
consists of amino acids 45-51 or 36-52 of mature SDF-1. In
addition, fragments of SDF-1 comprising amino acids 45-51 or 36-52
are provided, as well as such fragments with conservative
substitutions. In another preferred embodiment, the chemokine
derivative or analog is a molecule that comprises the amino acid
sequence Lys-Asn-Arg-Gln-Val (SEQ ID NO:3, amino acids 45-49 of
mature RANTES (Schall, 1991, Cytokine 3:165-183)) which is believed
by the inventors to bind CC CKR-5, with the proviso that the
molecule is less than 58, 55, 50, 45, 40, 35, 30, 20, 15, 14, 13,
12 ,11, 10, 9, or 8 amino acids. In a particular embodiment, the
chemokines, chemokine derivatives and/or chemokine analogs is a
molecule that comprises the amino acid sequence
Cys-Ser-Asn-Pro-Ala-Val-V-
al-Phe-Val-Thr-Arg-Lys-Asn-Arg-Gln-Val-Cys (SEQ ID NO:4, amino
acids 34-50 of mature RANTES (Schall, 1991, Cytokine 3:165-183)).
In a particular embodiment, a fusion protein is provided wherein an
RANTES sequence is found fused via a peptide bond to a different
sequence and wherein the entire RANTES sequence contained in the
fusion protein consists of amino acids 45-49 or 34-50 of mature
RANTES, or comprises amino acids 45-49 or 34-50 of mature RANTES
but has less than 58, 55, 50, 45, 40, 35, 30, 20, 15, 14, 13, 12
,11, 10, 9, 8, or 7 contiguous amino acids of the RANTES sequence
where applicable, or with conservative substitutions therein. In
another embodiment, a peptide is provided whose amino acid sequence
consists of amino acids 45-49 or 34-50 of mature RANTES. In
addition, fragments of SDF-1 comprising amino acids 45-49 or 34-50
of mature RANTES are provided, as well as such fragments with
conservative substitutions. In specific embodiments described
infra, RANTES and/or SDF-1 derivatives or analogs comprising the
amino acid sequences listed above are joined at amino or
carboxy-termini via a peptide bond to an amino acid sequence of a
different protein to form a chimeric, or fusion protein.
[0071] In a specific embodiment of the invention, proteins
consisting of or comprising a fragment of a chemokine consisting of
at least 5 (continuous) amino acids of the chemokine is provided.
In other embodiments, the fragment consists of at least 6, 7, 8, 9,
10, 15, 20, 30, 40, 50, 60, 70 or 80 amino acids of the chemokine.
In specific embodiments, such fragments are not larger than 10, 20,
30, 40, 50, 60, 70 or 80 amino acids. Derivatives or analogs of a
chemokine include but are not limited to those molecules exhibiting
antiviral activity and that comprise regions that are substantially
homologous to a chemokine or fragment thereof (e.g., in various
embodiments, at least 60% or 70% or 80% or 90% or 95% identity over
an amino acid sequence of identical size or when compared to an
aligned sequence wherein the alignment is done by a computer
homology program known in the art) or whose encoding nucleic acid
is capable of hybridizing to a coding chemokine sequence, under
high stringency, moderately high stringency, or low stringency
conditions. In a specific embodiment, the chemokine derivative
retains the antigenicity (ability to bind to an anti-chemokine
antibody) or immunogenicity of the chemokine. Fragments and other
derivatives of a chemokine that retain the ability to bind to a
chemokine receptor are preferred.
[0072] By way of example and not limitation, procedures using
conditions of low stringency are as follows (see also Shilo and
Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters
containing DNA are pretreated for 6 h at 40.sub.EC in a solution
containing 35% formamide, 5.times. SSC, 50 mM Tris-HCl (pH 7.5), 5
mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA. Hybridizations are carried out in the same
solution with the following modifications: 0.02% PVP, 0.02% Ficoll,
0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10% (wt/vol) dextran
sulfate, and 5-20.times.10.sup.6 cpm .sup.32P-labeled probe is
used. Filters are incubated in hybridization mixture for 18-20 h at
40.sub.EC, and then washed for 1.5 h at 55.sub.EC in a solution
containing 2.times. SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and
0.1% SDS. The wash solution is replaced with fresh solution and
incubated an additional 1.5 h at 60.sub.EC. Filters are blotted dry
and exposed for autoradiography. If necessary, filters are washed
for a third time at 65-68.sub.EC and reexposed to film. Other
conditions of low stringency which may be used are well known in
the art.
[0073] By way of example and not limitation, procedures using
conditions of high stringency are as follows: prehybridization of
filters containing DNA is carried out for 8 h to overnight at
65.sub.EC in buffer composed of 6.times. SSC, 50 mM Tris-HCl (pH
7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48
h at 65.sub.EC in prehybridization mixture containing 100 .mu.g/ml
denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of
.sup.32P-labeled probe. Washing of filters is done at 37.sub.EC for
1 h in a solution containing 2.times. SSC, 0.01% PVP, 0.01% Ficoll,
and 0.01% BSA. This is followed by a wash in 0.1.times. SSC at
50.sub.EC for 45 min before autoradiography. Other conditions of
high stringency which may be used are well known in the art.
[0074] By way of example and not limitation, procedures using
conditions of moderately high stringency are as follows: filters
containing DNA are pretreated for 6 hours to overnight at 55.sub.EC
in buffer composed of 6.times. SSC, 5.times. Denhart's 0.5% SDS,
100 mg/mL salmon sperm DNA. Hybridizations are carried out in the
same solution upon adding 5-20.times.10.sup.6 cpm of
.sup.32P-labeled probe and incubated 8-48 hours at 55.sub.EC.
Washing of filters is done at 60.sub.EC in 1.times. SSC, 0.1% SDS,
with two exchanges after 30 minutes. Other conditions for
moderately high stringency screening are known in the art. For
further guidance regarding hybridization conditions see, for
example, Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,
Current Protocols in Molecular Biology, Green Publishing Associates
and Wiley Interscience, N.Y.
[0075] The invention also relates to chemokine derivatives or
analogs made by altering the chemokine sequence by substitutions,
additions or deletions that provide for molecules with anti-viral
activity (e.g., inhibit infection or replication of an
immunodeficiency virus, preferably HIV) or demonstrate the ability
to bind to a chemokine receptor. Thus, the chemokine derivatives
include polypeptides containing, as a primary amino acid sequence,
all or part of the chemokine amino acid sequence including altered
sequences wherein functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a
polypeptide which is functionally active. For example, one or more
amino acid residues within the sequence can be substituted by
another amino acid of a similar polarity which acts as a functional
equivalent, resulting in a silent alteration. Conservative
substitutions for an amino acid within the sequence may be selected
from other members of the class to which the amino acid belongs.
For example, the nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan and methionine. The polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine. The positively charged (basic) amino acids include
arginine, lysine and histidine. The negatively charged (acidic)
amino acids include aspartic acid and glutamic acid. Such chemokine
derivatives can be made either by chemical peptide synthesis or by
recombinant production from nucleic acid encoding the chemokine
which nucleic acid has been mutated. Any technique for mutagenesis
known in the art can be used, including but not limited to,
chemical mutagenesis, in vitro site-directed mutagenesis
(Hutchinson, C., et al., 1978, J. Biol. Chem 253:6551), use of
TAB.sub.7 linkers (Pharmacia), etc.
[0076] In one embodiment, the only non-conservative amino acid
substitutions in the RANTES derivatives of the invention are the
substitution of leucine and isoleucine in place of the tyrosine
residues at amino acid numbers 27 and 29 (Schall, 1991, Cytokine
3:165-183), respectively.
[0077] In another embodiment, the only non-conservative amino acid
substitutions in the SDF-1 derivatives of the invention are the
substitution of tyrosine in place of the leucine and isoleucine
residues at amino acid numbers 28 and 30 (SWISS-PROT:P48061, Jun.
1, 1996), respectively.
[0078] In a specific embodiment, the chemokine, derivatives or
analogs of the invention comprise the sequence
Lys-Asn-X-Arg-Gln-Val (SEQ ID NO:5), where X is any amino acid, but
is preferably asparagine or another polar neutral amino acid. A
hexapeptide having the sequence of SEQ ID NO:5 is also
provided.
[0079] Both .beta. and .alpha. chemokines are active as monomers as
well as dimers, and there is evidence that monomeric .beta.
chemokines can inhibit HIV infection and replication (DeVico et
al., personal observation). It is therefore possible to construct a
monomeric chimeric molecule that displays a wide range of receptor
binding that will inhibit the infection or replication of a variety
of HIV isolates. Accordingly, in a specific embodiment, the
chemokine derivative or analog is a chimeric, or fusion, protein
containing the polypeptide sequence of a chemokine that binds the
chemokine receptor (preferably consisting of at least 5, 6, 7, 8,
9, 10, 15, 20, 30, 40, 50 60, 70 or 80 amino acids of the
chemokine) joined at its amino- or carboxy-terminus via a peptide
bond to an amino acid sequence of a different protein. In one
embodiment, such a chimeric protein is produced by recombinant
expression of a nucleic acid encoding the protein (comprising a
chemokine amino acid sequence which binds the chemokine receptor
joined in-frame to a coding sequence for a different protein). Such
a chimeric product can be made by ligating the appropriate nucleic
acid sequences encoding the desired amino acid sequences to each
other by methods known in the art, in the proper coding frame, and
expressing the chimeric product by methods commonly known in the
art. Alternatively, such a chimeric product may be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer. In a
specific embodiment, a chimeric nucleic acid encoding a chemokine
with a heterologous signal sequence is expressed such that the
chimeric protein is expressed and processed by the cell to the
mature chemokine.
