U.S. patent application number 12/044845 was filed with the patent office on 2008-10-02 for cc chemokine receptor 5 dna, new animal models and therapeutic agents for hiv infection.
This patent application is currently assigned to The Gov't of the U.S.A. as represented by the Secretary of the Dept. of Health & Human Services. Invention is credited to Ghalib Alkhatib, Edward A. Berger, Christopher C. Broder, Christophe Combadiere, Yu Feng, Paul E. Kennedy, Philip M. Murphy.
Application Number | 20080241167 12/044845 |
Document ID | / |
Family ID | 21788278 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241167 |
Kind Code |
A1 |
Combadiere; Christophe ; et
al. |
October 2, 2008 |
CC CHEMOKINE RECEPTOR 5 DNA, NEW ANIMAL MODELS AND THERAPEUTIC
AGENTS FOR HIV INFECTION
Abstract
The susceptibility of human macrophages to human
immunodeficiency virus (HIV) infection depends on cell surface
expression of the human CD4 molecule and CC cytokine receptor 5.
CCR5 is a member of the 7-transmembrane segment superfamily of
G-protein-coupled cell surface molecules. CCR5 plays an essential
role in the membrane fusion step of infection by some HIV isolates.
The establishment of stable, nonhuman cell lines and transgenic
mammals having cells that coexpress human CD4 and CCR5 provides
valuable tools for the continuing research of HIV infection. In
addition, antibodies which bind to CCR5, CCR5 variants, and
CCR5-binding agents, capable of blocking membrane fusion between
HIV and target cells represent potential anti-HIV therapeutics for
macrophage-tropic strains of HIV.
Inventors: |
Combadiere; Christophe;
(Paris, FR) ; Feng; Yu; (San Diego, CA) ;
Alkhatib; Ghalib; (Carmel, IN) ; Berger; Edward
A.; (Rockville, MD) ; Murphy; Philip M.;
(Rockville, MD) ; Broder; Christopher C.;
(Rockville, MD) ; Kennedy; Paul E.; (Silver
Spring, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Gov't of the U.S.A. as
represented by the Secretary of the Dept. of Health & Human
Services
|
Family ID: |
21788278 |
Appl. No.: |
12/044845 |
Filed: |
March 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11594375 |
Nov 7, 2006 |
7374872 |
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12044845 |
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10700313 |
Oct 31, 2003 |
7151087 |
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11594375 |
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08864458 |
May 28, 1997 |
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10700313 |
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60018508 |
May 28, 1996 |
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Current U.S.
Class: |
424/172.1 ;
435/29; 435/375; 435/440; 435/5; 435/7.2; 436/501; 514/44A |
Current CPC
Class: |
C07K 14/7158 20130101;
A01K 2217/05 20130101; A61K 38/00 20130101; A61P 31/18
20180101 |
Class at
Publication: |
424/172.1 ;
435/375; 436/501; 435/7.2; 435/5; 435/29; 514/44; 435/440 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/06 20060101 C12N005/06; G01N 33/566 20060101
G01N033/566; G01N 33/53 20060101 G01N033/53; C12Q 1/70 20060101
C12Q001/70; C12Q 1/02 20060101 C12Q001/02; A61K 31/7052 20060101
A61K031/7052; C12N 15/87 20060101 C12N015/87; A61P 31/18 20060101
A61P031/18 |
Claims
1. A method of inhibiting membrane fusion between HIV and a target
cell that expresses CCR5 or between an HIV-infected cell and a CD4
positive uninfected cell that expresses CCR5, comprising contacting
the target or CD4 positive cell with a fusion-inhibiting effective
amount of a CCR5 binding or blocking agent.
2. The method of claim 1, wherein the agent is an anti-CCR5
antibody or epitope binding fragment thereof.
3. The method of claim 2, wherein the antibody is a monoclonal
antibody or a polyclonal antibody.