[0080] In a preferred embodiment, the chimeric of the invention
comprises the amino acid sequence Lys-Asn-Asn-Asn-Arg-Gln-Val (SEQ
ID NO:1) or
Cys-Ala-Leu-Gln-Ile-Val-Ala-Arg-Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys
(SEQ ID NO:2) of mature SDF-1 joined at its amino- and/or
carboxy-terminus via a peptide bond to a different protein. In
another preferred embodiment, the chimeric comprises the amino acid
sequence Lys-Asn-Arg-Gln-Val (SEQ ID NO:3) or
Cys-Ser-Asn-Pro-Ala-Val-Val-Phe-Val-Thr-Arg-Lys-Asn-Arg-Gln-V-
al-Cys (SEQ ID NO:4) of mature RANTES (Schall, 1991, Cytokine
3:165-183) joined at its amino- and/or carboxy-terminus via a
peptide bond to a different protein.
[0081] In other specific embodiments, the chimeric of the invention
comprises a RANTES derivative wherein the amino acid sequence
Lys-Asn-Asn-Asn-Arg-Gln-Val (SEQ ID NO:1) or Lys-Asn-X-Arg-Gln-Val
(SEQ ID NO:5) is substituted for the sequence Lys-Asn-Arg-Gln-Val
(SEQ ID NO:3) in RANTES, or the amino acid sequence
Cys-Ala-Leu-Gln-Ile-Val-Ala-A-
rg-Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys (SEQ ID NO:2) is substituted
for the sequence
Cys-Ser-Asn-Pro-Ala-Val-Val-Phe-Val-Thr-Arg-Lys-Asn-Arg-Gln--
Val-Cys (SEQ ID NO:4) in RANTES. In other specific embodiments, the
chimeric of the invention comprises a SDF-1 derivative wherein the
amino acid sequence Lys-Asn-Arg-Gln-Val (SEQ ID NO:3) or
Lys-Asn-X-Arg-Gln-Val (SEQ ID NO:5) is substituted for the sequence
Lys-Asn-Asn-Asn-Arg-Gln-Val (SEQ ID NO:1) in SDF-1, or the amino
acid sequence
Cys-Ser-Asn-Pro-Ala-Val-Val-Phe-Val-Thr-Arg-Lys-Asn-Arg-Gln-Val-Cys
(SEQ ID NO:4) is substituted for the sequence
Cys-Ala-Leu-Gln-Ile-Val-Ala-Arg--
Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys (SEQ ID NO:2) in SDF-1.
Molecules comprising or consisting of this chimeric sequence are
provided, as are nucleic acids encoding the same.
[0082] In another embodiment, a chimeric of the invention contains
those coding portions of two chemokines that bind to two distinct
chemokine receptors. For example, a chimeric can be constructed
which contains a nucleotide sequence encoding the amino acid
sequence in RANTES that bind CC CKR-5 and a nucleotide sequence
encoding the amino acid sequence of SDF-1 that bind C.times.C CKR4.
The encoded protein of such a recombinant molecule could exhibit
properties associated with both chemokines, and portray a novel
profile of biological activities, including the ability to bind
both chemokine receptors. In specific embodiments, such chimerics
of the invention comprise a first amino acid sequence comprising
Lys-Asn-Asn-Asn-Arg-Gln-Val (SEQ ID NO:1) or
Cys-Ala-Leu-Gln-Ile-Val-Ala--
Arg-Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys (SEQ ID NO:2) of mature
SDF-1 joined at its amino- or carboxy-terminal to a second amino
acid sequence comprising Lys-Asn-Arg-Gln-Val (SEQ ID NO:3) or
Cys-Ser-Asn-Pro-Ala-Val-V-
al-Phe-Val-Thr-Arg-Lys-Asn-Arg-Gln-Val-Cys (SEQ ID NO:4) of mature
RANTES. Molecules comprising or consisting of this chimeric
sequence are provided, as are nucleic acids encoding the same.
[0083] In additional embodiments, the chimeric of the invention
comprises fragments of chemokines. In a specific embodiment the
chimeric of the invention comprises a fragment of SDF-1 comprising
the amino acid sequence Lys-Asn-Asn-Asn-Arg-Gln-Val (SEQ ID NO:1)
or
Cys-Ala-Leu-Gln-Ile-Val-Ala-Arg-Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys
(SEQ ID NO:2), wherein said SDF-1 fragment is less than 60 amino
acids in length; fused via a covalent bond to an amino acid
sequence comprising a fragment of RANTES comprising the amino acid
sequence Lys-Asn-Arg-Gln-Val (SEQ ID NO:3) or
Cys-Ser-Asn-Pro-Ala-Val-Val-Phe-Val-Thr-Arg-Lys-Asn-Arg--
Gln-Val-Cys (SEQ ID NO:4), wherein said RANTES fragment is less
than 55 amino acids in length. Optionally, one or both of the
fragment components of this chimeric are capable of binding one or
more chemokine receptors. Molecules comprising or consisting of
this chimeric sequence are provided, as are nucleic acids encoding
the same.
[0084] Chimeric chemokines of the invention may be synthetic
peptide fragments or full length synthetic chemokines wherein are
inserted specific sequences from .alpha., .beta., and/or .gamma.
chemokines which optionally bind a chemokine receptor or inhibit
immunodeficiency virus infection or replication, so as to bind one
or more chemokine receptors. The primary sequence of the chemokines
may also be used to predict tertiary structure of the molecules
using computer simulation (Hopp and Woods, 1981, Proc. Natl. Acad.
Sci. U.S.A. 78:3824-3828); chemokine/chemokine chimeric recombinant
genes can be designed in light of correlations between tertiary
structure and biological function.
[0085] Derivatives mentioned above also can be cyclized, e.g., as
described herein.
[0086] Antiviral activity of the chemokines, nucleic acids encoding
chemokines, or derivatives (including fragments and chimeric
proteins) or analogs thereof, for treatment or prevention of HIV
infection can be demonstrated by any of the methods disclosed in
herein or known to one skilled in the art.
[0087] 7.2 Preparation of Chemokines, Derivatives and Analogs
[0088] The chemokines, derivatives or analogs of the invention can
be obtained commercially or alternatively, purified from biological
tissue or cell culture, or produced by recombinant or synthetic
techniques known in the art.
[0089] Native chemokine preparations can be obtained from a variety
of sources. Recombinant RANTES, MIP-1.alpha. and MIP-1.beta. are
commercially available (Sigma Immunochemicals, St. Louis, Mo.;
R&D Systems, Minneapolis, Minn.; and PeproTech, Rocky Hills,
N.J.). Alternatively, standard methods of protein purification may
be used to isolate and purify, or partially purify, chemokines from
any source known to contain or produce the desired chemokine, e.g.,
RANTES, MIP-1.alpha., and MIP-1.beta. may be isolated from sources
such as CD8.sup.+ T cells, HTLV-I, II transformed cell lines such
as FC36.22, or uninfected immortalized cell lines. Such standard
protein purification techniques include, but are not limited to,
chromatography (e.g., ion exchange, affinity, gel
filtration/molecular exclusion chromatography and reversed phase
high performance liquid chromatography (RP-HPLC)), centrifugation,
differential solubility, and electrophoresis (for a review of
protein purification techniques, see, Scopes, Protein Purification;
Principles and Procedure, 2nd Ed., C. R. Cantor, Editor, Springer
Verlag, New York, N.Y. (1987), and Parvez et al., Progress in HPLC,
Vol. 1, Science Press, (1985) Utrecht, The Netherlands). For
example, antibodies to RANTES, MIP-1.alpha., and MIP-1.beta. are
available commercially (e.g., Sigma Immunochemicals (St. Louis,
Mo.); R&D Systems (Minneapolis, Minn.); and PeproTech, Inc.
(Rocky Hills, N.J.)) and can be used to prepare an affinity
chromatography column which can be used to purify the respective
chemokines by well-known techniques (see, e.g., Hudson & May,
1986, Practical Immunology, Blackwell Scientific Publications,
Oxford, United Kingdom).
[0090] Recombinant expression techniques can be applied to obtain
the chemokines, derivatives, and analogs of the invention (see,
e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, 2d Ed., Cold Spring Harbor,
N.Y., Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach,
MRL Press, Ltd., Oxford, U.K., Vol. I, II). The nucleic acid
sequences of the chemokines listed supra are known (for example,
see, Rossi et al., 1997, J. Immunol. 158:1033-1036; Kitaura et al.,
1996, J. Biol. Chem. 271:7725-7730; Ponath et al., 1996, J. Clin.
Invest. 97:604-612; Hromas, R., Jan. 16, 1997, Genbank Accession
Number U64197; Uguccioni et al., 1996, J. Exp. Med. 183:2379-2384;
Baggiolini et al., 1994, Advances in Immunology 55:97-179; Miller
and Krangel, 1992, Immunology 12:17-46; and Schall, 1991, Cytokine
3:165-183) and can be isolated using well-known techniques in the
art, such as screening a library, chemical synthesis, or polymerase
chain reaction (PCR). Other chemokines may be cloned using routine
recombinant techniques known in the art in combination with assays
which select for known biochemical properties of the chemokine of
interest. Cloned chemokine gene sequence can be modified by any of
numerous strategies known in the art.
[0091] To recombinantly produce a chemokines, chemokine derivatives
and/or chemokine analogs, a nucleic acid sequence encoding the
chemokine derivative or analog is operatively linked to a promoter
such that the chemokine, derivative, or analog is produced from
said sequence. For example, a vector can be introduced into a cell,
wherein the vector or a portion thereof is expressed, producing a
chemokine or a portion thereof. In a preferred embodiment, the
nucleic acid is DNA if the source of RNA polymerase is DNA-directed
RNA polymerase, but the nucleic acid may also be RNA if the source
of polymerase is RNA-directed RNA polymerase or if reverse
transcriptase is present in the cell or provided to produce DNA
from the RNA. Such a vector can remain episomal or become
chromosomally integrated, as long as it can be transcribed to
produce the desired RNA. Such vectors can be constructed by
recombinant DNA technology methods standard in the art.
[0092] A variety of host-vector systems may be utilized to express
the protein-coding sequence. These include but are not limited to
mammalian cell systems infected with virus (e.g., vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.,
baculovirus); microorganisms such as yeast containing yeast
vectors, or bacteria transformed with bacteriophage, DNA, plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in
their strengths and specificities and depending on the host-vector
system utilized, any one of a number of suitable transcription and
translation elements may be used.
[0093] Expression of a chemokine protein, derivative, or analog may
be controlled by any promoter/enhancer element known in the art.
Such promoters include, but are not limited to: the SV40 early
promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),
the promoter contained in the 3.sub.N long terminal repeat of Rous
sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the HSV-1
(herpes simplex virus-1) thymidine kinase promoter (Wagner et al.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory
sequences of the metallothionein gene (Brinster et al., 1982,
Nature 296:39-42); prokaryotic expression vectors such as the
.beta.-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.
Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer,
et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also
"Useful proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; plant expression vectors comprising the nopaline
synthetase promoter region (Herrera-Estrella et al., Nature
303:209-213) or the cauliflower mosaic virus 35S RNA promoter
(Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and the promoter
of the photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fungi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
7:425-515); insulin gene control region which is active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444), mouse mammary tumor virus control region which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., 1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region which is active in the liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94; myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-286), and gonadotropic releasing hormone gene
control region which is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-1378). The promoter element which is
operatively linked to the nucleic acid encoding chemokines,
chemokine derivatives and/or chemokine analogs can also be a
bacteriophage promoter with the source of the bacteriophage RNA
polymerase expressed from a gene for the RNA polymerase on a
separate plasmid, e.g., under the control of an inducible promoter,
for example, the nucleic acid encoding chemokine, derivative, or
analog, operatively linked to the T7 RNA polymerase promoter with a
separate plasmid encoding the T7 RNA polymerase.
[0094] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired.
Expression from certain promoters can be elevated in the presence
of certain inducers; thus, expression of the genetically engineered
chemokines, chemokine derivatives and/or chemokine analogs may be
controlled. Furthermore, different host cells have characteristic
and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, phosphorylation of proteins. Appropriate cell lines
or host systems can be chosen to ensure the desired modification
and processing of the foreign protein expressed. For example,
expression in a bacterial system can be used to produce an
unglycosylated core protein product. Expression in yeast will
produce a glycosylated product. Expression in mammalian cells can
be used to ensure "native" glycosylation of a heterologous protein.
Furthermore, different vector/host expression systems may effect
processing reactions to different extents.
[0095] The chemokine-encoding nucleic acid sequence can be mutated
in vitro or in vivo, to create and/or destroy translation,
initiation, and/or termination sequences, or to create variations
in coding regions. Any technique for mutagenesis known in the art
can be used, including but not limited to, in vitro site-directed
mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem 253:6551),
use of TAB.sub.7 linkers (Pharmacia), etc.
[0096] The experimentation involved in mutagenesis consists
primarily of site-directed mutagenesis followed by phenotypic
testing of the altered gene product. Some of the more commonly
employed site-directed mutagenesis protocols take advantage of
vectors that can provide single stranded as well as double stranded
DNA, as needed. Generally, the mutagenesis protocol with such
vectors is as follows. A mutagenic primer, i.e., a primer
complementary to the sequence to be changed, but consisting of one
or a small number of altered, added, or deleted bases, is
synthesized. The primer is extended in vitro by a DNA polymerase
and, after some additional manipulations, the now double-stranded
DNA is transfected into bacterial cells. Next, by a variety of
methods, the desired mutated DNA is identified, and the desired
protein is purified from clones containing the mutated sequence.
For longer sequences, additional cloning steps are often required
because long inserts (longer than 2 kilobases) are unstable in
those vectors. Protocols are known to one skilled in the art and
kits for site-directed mutagenesis are widely available from
biotechnology supply companies, for example from Amersham Life
Science, Inc. (Arlington Heights, Ill.) and Stratagene Cloning
Systems (La Jolla, Calif.).
[0097] In other specific embodiments, the chemokine derivative or
analog may be expressed as a fusion, or chimeric protein product
(comprising the protein, fragment, analog, or derivative joined via
a peptide bond to a heterologous protein sequence (of a different
protein)). Such a chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other by methods known in the art, in the proper
coding frame, and expressing the chimeric product by methods
commonly known in the art.
[0098] In addition, chemokines, derivatives (including fragments
and chimeric proteins), and analogs can be chemically synthesized.
See, e.g., Clark-Lewis et al., 1991, Biochem. 30:3128-3135 and
Merrifield, 1963, J. Amer. Chem. Soc. 85:2149-2156. For example,
chemokines, derivatives and analogs can be synthesized by solid
phase techniques, cleaved from the resin, and purified by
preparative high performance liquid chromatography (e.g., see
Creighton, 1983, Proteins, Structures and Molecular Principles, W.
H. Freeman and Co., N.Y., pp. 50-60). Chemokines, derivatives and
analogs can also be synthesized by use of a peptide synthesizer.
The composition of the synthetic peptides may be confirmed by amino
acid analysis or sequencing (e.g., the Edman degradation procedure;
see Creighton, 1983, Proteins, Structures and Molecular Principles,
W. H. Freeman and Co., N.Y., pp. 34-49). Furthermore, if desired,
nonclassical amino acids or chemical amino acid analogs can be
introduced as a substitution or addition into the chemokines,
chemokine derivatives and/or chemokine analogs. Non-classical amino
acids include but are not limited to the D-isomers of the common
amino acids, 2,4-diaminobutyric acid, .alpha.-amino isobutyric
acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, .gamma.-Abu,
.epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer
amino acids such as .beta.-methyl amino acids, C.sub..alpha.-methyl
amino acids, N.sub..alpha.-methyl amino acids, and amino acid
analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[0099] By way of example but not by way of limitation, proteins
(including peptides) of the invention can be chemically synthesized
and purified as follows: chemokines, derivatives and analogs can be
synthesized by employing the N-.alpha.-9-fluorenylmethyloxycarbonyl
or Fmoc solid phase peptide synthesis chemistry using a Rainin
Symphony Multiplex Peptide Synthesizer. The standard cycle used for
coupling of an amino acid to the peptide-resin growing chain
generally includes: (1) washing the peptide-resin three times for
30 seconds with N,N-dimethylformamide (DMF); (2) removing the Fmoc
protective group on the amino terminus by deprotection with 20%
piperdine in DMF by two washes for 15 minutes each, during which
process mixing is effected by bubbling nitrogen through the
reaction vessel for one second every 10 seconds to prevent
peptide-resin settling; (3) washing the peptide-resin three times
for 30 seconds with DMF; (4) coupling the amino acid to the peptide
resin by addition of equal volumes of a 250 mM solution of the Fmoc
derivative of the appropriate amino acid and an activator mix
consisting or 400 mM N-methylmorpholine and 250 mM
(2-(1H-benzotriazol-1-4))-1,1,3,3-tetrameth- yluronium
hexafluorophosphate (HBTU) in DMF; (5) allowing the solution to mix
for 45 minutes; and (6) washing the peptide-resin three times for
30 seconds of DMF. This cycle can be repeated as necessary with the
appropriate amino acids in sequence to produce the desired
polypeptide. Exceptions to this cycle program are amino acid
couplings predicted to be difficult by nature of their
hydrophobicity or predicted inclusion within a helical formation
during synthesis. For these situations, the above cycle can be
modified by repeating step 4 a second time immediately upon
completion of the first 45 minute coupling step to "double couple"
the amino acid of interest. Additionally, in the first coupling
step in polypeptide synthesis, the resin can be allowed to swell
for more efficient coupling by increasing the time of mixing in the
initial DMF washes to three 15 minute washes rather than three 30
second washes. After polypeptide synthesis, the peptide can be
cleaved from the resin as follows: (1) washing the
polypeptide-resin three times for 30 seconds with DMF; (2) removing
the Fmoc protective group on the amino terminus by washing two
times for 15 minutes in 20% piperdine in DMF; (3) washing the
polypeptide-resin three times for 30 seconds with DMF; and (4)
mixing a cleavage cocktail consisting of 95% trifluoroacetic acid
(TFA), 2.4% water, 2.4% phenol, and 0.2% triisopropysilane with the
polypeptide-resin for two hours, then filtering the peptide in the
cleavage cocktail away from the resin, and precipitating the
peptide out of solution by addition of two volumes of ethyl ether.
To isolate the polypeptide, the ether-peptide solution can be
allowed to sit at -20.sub.EC for 20 minutes, then centrifuged at
6,000.times.G for 5 minutes to pellet the polypeptide, and the
polypeptide can be washed three times with ethyl ether to remove
residual cleavage cocktail ingredients. The final polypeptide
product can be purified by reversed phase high pressure liquid
chromatography (RP-HPLC) with the primary solvent consisting of
0.1% TFA and the eluting buffer consisting of 80% acetonitrile and
0.1% TFA. The purified polypeptide can then be lyophilized to a
powder.
[0100] In one embodiment, the polypeptide is a cyclic peptide.
Cyclized polypeptides can be prepared by any method known in the
art. For example, but not by way of limitation, disulfide bridge
formation can be achieved by (1) dissolving the purified peptide at
a concentration of between 0.1.-0.5 mg/ml in 0.01 M ammonium
acetate, pH 7.5; (2) adding 0.01 M potassium ferricyanide to the
dissolved peptide dropwise until the solution appears pale yellow
in color and allowing this solution to mix for 24 hours; (3)
concentrating the cyclized polypeptide to 5-10 ml of solution,
repurifying the polypeptide by reverse phase-high pressure liquid
chromatography (RP-HPLC) and finally lyophilizing the polypeptide.