4. The method of claim 1, wherein the contacting is by in vivo
administration to a subject.
5. The method of claim 2, wherein the anti-CCR5 antibody is
administered by intravenous, intramuscular or subcutaneous
injections.
6. The method of claim 4, wherein the anti-CCR5 antibody is
administered within a dose range of 0.1 .mu.g/kg to 100 mg/kg.
7. The method of claim 5, wherein the antibody is formulated in a
pharmaceutically acceptable carrier.
8. A method for identifying a compound which blocks HIV-envelope
mediated membrane fusion, comprising: a) incubating components
comprising the compound and a CCR5 polypeptide under conditions
sufficient to allow the components to interact; and b) measuring
the binding of the compound to CCR5 polypeptide.
9. The method of claim 8, wherein the compound is a peptide.
10. The method of claim 8, wherein the compound is a
peptidomimetic.
11. The method of claim 8, wherein the CCR5 polypeptide is
expressed in a cell.
12. The method of claim 11 wherein the cell is a recombinant cell
line that expresses CCR5 polypeptide.
13. The method of claim 8, wherein the compound comprises an
antibody.
14. The method of claim 13, wherein the antibody comprises a
polyclonal antibody, monoclonal antibody, or fragment thereof.
15. The method of claim 13, wherein the antibody is specific for an
extracellular region of the CCR5 polypeptide.
16. The method of claim 13, wherein the antibody comprises a
humanized monoclonal antibody.
17. The method of claim 8, wherein the compound comprises an
antisense molecule.
18. The method of claim 8, wherein the method further comprises:
(c) incubating components comprising the compound with a CCR5
positive cell under conditions sufficient to allow the components
to interact with the CCR5 positive cell; (d) contacting the
components of (c) with HIV or an HIV-infected cell; and (e)
measuring the ability of the compound to block membrane fusion
between the HIV and the CCR5 positive cell or between the
HIV-infected cell and the CCR5 cell.
19. The method of claim 8, wherein the method further comprises:
(c) incubating a first eukaryotic cell expressing CCR5 and CD4 with
a second eukaryotic cell expressing an HIV envelope protein under
conditions that permit fusion of the first eukaryotic and second
eukaryotic cells; (d) contacting components comprising the compound
with the first eukaryotic and second eukaryotic cells; and (e)
detecting fusion between the first eukaryotic and second eukaryotic
cells.
20. The method of claim 19, wherein contacting components
comprising the compound with the first eukaryotic and second
eukaryotic cells is performed before the first eukaryotic and
second eukaryotic cells are incubated.
21. The method of claim 19, wherein contacting components
comprising the compound with the first eukaryotic and second
eukaryotic cells is performed after the first eukaryotic and second
eukaryotic cells are incubated.
22. A method for identifying a compound which blocks HIV-envelope
mediated membrane fusion, comprising: incubating components
comprising the compound with a CCR5 positive cell under conditions
sufficient to allow the components to interact with the CCR5
positive cell; contacting the components and the CCR5 positive
cells with HIV or an HIV-infected cell; and measuring the ability
of the compound to block membrane fusion between the HIV and the
CCR5 positive cell or between the HIV-infected cell and the CCR5
cell.
23. The method of claim 22, wherein the CCR5 positive cell
expresses CD4.
24. A method for identifying a compound which blocks HIV-envelope
mediated membrane fusion, comprising: incubating a first eukaryotic
cell expressing CCR5 and CD4 with a second eukaryotic cell
expressing an HIV envelope protein under conditions that permit
fusion of the first eukaryotic and second eukaryotic cells;
contacting components comprising the compound with the first
eukaryotic and second eukaryotic cells; and detecting fusion
between the first eukaryotic and second eukaryotic cells.
25. A method of treating a subject having an HIV-related disorder
associated with expression of CCR5 comprising administering to an
HIV infected or susceptible cell of the subject, an agent that
suppresses CCR5.