In a specific embodiment, wherein the polypeptide does not contain
two appropriately situated cysteine residues, cysteine residues can
be introduced at the amino-terminus and/or carboxy-terminus and/or
internally such that the polypeptide to be cyclized contains two
cysteine residues spaced such that the residues can form a
disulfide bridge. Alternatively, a cyclic polypeptide can be
obtained by generating an amide linkage. An amide linkage can be
achieved by, for example, but not limited to, the following
procedure: An allyl protected amino acid, such as aspartate,
glutamate, asparagine or glutamine, can be incorporated into the
polypeptide as the first amino acid, and then the remaining amino
acids coupled on. The allyl protective group can be removed by a
two hour mixing of the polypeptide-resin with a solution of
tetrakistriphenylphophine palladium (0) in a solution of chloroform
containing 5% acetic acid and 2.5% N-methylmorpholine. The
polypeptide resin can be washed three times with 0.5%
N,N-diisopropylethylamine (DIEA) and 0.5% sodium
diethyldithiocabamate in DMF. The amino terminal Fmoc group on the
polypeptide chain can be removed by two incubations for 15 minutes
each in 20% piperdine in DMF, and washed three times with DMF for
30 seconds each. The activator mix, N-methylmorpholine and HBTU in
DMF, can be brought onto the column and allowed to couple the free
amino terminal end to the carboxyl group generated by removal of
the allyl group to cyclize the polypeptide. The polypeptide can
cleaved from the resin as described in the general description of
chemical polypeptide synthesis above and the polypeptide purified
by reverse phase-high pressure liquid chromatography (RP-HPLC). In
a specific embodiment, wherein the polypeptide to be cyclized does
not contain an allyl protected amino acid, an allyl protected amino
acid can be introduced into the sequence of the polypeptide, at the
amino-terminus, carboxy-terminus or internally, such that the
polypeptide can be cyclized.
[0101] In another embodiment, a polypeptide that is a chemokine or
derivative thereof is synthesized to contain disulfide brides
corresponding to those observed in the native chemokine. Techniques
known in the art may be applied to achieve selective disulfide
bridge formation, including but not limited to the use of cysteine
residues that having different protection groups. For example, full
length RANTES may be synthesized as described above, but
incorporating acetamidomethyl (Acm) protected cysteine amino acids
at positions 10 and 37 of the polypeptide. After synthesis is
completed, the polypeptide is cleaved off the support resin. The
cleavage, deprotection reaction is for 2 hours in 88%
trifluoracetic acid, 5% water, 5% phenol, and 2%
triisopropylsilane. This step additionally deprotects the initial
trityl groups from Cys11 and Cys50. The polypeptides are purified
by reverse phase HPLC to >95% purity. The polypeptide is diluted
in 0.1M NaOH, pH 8.0 at a concentration of 0.25 mg/ml and allowed
to mix 24 hours at room temperature to create a disulfide bridge
between amino acids Cys11 and Cys50. Disulfide linked polypeptide
is repurified by reverse phase HPLC. The polypeptide is dissolved
in 80% acetic acid at a concentration of 0.25 mg/ml and solid
iodine is added at a concentration of 0.25 mg/ml and allowed to mix
for 24 hours at room temperature. This step deprotects the Acm
groups from Cys10 and Cys34 amino acids to form the final disulfide
bridge. An equal volume of water is added to the mixture which is
extracted four times with 50 mls of CCl.sub.4 to remove the iodine.
The product, now containing disulfide bridges between amino acids
Cys11 and Cys50 and between amino acids Cys10 and Cys34, is
lyophilized.
[0102] The chemokines, derivatives, or analogs of the invention may
be synthesized in their entirety by the sequential addition of
amino acid residues or alternatively as fragment subcomponents
which may be combined using techniques well known in the art, such
as, for example, fragment condensation (Shin et al., 1992, Biosci.
Biotech. Biochem. 56:404-408; Nyfeler et al., 1992, Peptides, Proc.
12th Amer. Pep. Soc., Smith and Rivier (eds), Leiden, pp 661-663);
and Nokihara et al., 1990, Protein Research Foundation, Yanaihara
(ed), Osaka, pp 315-320).
[0103] Also included within the scope of the invention are
chemokines, derivatives, and analogs which are differentially
modified during or after synthesis, e.g., by benzylation,
glycosylation, acetylation, phosphorylation, amidation, pegylation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. In specific embodiments, the chemokines, derivatives, or
analogs are acetylated at the N-terminus and/or amidated at the
C-terminus. Any of numerous chemical modifications may be carried
out by known techniques, including but not limited to acetylation,
formylation, oxidation, reduction; metabolic synthesis in the
presence of tunicamycin; etc. These modifications may serve to
increase the stability, bioavailability and/or inhibitory action of
the peptides of the invention.
[0104] In a less preferred embodiment, chemokine derivatives can be
obtained by proteolysis of the chemokine followed by purification
using standard methods such as those described above (e.g.,
immunoaffinity purification).
[0105] Any of the chemokines, derivatives or analogs described
above may, additionally, have a non-peptide macromolecular carrier
group covalently attached to its amino and/or carboxy termini. Such
macromolecular carrier groups may include, for example, lipid-fatty
acid conjugates or carbohydrates.
[0106] 7.3 Assays for Receptor Binding and Inhibition of Viral
Infection or Replication by Chemokine Proteins, Derivatives and
Analogs
[0107] The ability of chemokines or the derivatives or analogs
thereof to bind chemokine receptors and thereby interfere with
viral infection or replication can be assayed by various
methods.
[0108] In a preferred embodiment, the chemokine derivatives
(including fragments and chimeric proteins) or analogs, bind
protein sequences contained in the extracellular domain of a
chemokine receptor. Binding can be assayed by means well-known in
the art. For example, bioassays may be performed wherein cells
known to be expressing a chemokine receptor are exposed to the
chemokine derivative or analog to be tested and assayed for a known
effect (e.g., signal transduction). Alternatively, chemokines,
derivatives or analogs can be tested for the ability to bind
chemokine receptors by procedures, including but not limited to,
protein affinity chromatography, affinity blotting,
immunoprecipitation, cross-linking, and library based methods such
as protein probing, phage display, and the two-hybrid system (see,
generally, Phizicky et al., 1995, Microbiol. Rev. 59:94-123).
Further, where DNA encoding a chemokine receptor has been
identified, this sequence may be routinely manipulated in known
assays to identify chemokine derivatives or analogs which bind to
the extracellular domain of the receptor. Such assays include, but
are limited to, in vitro cell aggregation and interaction trap
assays. Nucleic acids encoding CC CKR-1 (Neote et al., 1993, Cell
72:415-425); CC CKR-2A and CC CKR-2B (Chavo et al., 1994, Proc.
Natl. Acad. Sci. 91:2752-2756); CC CKR-3 (Daugherty et al., 1996,
J. Exp. Med. 183:2349-2354 and Ponath et al., 1996, J. Exp. Med.
183:1-12); CC CKR-4 (Power et al., 1995, J. Biol. Chem.
270:19495-19500); CC CKR-5 (Samson et al., 1996, Biochemistry
35:3362-3367); C.times.C CKR4 (Feng et al., 1996, Science
272:872-877); IL-8RA and IL-8RB (Kunz et al., 1991, J. Biol. Chem.
267:9101-9106 and Gerard et al., 1994, Corr. Opin. Immunol.
6:140-145); Duffy antigen (Horuk et al., 1994, J. Biol. Chem.
269:1770-1773; Neote et al., 1994, Blood 84:44-52; and Neote et
al., 1993, J. Biol. Chem. 268:12247-12249); and Mig receptor and
.gamma.IP-10 receptor (Loestcher et al., 1996, J. Exp. Med.
184(3):963-969) have been isolated and cloned.
[0109] High throughput screening for chemokines, chemokine
derivatives and/or chemokine analogs receptor binding may be
performed by methods known in the art, including but not limited to
flow cytometry. According to this method, cells that express human
CD4 and one of the HIV co-receptors (e.g., CC CKR-5, C.times.C
CKR4, etc.) are treated with biotinylated chemokine, derivative, or
analog and cell surface binding to each cell type is detected with
an avidin FITC conjugate. Alternatively, other methods for labeling
or detecting binding of the chemokines, chemokine derivatives
and/or chemokine analogs, such as antibodies, may be used. The same
flow cytometry system may be used to assess receptor binding
specificity, by testing for competitive binding between the
chemokines, chemokine derivatives and/or chemokine analogs and
known ligands.
[0110] The antiviral activity exhibited by the chemokine,
derivative and/or analog of the invention may be measured, for
example, by easily performed in vitro assays, which can test the
compound's ability to inhibit syncytia formation or to inhibit
infection by cell-free virus and assess the effects of the compound
on cell proliferation and viability. Applying these assays, the
relative antiviral activity that a chemokine, derivative and/or
analog exhibits against a given virus or strain of immunodeficiency
virus and chemokine, derivative, and/or analog combination
formulation best suited for viral and strain specific inhibitory
activity can be determined.
[0111] In one embodiment, a cell fusion assay is used to test the
ability of chemokines, chemokine derivatives and/or chemokine
analogs, to inhibit HIV-induced syncytia formation in vitro. Such
an assay involves culturing uninfected CD4.sup.+ cells in the
presence of chronically HIV-infected cells and the composition
containing a chemokines, chemokine derivatives and/or chemokine
analogs to be assayed. For each, a range of concentrations may be
tested. This range should include a control culture wherein no
chemokine, derivative and/or analog has been added. Standard
conditions for culturing, well known to those of ordinary skill in
the art, are used. After incubation for an appropriate period, such
as, for example, 24 hours at 37.sub.EC, the culture is examined
microscopically for the presence of multinucleated giant cells,
which are indicative of cell fusion and syncytia formation.
[0112] In one embodiment, an in vitro cell-free infectivity assay
is performed using primary macrophages and the macrophage-tropic
isolate HIV-1.sub.BaL, the first described macrophage-tropic HIV-1
isolate (see, Gartner et al., 1986, Science 233:215). According to
this assay, primary macrophage cells isolated according to methods
known in the art are infected with HIV-1.sub.BaL that has been
propagated and maintained only in primary macrophages. The input
immunodeficiency virus is incubated with primary macrophages in the
presence of concentrations of the chemokine, derivative, or analog
to be tested. After a defined period of infection, unbound virus is
removed by washing, and the cells are placed in culture. The level
of virus replication in this assay may be assessed by techniques
known in the art, including but not limited to, measuring reverse
transcriptase (RT) levels, or the release of extracellular p24 core
antigen at different days post-infection. A constant level of
inhibition of viral infection or replication is determined by
measuring output HIV p24 levels (or another indicator of viral
infection or replication, such as for example, RT) relative to
control assays performed in the absence of the chemokines,
chemokine derivatives and/or chemokine analogs. Preferably, the
chemokine derivative or analog reduces levels of virus, as measured
by, for example, p24, by 50% relative to control assays carried out
in the absence of test compound. The presence of p24 may be
determined using methods known in the art, such as commercially
available enzyme-linked immunosorbent assays (Coulter, Hialeah,
Fla.; Abbott Laboratories, Hvalstad, Norway). Alternatively, RT
activity may be tested by monitoring cell-free supernatant using
standard techniques such as those described by, for example, Goff
et al. (Goff et al., 1981, J. Virol. 38:239-248) and Willey et al.
(Willey et al., 1988, J. Virol. 62:139-147).
[0113] In addition to evaluating the antiviral activity of a
chemokines, chemokine derivatives and/or chemokine analogs, the
primary macrophage/HIV-1.sub.BaL cell-free infectivity assay test
system may also be used to determine the ability of combinations of
chemokines, derivatives, and/or analogs to suppress HIV infection
or replication. Furthermore, the assay may routinely be modified to
use other macrophage-tropic strains that have been propagated and
maintained in macrophages to identify chemokines, derivatives
and/or analogs effective in inhibiting infection or replication of
one or multiple M-tropic viral isolates.
[0114] In a preferred embodiment, an in vitro cell-free infectivity
assay is performed using activated primary CD4.sup.+ peripheral
blood mononuclear cells (PBMC's) that have been isolated according
to methods known in the art; such as for example, (+) or (-)
selection by immunomagnetic beads (Dynal A. S., Norway) and Ficoll
gradient centrifugation. Techniques for activating primary PBMC
with such compounds as phytohemagglutinin (PHA) or the monoclonal
antibody OKT3 are also known in the art (see, e.g., Cocchi et al.,
1995, Science, 270:1811-1815). The activated primary PBMC are
incubated with 10-50 TCID.sub.50 (half-maximal tissue-culture
infectious dose) primary syncytia-inducing or
non-syncytia-inducing, T-tropic, viral stocks that have been
obtained from the NIH AIDS Research and Reference Reagent Program
or isolated according to methods known in the art, such as for
example, that described in herein. Primary virus stocks may also be
generated from lymph node T cells (via lymph node aspirate or
biopsy). The procedure for isolating virus from lymph node material
is the same as that used to isolate virus from PBMC's.
[0115] As above, the input immunodeficiency virus is incubated with
target cells in the presence of various quantities of the test
chemokine, derivative, or analog to be tested. After a defined
period of infection, unbound virus is removed by washing, and the
cells are placed in culture. As above, the level of virus
replication in this assay may be assessed by techniques known in
the art, including but not limited to, measuring reverse
transcriptase levels or the release of extracellular p24 core
antigen at different days post-infection. A constant level of
inhibition of viral infection or replication is determined by
measuring output HIV p24 levels (or another indicator) relative to
control assays performed in the absence of the chemokines,
chemokine derivatives and/or chemokine analogs. Preferably, the
chemokine derivative or analog reduces levels of virus, as measured
by, for example, p24, by 50% relative to control assays carried out
in the absence of test compound.
[0116] In addition to evaluating the antiviral activity of a
chemokines, chemokine derivatives and/or chemokine analogs, the
primary CD4.sup.+ PBMC/HIV-1 assay may be used to formulate
pharmaceutical compositions containing combinations of chemokines,
derivatives and/or chemokine analogs, effective in inhibiting
infection or replication of the viral isolates assayed and may be
applied to formulate a pharmaceutical composition effective in
inhibiting infection or replication of a plurality of T-tropic
strains.
[0117] In an additional embodiment, an in vitro cell-free
infectivity assay is performed using PM1 cells and HIV.sub.BaL. As
above, the input immunodeficiency virus is incubated with target
cells (PM1) in the presence of various quantities of the test
chemokine, derivative, or analog to be tested. After a defined
period of infection, unbound virus is removed by washing, and the
cells are placed in culture. As above, the level of
immunodeficiency virus replication in this assay may be assessed by
techniques known in the art, including but not limited to,
measuring reverse transcriptase levels or the release of
extracellular p24 core antigen at different days post-infection. A
constant level of inhibition of viral infection or replication is
determined by measuring output HIV p24 levels (or another
indicator) relative to control assays performed in the absence of
the chemokines, chemokine derivatives and/or chemokine analogs.
Preferably, the chemokine derivative or analog reduces levels of
virus, as measured by, for example, p24, by 50% relative to control
assays carried out in the absence of test compound.
[0118] In another embodiment, an assay is performed using cells
from HIV.sup.+ individuals. According to this assay, HIV.sup.+
CD4.sup.+ peripheral blood cells are recovered from an infected
individual using techniques known in the art and incubated in the
presence and absence of test chemokines, chemokine derivatives
and/or chemokine analogs. Optionally, the cells are co-cultured
with uninfected allogeneic CD4.sup.+ PBMC's. According to this
assay, the input immunodeficiency virus is incubated with target
cells in the presence of various concentrations of the test
chemokine, derivative, or analog that are maintained throughout
culture. Culture supernatant samples are removed periodically
(every 1-3 days) and tested for virus expression by techniques
known in the art, such as by measuring the release of extracellular
p24 core antigen, or another indicator of viral infection or
replication, at different days post-infection. Virus is usually
detected by day 7 of culturing. A constant level of inhibition of
viral infection or replication is determined relative to control
assays performed in the absence of the chemokines, chemokine
derivatives and/or chemokine analogs. For many individuals with
advanced infection, CD4.sup.- cell levels are very low. In these
cases, CD4.sup.- cells isolated from the HIV.sup.+ individual are
optionally incubated with uninfected CD4.sup.+ target cells. This
assay models the rapid viral replication and cytopathic effects
contributing to the loss of CD4.sup.+ cells in vivo by utilizing
primary target cells and primary viral isolates and is exemplified
herein. In addition to evaluating the antiviral activity of a
chemokines, chemokine derivatives and/or chemokine analogs, this
assay may be used to determine the ability of combinations of
chemokines, derivatives and/or analogs to inhibit transmission of
isolates specific to the patient at a given time.
[0119] In another embodiment, chemokine(s), derivative(s) and/or
analog(s) are identified by their ability to inhibit the isolation
of primary immunodeficiency virus isolates from primary target
cells removed from an infected individual. According to this
embodiment, CD4.sup.- target cells isolated from an HIV.sup.+
individual using techniques known in the art are exposed to one or
more chemokines, derivatives, and/or analogs and known techniques,
such as those described infra, are applied to isolate the virus
from the cells. In a preferred embodiment, these chemokines,
derivatives and/or analogs are known or indicated by the in vitro
assays described herein to inhibit the infection or replication of
one or more HIV-1 strains. Parallel control experiments are
performed wherein the same virus isolation technique is performed
in the absence of chemokines, derivatives, and/or analogs. An
inability or reduced ability to isolate immunodeficiency virus in
the test samples, but not the control sample indicates that the
primary immunodeficiency virus isolates are sensitive to the test
chemokines, derivatives, and/or analogs.
[0120] The chemokine protein, derivative, or analog compositions
may then be combined with suitable pharmaceutically acceptable
carriers and administered by techniques known in the art, such as
those described herein.
[0121] Techniques known in the art may be applied to formulate
compositions displaying minimal toxicity. For each in vitro test of
chemokines, derivatives and/or analogs of the invention, it is
important to determine the effects on cell proliferation and
viability. Methods for assessing effects of the compounds tested on
cell proliferation include, but are not limited to, assaying for
thymidine uptake and counting cells (using, for example, a
hemocytometer or flow cytometer). Methods for assessing cell
viability include, but are not limited to, trypan blue dye
exclusion. In a specific embodiment, an assay is performed wherein
the proliferative response of stimulated target cells to a range of
concentrations of the test composition(s) is assessed by monitoring
[.sup.3H]-Thymidine incorporation.
[0122] Other methods for assaying the antiviral activity of
chemokines, derivatives and/or analogs will be known to the skilled
artisan and are within the scope of the invention.
[0123] The assays described herein may be applied to routinely
predict which chemokines, chemokine derivatives and/or chemokine
analogs will display an antiviral effect in vivo and the optimal
concentration for doing so. Chemokines, derivatives, and analogs
displaying anti-viral activity are optionally combined.
[0124] The in vitro assays described herein can further be applied
to screen numerous primary and established viral isolates with
chemokines, derivatives, and/or analogs for formulation of a
pharmaceutical composition containing as active ingredients,
chemokines, derivatives, and/or analogs that are able to inhibit
infection or replication of a plurality of viral isolates. In a
specific embodiment, this pharmaceutical composition inhibits
infection or replication of at least 3, 4, 5, 6, 7, 8, 9, 10, 15,
or 20 distinct immunodeficiency virus isolates. While the
pharmaceutical composition formulated according to this method is
ideally suited for prophylactic administration, therapeutic
administration of the pharmaceutical composition is also
envisioned.
[0125] The invention provides for treatment or prevention of
diseases and disorders associated with infection by an
immunodeficiency virus, particularly, HIV, by administration of a
therapeutic compound ("therapeutic"). The therapeutics of the
invention are as described herein, e.g., chemokines and
therapeutically and/or prophylactically effective chemokine
derivatives and/or analogs, i.e., those derivatives and analogs
which prevent or treat HIV infection (e.g. as demonstrated in vitro
assays described infra), as well as nucleic acids encoding such
chemokines, derivatives and analogs thereof.
[0126] The invention also provides methods for treating or
preventing viral infections, by administering an effective amount
of a therapeutic of the invention that binds to one or more
chemokine receptors. In one embodiment, the therapeutic is a
chemokine, derivative, or analog that binds to an .alpha. and
.beta. chemokine receptor. In further embodiments, the therapeutic
is a chemokine, chemokine derivative and/or chemokine analog that
binds to 3, 4, 5, 6, 7, 8, 9, or 10 chemokine receptors. In a
preferred embodiment, the therapeutic is able to bind CC CKR-5 and
C.times.C CKR4. In another embodiment, the pharmaceutical
composition contains a plurality of therapeutic chemokines,
derivatives, and/or analogs.