26. The method of claim 25, wherein the agent is an anti-CCR5
antibody.
27. The method of claim 25, wherein the agent is an antisense
nucleic acid that hybridizes to a CCR5 nucleic acid.
28. The method of claim 25, wherein the agent is introduced into
the cell using a carrier.
29. The method of claim 25, wherein the carrier is a vector.
30. The method of claim 25, wherein the administering is ex
vivo.
31. The method of claim 25, wherein the administering is in vivo.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. patent application Ser. No.
11/594,375, filed Nov. 7, 2006, which is a divisional of U.S.
patent application Ser. No. 10/700,313, filed Oct. 31, 2003, now
U.S. Pat. No. 7,151,087, which is a continuation of U.S. patent
application Ser. No. 08/864,458 (abandoned), filed May 28, 1997,
which in turn claims the benefit of U.S. Provisional Application
60/018,508, filed May 28, 1996. All applications are incorporated
herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to in vitro and in vivo models
for the study of human immunodeficiency virus (HIV) infection and
the effectiveness of anti-HIV therapeutics. The invention more
specifically relates to cell surface proteins that participate in
HIV infection and which are useful for the development of animal
models.
BACKGROUND OF THE INVENTION
[0003] An HIV infection cycle begins with the entry of an HIV virus
into a target cell. Entry commences when an HIV envelope
glycoprotein (env) binds to a human CD4 molecule in a target cell
membrane. This binding leads to fusion of virus and cell membranes,
which in turn facilitates virus entry into the host. The
HIV-infected host cell eventually expresses env on its surface.
This expression allows the infected cell to fuse with uninfected,
CD4-positive cells, thereby spreading the virus.
[0004] Recent studies have shown that the HIV fusion process occurs
with a wide range of human cell types that either express human CD4
endogenously or that have been engineered to express human CD4. The
fusion process, however, does not occur with nonhuman cell types
engineered to express human CD4 even though these nonhuman cells
still can bind env. The disparity between human and nonhuman cell
types exists because membrane fusion requires the coexpression of
human CD4 and one or more cofactors specific to human cell types.
Nonhuman cell types that have been engineered to express human CD4
but not the additionally required factor(s) are incapable of
membrane fusion, and therefore are nonpermissive for HIV
infection.
[0005] Some individual HIV isolates, designated
"macrophage-tropic," efficiently infect primary macrophages but not
immortalized T-cell lines. Other isolates, designated "T-cell
line-tropic," have the opposite property and infect immortalized
T-cell lines more efficiently than they infect primary macrophages.
Both types of isolates readily infect primary T-cells from the
body, however. The selective tropism of these two types of isolates
is thought to be due to their requirements for distinct cofactors
that are differentially expressed on different CD4 positive cell
types. It should be understood that other HIV strains are
"dual-tropic" and have the ability to infect both macrophages and
immortalized T-cell lines and are believed to be able to use more
than one cofactor.
[0006] Recently a cofactor required for fusion of virus and cell
membranes has been described. Feng et al., Science 272: 872-7
(1996). This factor, called "fusin," (also known as CXCR4) permits
cells that contain human CD4 to fuse with the surface of an HIV
virus. Fusin functions preferentially for T-cell line-tropic HIV-1
isolates and much less well for macrophage-tropic HIV-1
isolates.
[0007] The discovery of fusin allows the creation of a successful
small animal model. Such a model is crucial for studies of HIV
infection and of the effectiveness of anti-HIV therapeutics. But
the presence of fusin enables the study of T-cell line-tropic but
not macrophage-tropic isolates. This is an important distinction
because macrophage-tropic isolates represent the predominant type
of isolates obtained from infected individuals. Macrophage-tropic
isolates also appear to be preferentially transmitted between
individuals. A putative cofactor that is necessarily expressed with
CD4 to allow entry of macrophage-tropic isolates remains
unknown.
[0008] In recent years, researchers have bred transgenic animals
that contain cells which express