[0127] The therapeutics of the invention can also be tested in vivo
for toxicity and/or the desired therapeutic or prophylactic
activity. For example, such compounds can be tested in suitable
animal models including but not limited to rats, mice, chickens,
cows, sheep, dogs, cats, monkeys, apes, rabbits, etc. For in vivo
testing, prior to administration to humans, any animal model system
known in the art may be used.
[0128] 7.4 Formulating a Patient-Specific Pharmaceutical
Composition
[0129] In particular embodiments, the invention provides methods
for formulating a pharmaceutical composition which comprises the
chemokines, derivatives, and/or analogs that inhibit replication
and/or infection of immunodeficiency virus isolate(s) found in an
individual at a given time and preferably does not comprise
chemokines or derivatives and/or analogs thereof that do not
inhibit replication and/or infection of such isolate(s). Such
methods are achieved by isolating primary immunodeficiency
virus(es) from PBMC's and/or lymph nodes of a patient, and testing
the virus(es) against a panel of chemokines (or derivatives or
analogs thereof) to determine which particular chemokines are
active in inhibiting the infection and/or replication of such viral
isolate(s). The chemokines thereby that are identified as having
activity in inhibiting replication and/or infection of the
immunodeficiency virus isolate(s) are then used as components of a
pharmaceutical composition comprising them to treat the
patient.
[0130] The ability to formulate a therapeutic composition to
contain only those chemokines, derivatives, and/or analogs that are
effective in inhibiting viral infection or replication in the
patient and further, to contain only those compounds demonstrating
high viral infection or replication inhibiting activity is
extremely valuable since the risk of serious side effects increases
with chemokine concentration.
[0131] Techniques that can be used for isolating a primary
immunodeficiency virus from PBMC's and/or lymph nodes of a patient
are known in the art. In one embodiment, primary viruses are
propagated in allogeneic CD4.sup.+ PBMC's prior to isolation, and
then tested in in vitro assays, such as those described infra which
use primary macrophages and CD4.sup.+ PBMC's as target cells, for
inhibiting viral infection or replication in the presence of
chemokines, derivatives and/or analogs. In specific embodiments,
the immunodeficiency virus isolate is HIV.
[0132] Once a immunodeficiency virus is isolated, the ability of
one, two, three, or more, preferably a panel of at least 5
chemokines, derivatives, and/or analogs to inhibit infection or
replication of the isolate may be tested according to the assays
described herein. Chemokines, derivatives, and analogs found to be
effective at inhibiting infection or replication of the
immunodeficiency virus isolate are preferably then tested over a
range of concentrations to determine optimum antiviral
concentrations using techniques known in the art.
[0133] In a specific embodiment, the pharmaceutical composition of
the invention comprises a plurality of chemokines, derivatives,
and/or analog determined to be effective in inhibiting infection
and/or replication of an immunodeficiency virus isolate of
interest. In particular the pharmaceutical composition comprises 2,
3, 4, 5, 6, 7, 8, 9, or 10 chemokines, derivatives, and or analogs
determined to have antiviral activity. Assays described infra, may
be used to determine optimum relative concentrations of the
chemokine, derivative, and/or analog components of the
pharmaceutical composition.
[0134] The invention therefore provides methods by which to
identify chemokine(s), derivative(s), or analog(s) that inhibit
infection or replication of a specific isolate of an
immunodeficiency virus, particularly an HIV-1 isolate, and by which
pharmaceutical compositions containing these chemokines or
therapeutically or prophylactically effective derivatives, or
analogs, alone or in combination, are routinely formulated. The
invention further provides methods for treating or preventing
immunodeficiency virus infections, in particular HIV infection, in
mammals, including humans, by administering the therapeutic
compositions of the invention.
[0135] The invention thus provides methods for formulating on a
patient-to-patient basis, a pharmaceutical composition comprising
chemokines, derivatives and/or analogs that are known to be
effective against isolate(s) of an immunodeficiency virus present
in an individual at a given time.
[0136] 7.5 Therapeutic Uses
[0137] The invention provides for treatment or prevention of
diseases and disorders associated with infection by an
immunodeficiency virus, particularly, HIV, by administration of a
therapeutic of the invention. Such therapeutics include, but are
not limited to: chemokines and therapeutically and prophylactically
effective chemokine derivatives and/or analogs, i.e., those
derivatives and analogs which prevent or treat HIV infection (e.g.
as demonstrated in vitro assays described infra), as well as
nucleic acids encoding such chemokines, derivatives and analogs
thereof (e.g., for use in gene therapy). Examples of therapeutics
are those chemokines, derivatives and analogs described herein and
nucleic acids encoding such proteins. Preferred assays to determine
the utility of a specific therapeutic and whether its
administration is indicated for treatment are described herein.
[0138] A preferred embodiment of the invention is directed to
methods of using a therapeutic for treatment and prevention of HIV
infection, preferably HIV-1 infection, in a human subject.
[0139] Therapeutic compositions of the invention have application
in treating and preventing disorders associated with
immunodeficiency viruses, including but not limited to types of
HIV, e.g., HIV-1 and HIV-2. A preferred embodiment of the invention
relates to methods of using a therapeutic for treatment or
prevention of HIV infection, preferably HIV-1, in a human subject.
In the treatment of HIV infection, the therapeutic of the invention
can be used to prevent progression of HIV-1 infection to acquired
immune deficiency syndrome AIDS or to AIDS-related complex (ARC) in
a human patient, or to treat a human patient with ARC or AIDS.
[0140] Therapeutic compositions of the invention also have
application in treating and preventing disorders associated with
non-human immunodeficiency viruses, including but not limited to
simian immunodeficiency virus.
[0141] In vitro assays which can be used to determine whether
administration of a specific composition containing one or more
chemokines, derivatives or analogs, inhibits viral infection or
replication are discussed infra. These assays can indicate which
chemokine, derivative, or analog has the desired therapeutic
efficacy in inhibiting infection or replication of a particular
viral isolate and additionally may be used to formulate the
appropriate pharmaceutical combination of chemokines, derivatives,
and/or analogs that demonstrates antiviral activity against one or
multiple viral strains.
[0142] In a specific embodiment, the therapeutic method of the
invention is carried out as monotherapy, i.e., as the only agent
provided for treatment or prevention of HIV. In another embodiment,
the therapeutic is administered in combination with one or more
anti-viral compounds, for example, protease inhibitors (e.g.,
sequinavir) and/or reverse transcriptase inhibitors (e.g.,
azidothymidine (AZT), lamioridine (3TC), dideoxyinosine (ddI),
dideoxycytidine (ddC)). The therapeutic may also be administered in
conjunction with chemotherapy (e.g., treatment with adriamycin,
bleomycin, vincristine, vinblastine, doxorubicin and/or Taxol) or
other therapies known in the art.
[0143] 7.5.1 Gene Therapy
[0144] In a specific embodiment, nucleic acids comprising a
sequence encoding chemokine, protein derivative or protein analog,
effective at inhibiting HIV replication and/or infection in vitro
are administered for treatment or prevention of HIV infection, by
way of gene therapy. Gene therapy refers to therapy performed by
the administration of a nucleic acid to a subject. In this
embodiment of the invention, the nucleic acid produces its encoded
protein that mediates a therapeutic effect by preventing or
treating HIV infection. For example, any of the methods for gene
therapy available in the art can be used according to the present
invention. Exemplary methods are described below.
[0145] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215. Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0146] In a preferred aspect, the nucleic acid encoding chemokines,
chemokine derivatives and/or chemokine analogs is part of an
expression vector that produces chemokines, chemokine derivatives
and/or chemokine analogs in a suitable host. In particular, such a
nucleic acid has a promoter operably linked to the nucleic acid
sequence coding for chemokines, chemokine derivatives and/or
chemokine analogs, said promoter being inducible or constitutive,
and, optionally, tissue-specific. In another particular embodiment,
a nucleic acid molecule is used wherein the chemokine, derivative,
or analog sequences and any other desired sequences are flanked by
regions that promote homologous recombination at a desired site in
the genome, thus providing for intrachromosomal expression of
chemokine, derivative, or analog (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0147] Delivery of the nucleic acid into a patient may be either
direct, wherein case the patient is directly exposed to the nucleic
acid or nucleic acid-carrying vector, or indirect, wherein case,
cells are first transformed with the nucleic acid in vitro, then
administered to the patient. These two approaches are known,
respectively, as in vivo or ex vivo gene therapy.
[0148] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering it in linkage to a peptide which
is known to enter the cell or nucleus, e.g., by administering it in
linkage to a ligand subject to receptor-mediated endocytosis (see
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be
used to target cell types specifically expressing the receptors),
etc. In a specific embodiment, the nucleic acid can be targeted in
vivo for cell specific uptake and expression, by targeting a
specific receptor (see, e.g., PCT Publications WO 92/06180 dated
Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson
et al.); WO 92/20316 dated Nov. 26, 1992 (Findeis et al.); WO
93/14188 dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated
Oct. 14, 1993 (Young)). In another embodiment, a nucleic
acid-ligand complex can be formed wherein the ligand comprises a
fusogenic viral peptide to disrupt endosomes, allowing the nucleic
acid to avoid lysosomal degradation. Alternatively, the nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination (Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al., 1989, Nature 342:435-438).
[0149] In a specific embodiment, a viral vector that contains the
nucleic acid sequence encoding a chemokines, chemokine derivatives
and/or chemokine analogs is used. For example, a retroviral vector
can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599).
These retroviral vectors have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome.
Retroviral vectors are maintained in infected cells by integration
into genomic sites upon cell division. The nucleic acid to be used
in gene therapy is cloned into the vector, which facilitates
delivery of the gene into a patient. More detail about retroviral
vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302,
which describes the use of a retroviral vector to deliver the mdr1
gene to hematopoietic stem cells in order to make the stem cells
more resistant to chemotherapy. Other references illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., 1994,
J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0150] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234.
[0151] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300.) Herpes viruses are other viruses that can also be
used.
[0152] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0153] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including, but not limited to, transfection,
electroporation, microinjection, infection with a viral vector
containing the nucleic acid sequences, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see e.g.,
Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al.,
1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0154] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In a preferred
embodiment, recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are administered intravenously. Additionally,
epithelial cells can be injected, e.g., subcutaneously, or
recombinant skin cells (e.g., keratinocytes) may be applied as a
skin graft onto the patient. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0155] In an embodiment wherein recombinant cells are used in gene
therapy, a nucleic acid sequence coding for chemokine, or
therapeutically or prophylactically effective derivative, or analog
is introduced into the cells such that it is expressible by the
cells or their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect. In a specific
embodiment, stem or progenitor cells, preferably hematopoietic stem
or progenitor cells, are used. Any stem and/or progenitor cells
which can be isolated and maintained in vitro can potentially be
used in accordance with this embodiment of the present
invention.
[0156] 7.5.2 Demonstration of Therapeutic Utility
[0157] The therapeutics of the invention are preferably tested in
vitro, and then in vivo for the desired therapeutic or prophylactic
activity and/or for toxicity, prior to use in humans. While any in
vitro or in vivo assays known in the art may be utilized to test
the efficacy of a therapeutic of the invention, it is preferred
that such is determined by applying one or more of the in vitro
assays described infra.
[0158] 7.5.3 Prophylactic Uses
[0159] The therapeutics of the invention can be administered to
prevent viral replication or infection. The prophylactic methods of
the invention can be used not only to prevent viral infection, but
also to prevent post-infection viral replication or further
infection that precedes disease development. It is particularly
envisioned that administration can follow shortly after an
individual engages in behavior that may expose such individual to
the viral agent or otherwise render the individual at high risk for
developing an immunodeficiency virus infection. Administration of
the compositions of the invention may be used as a prophylactic
measure in previously uninfected individuals after acute exposure
to an HIV virus. Examples of such prophylactic use of the
therapeutic of the invention may include, but is not limited to,
prevention of immunodeficiency virus transmission from mother to
fetus or infant (e.g., at parturition or through breast milk) and
other settings where the likelihood of HIV transmission exists,
such as, for example, accidents in health care settings wherein
workers are exposed to HIV-containing blood products. Such
administration is indicated where the therapeutic is shown in
assays, as described supra, to have utility for treatment or
prevention of the transmission of one or more immunodeficiency
virus strains, preferably HIV.
[0160] 7.6 Therapeutic/Prophylactic Compositions and Methods of
Administering
[0161] The invention provides methods of treatment and prevention
by administration to a subject wherein such treatment or prevention
is desired a therapeutically or prophylactically effective amount
of a therapeutic of the invention. The subject is preferably an
animal, including, but not limited to, animals such as monkeys,
sheep, cows, pigs, horses, chickens, cats, dogs, etc., and is
preferably a mammal, and most preferably human. The subject can be
a fetus, child, or adult. In a preferred aspect, the therapeutic is
substantially purified.
[0162] Formulations and methods of administration that can be
employed when the therapeutic comprises a nucleic acid are
described above; additional appropriate formulations and routes of
administration can be selected from among those described
hereinbelow.
[0163] Various delivery systems are known and can be used to
administer a therapeutic of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the therapeutic, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
of a therapeutic nucleic acid as part of a retroviral or other
vector, etc. Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
may be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
therapeutic compositions of the invention into the central nervous
system by any suitable route, including intraventricular and
intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent.
[0164] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved, for example and
not by way of limitation, by topical application, by injection, by
means of a catheter, by means of a suppository, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or
fibers.
[0165] In another embodiment, the therapeutic can be delivered in a
vesicle, in particular a liposome (see Langer, 1990 Science
249:1527-1533; Treat et al., 1989, in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327;
see generally ibid.)
[0166] In yet another embodiment, the therapeutic can be delivered
in a controlled release system. In one embodiment, a pump may be
used (see Langer, 1990, Science 249:1527-1533; Sefton 1987, CRC
Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980; Surgery
88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, 1974, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability, Drug
Product Design and Performance, 1984, Smolen and Ball (eds.),
Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;
During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 71:105). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Medical
Applications of Controlled Release, 1984, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla., vol. 2, pp. 115-138).
[0167] Other controlled release systems are discussed in the review
by Langer (1990, Science 249:1527-1533).
[0168] In a specific embodiment where the therapeutic is a nucleic
acid encoding a protein therapeutic, the nucleic acid can be
administered by gene therapy methods as described supra.
[0169] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a therapeutic, and a therapeutically acceptable
carrier. In a specific embodiment, the term "therapeutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Therapeutic Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the therapeutic, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0170] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0171] The therapeutics of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0172] The amount of the therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vivo
and/or in vitro assays may optionally be employed to help predict
optimal dosage ranges. The precise dose to be employed in the
formulation will also depend on the route of administration, and
the seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each patient's
circumstances. However, suitable dosage ranges for intravenous
administration are generally about 1-1000 micrograms of active
compound per kilogram body weight. Suitable dosage ranges for
intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0173] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0174] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
8. EXAMPLES
[0175] The invention is illustrated by the following non-limiting
examples.
[0176] 8.1 Primary Macrophage/HIV-1.sub.BaL Cell Free Infectivity
Assay for Chemokine Suppression
[0177] The following assay is used to determine the ability of a
chemokines, chemokine derivatives and/or chemokine analogs to
interfere with the infection or replication of HIV.sub.BaL.
Peripheral blood mononuclear cells (PBMC's) (2.times.10.sup.6) are
added to triplicate assay wells of a 48 well culture plate and
cultured in 10 ng/ml recombinant human GM-CSF to mature the
monocytes into macrophages. After 48 hours the nonadherent cells
are washed away and the adherent cells cultured in GM-CSF for an
additional 96 hours resulting in mature macrophages. The wells are
again washed and then infected with 50 TCID.sub.50 of HIV-1.sub.BaL
(available from the NIH AIDS Research and Reference Reagent
Program) that has been propagated in primary macrophages in the
presence of chemokines, chemokine derivatives and/or chemokine
analogs in a total volume of 200 .mu.l (GM-CSF is no longer
present). Each chemokines, chemokine derivatives and/or chemokine
analogs concentration is tested in triplicate wells. Throughout the
course of the experiment, controls are maintained wherein the cell
medium containing the virus does not contain the test chemokines,
chemokine derivatives and/or chemokine analogs. After an overnight
(18 hour) incubation each well is washed to remove virus and
replenished with fresh medium containing the corresponding amount
of chemokine derivative, or analog. After 48 hours in culture the
cells are refed with 200 .mu.l of fresh medium with the
corresponding concentration of chemokines, chemokine derivatives
and/or chemokine analogs. Infectivity is determined by commercial
HIV-1 p24 antigen capture ELISA on culture well supernatants were
collected 5-7 days post-infection (Coulter, Hialeah, Fla.).
[0178] Reduced levels of virus in test samples as indicated by
reduced levels of p24 in the ELISA relative to the control
indicates that the chemokine, derivative, or analog interferes with
the infection or replication of HIV-1.sub.BaL in primary macrophage
cells at the concentration tested. Preferably, the chemokine
derivative or analog reduces levels of virus, as measured by, for
example, p24, by 50% relative to control assays carried out in the
absence of test compound.
[0179] 8.2 Primary CD4.sup.+ PBMC/Primary HIV-1 Isolate Cell-Free
Infectivity Assay for Chemokine Suppression
[0180] The following assay is used to determine the ability of a
chemokines, chemokine derivatives and/or chemokine analogs to
interfere with the infection or replication of a primary HIV-1
isolate in primary CD4.sup.+ cells. Target cells can either be
peripheral blood mononuclear cells (PBMC's) depleted of CD8.sup.+
cells using anti-CD8 immunomagnetic beads or CD4.sup.+ PBMC's
purified with anti-CD4 immunomagnetic beads. Immunomagnetic bead
depletion/purification protocols are carried out according to
manufacturer's instructions (Dynal A. S., Norway).
[0181] Viruses are isolated according to procedures known in the
art. Briefly, isolates are obtained by co-culturing of 1 to
2.times.10.sup.6 PBMC's from HIV-1 infected individuals with
phytohemagglutinin (PHA)-stimulated PBMC from two HIV-1 negative
blood donors. The cultures are maintained in complete RPMI 1640
medium (Gibco) containing 10% fetal calf serum (FCS), 5U/ml of rIL2
(R & D Systems, Minneapolis, Minn.), 2 .mu.g/ml polybrene
(Sigma, St. Louis, Mo.) and antibiotics. Virus antigen production
is measured in supernatants twice weekly using an HIV-1 p24 antigen
capture ELISA (Coulter, Hialeah, Fla.). Virus stocks are generated
from the p24 antigen capture assay positive supernatants by
passaging of the virus isolates once or twice in PHA stimulated
blood donor PBMC's. The virus containing supernatants are
aliquotted and cryopreserved at -75.sub.EC.
[0182] The primary isolates are titered before use so that known
doses can be assayed. To determine the 50% tissue culture
infectious dose (TCID.sub.50) of virus stocks, the PBMC's from one
donor are activated with PHA and cultured for three days in
complete medium of RPMI. The activated PBMC's are thereafter
aliquotted in fetal calf serum containing 10% DMSO and
cryopreserved at -15.sub.EC until use. At the time of virus stock
titration and/or chemokine inhibition experiments, the activated
PBMC's are thawed and expanded for 2-3 days in complete RPMI 1640
medium. As described in the protocol provided by the manufacturer
(Dynal A. S., Norway), CD8.sup.+ T cells are depleted from the
activated PBMC's using Dynabeads M-450 CD8. CD8.sup.+ T-cell
depleted PBMC's at a concentration of 1.times.10.sup.5 cells per
well in complete medium are seeded in each well in microtiter
plates (96 wells, Nunc, Denmark). Virus stocks are thawed and
serially diluted in five fold steps starting from a dilution of
1:2. Each dilution of virus inoculum prepared in complete medium is
added to the seeded cell suspension in equal volumes following
incubation at 37.sub.EC. After one hour incubation, complete RPMI
1640 is added to each well so that total volume per one well is 250
.mu.l. The old medium is removed and new complete medium is added
at day three post infection. The harvested culture medium is
evaluated for HIV-1 p24 at day seven. The TCID.sub.50 value is
defined as the reciprocal of the virus dilution resulting in 50%
positive wells using Reed-Muench calculation or the Spearman-Karber
equation.
[0183] Phytohemagglutinin (PHA)-activated target cells
(2.times.10.sup.5) are incubated for 1-2 hours with 10-50
TCID.sub.50 of a primary isolate of non-syncytium inducing (NSI) or
syncytium-inducing (SI) HIV-1 (which has been obtained from a
patient as described above and propagated only in primary PBMC's)
in the presence of chemokines, derivatives or analogs in a total
volume of 200-1000 .mu.l.
[0184] Controls consist of wells containing the cells, primary
HIV-1 isolate, and culture medium in place of chemokine derivative
or analog. The cells are then washed to remove virus and
replenished with fresh medium containing the corresponding amount
of chemokine. After 48 hours in culture the cells are refed with
200 .mu.l of fresh medium with the corresponding concentration of
chemokine. Infectivity is determined by HIV-1 p24 antigen capture
ELISA of culture well supernatants were collected 5-7 days
post-infection (Coulter, Hialeah, Fla.).
[0185] Reduced levels of virus in test samples as indicated by
reduced levels of p24 in the ELISA relative to the controls
indicate that the chemokines, chemokine derivatives and/or
chemokine analogs interferes with the infection of the primary HIV
isolate in primary CD4.sup.+ cells.
[0186] 8.3 PM1/HIV-1.sub.BaL Cell-Free Infectivity Assay for
Chemokine Suppression
[0187] The following assay is used to determine the ability of a
chemokines, chemokine derivatives and/or chemokine analogs to
interfere with the infection or replication of HIV-1.sub.BaL in the
CD4.sup.+ T-cell clone (PM1) which is susceptible to both primary
and macrophage-tropic HIV-1 isolates. The PM1/HIV-1.sub.BaL test
system is standardized in 48-well microliter plates using PM1 cells
(available from the NIH AIDS Research and Reference Reagent
Program) acutely infected with HIV-1.sub.BaL. PM1 cells
(2.times.10.sup.5/test) are infected with HIV-1.sub.BaL (10-50
TCID.sub.50/1.times.10.sup.6 cells) for 2 hr at 37.sub.EC, then
washed three times with pre-warmed phosphate buffered saline (PBS)
and resuspended in complete culture medium (250 .mu.l per test)
containing different dilutions of the chemokine, derivative and/or
analog composition to be assayed. At least four untreated controls,
resuspended in complete medium, with or without exogenous
interleukin-2 (IL-2), are always handled in parallel to treated
cultures. The controls do not contain the chemokines, chemokine
derivatives and/or chemokine analogs. At day 3 post-infection, 250
.mu.l of fresh chemokines, chemokine derivatives and/or chemokine
analogs composition containing the same original concentration of
the respective test composition is added to each culture. The level
of virus replication is assessed by measuring the release of
extracellular p24 core antigen at different days postinfection.
Five to nine days postinfection, the cultures are harvested,
centrifuged to remove the cells and tested for HIV-1 p24 antigen by
a commercial ELISA test (Coulter, Hialeah, Fla.).
[0188] Reduced levels of virus in test samples as indicated by
reduced levels of p24 in the ELISA relative to the controls
indicate that the chemokine, derivative, or analog interferes with
HIV.sub.BaL infection of in PM1 cells at the concentration tested.
Preferably, the chemokine derivative or analog reduces levels of
virus, as measured by, for example, p24, by 50% relative to control
assays carried out in the absence of test compound.
[0189] 8.4 Assay of HIV.sup.+ PBMCS for Suppression of HIV in the
Presence of Chemokines
[0190] The following assay is used to determine the ability of a
chemokines, chemokine derivatives and/or chemokine analogs to
interfere with the ability of a primary HIV-1 isolate from HIV
peripheral blood mononuclear cells to replicate and/or infect other
CD4.sup.+ cells.
[0191] CD4.sup.+ T cells (1.times.10.sup.5) from uninfected
individuals (purified with anti-CD4 immunomagnetic beads) or
CD8-depleted PBMC's (cells removed by anti-CD8 immunomagnetic
beads) are incubated with 1000 CD4.sup.+ peripheral blood cells
from the infected individual in the presence of different
concentrations e.g., 1 ng/ml to 1 .mu.g/ml of test chemokines,
chemokine derivatives and/or chemokine analogs in culture wells.
Controls consist of CD4.sup.- infected and non-infected incubations
wherein chemokine has not been added. For many individuals with
advanced infection, CD4.sup.+ T cell levels are very low. In these
cases, as many cells as possible are incubated with the uninfected
CD4.sup.+ target cells. The test chemokine concentration is
maintained throughout the duration of culture. Culture supernatant
samples are removed periodically (every 2-3 days) and tested for
virus expression by commercial HIV-1 p24 antigen capture ELISA.
Virus is usually detected by day 7.
[0192] Reduced levels of virus in the test sample relative to the
CD4.sup.+ infected controls as indicated by reduced levels of p24
in the ELISA indicate that the chemokines, chemokine derivatives
and/or chemokine analogs interferes with infection or replication
of the HIV.sup.+ peripheral blood cell HIV-1 isolate in CD4.sup.+
cells. Preferably, the chemokine derivative or analog reduces
levels of virus, as measured by, for example, p24, by 50% relative
to control assays carried out in the absence of test compound.
[0193] 8.5 Assay for the Effect of Compositions of the Invention on
Cellular Proliferation and Viability
[0194] To rule out the possibility that the antiviral activity of
the compositions assayed as described above may be due to a
negative effect on cellular viability or proliferation, the effect
of these compositions on the proliferative response and viability
of the target cells is determined for every in vitro test. For
example, the effect of the chemokine, derivative, or analog tested
in the primary CD4.sup.+ PBMC/primary HIV-1.sub.BaL isolate
cell-free infectivity assay on the proliferative response of
primary CD4.sup.+ PBMC may be determined. Peripheral blood
mononuclear cells are separated by Ficoll gradient centrifugation
and placed in round-bottom 96-well plates (10.sup.5 cells/well).
[.sup.3H]-Thymidine incorporation by stimulated cells is monitored
in the presence of concentrations of the compositions corresponding
to that used in the in vitro suppression assay and compared with
[.sup.3H]-Thymidine incorporation in controls that have not been
treated with the test composition. The test sample average
corrected counts per minute from triplicate cultures and the
percent radionucleotide incorporation is compared with that
observed for the control. Comparable levels of [.sup.3H]-Thymidine
incorporation in the test and control samples is indicative that
antiviral activity observed in the cell free infectivity assay is
not due to the suppression of cellular proliferation.
[0195] The effect of the chemokine, derivative, or analog tested on
the viability of primary CD4.sup.+ PBMC is determined applying
techniques known in the art using trypan blue dye exclusion.
[0196] 8.6 Mixture of MDC with I-309 is Especially Potent and
Titratable
[0197] HIV IIIB was mixed with PBMC at a multiplicity of infection
of 0.001-0.0025 and incubated for 2 hours at 37.degree. C. in RPMI
1640 medium containing 10% fetal calf serum (complete medium). The
cells were then washed and placed in microtiter culture wells with
the indicated amounts of chemokines in complete medium. Cells were
then cultured in a CO.sub.2 incubator at 37.degree. C. Culture
supernatants were collected 4 days post-infection and tested for
infection by HIV p24 capture ELISA.
[0198] For the antibody experiments, (FIGS. 2-4) primary CD8.sup.-
T cells from a series of HIV-seronegative donors were activated by
PHA and 10 ng/ml IL-2 in complete medium for 3 days. The cells were
then washed and cultured in complete medium with 10 ng/ml IL-2 in a
CO.sub.2 incubator. Eight days post-activation, culture
supernatants were collected. A portion of each supernatant was
tested directly for HIV-inhibiting activity in the infectivity
assay above. Another portion was treated with the indicated
concentrations of neutralizing anti-chemokine antibodies and tested
in parallel. If the chemokines contribute to the inhibitory
activity in the supernatants, treatment with the antibodies should
restore infection.
[0199] FIG. 1 demonstrates how mixtures of chemokines at the low
concentrations released by primary activated CD8+ T cells (as
determined by ELISA) block both R5 and X4 HIV infection. The
mixture of MDC with I-309 is especially potent and titratable. The
figure also shows that even at much higher concentrations, either
I-309 or MDC alone have much less antiviral effect, so they must
cooperate or synergize to mediate potent antiviral activity in the
mix. Also presented for comparison are tests (right panel) with
four randomly selected supernatants from activated CD8+ T cells.
Two test dilutions are shown. The "50%" sups contain the levels of
the chemokines used in the mix. As is apparent, the mix is as
potent as the supernatants; therefore the mix recapitulates the
natural antiviral activity produced by primary cells.
[0200] FIG. 2 shows the contribution of MDC to soluble HIV.sub.IIIB
suppressive activity produced by primary CD8+ T cells. Supernatants
from the cells that block HIV were treated with an antibody that
blocks MDC activity. The reduction in antiviral activity was then
plotted versus the amount of MDC present (as determined by ELISA)
in the same supernatant that was treated. The figure demonstrates
that the more MDC the cells make, the more it contributes to the
antiviral effect. FIG. 3 shows the same form of analysis on I-309,
using an anti-I-309 antibody. FIG. 4 shows the same analyses using
a mixture of antibodies to I-309 and MDC. This figure shows that
the two chemokines contribute very significantly to the natural
activity produced by primary CD8+ T cells.
[0201] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0202] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *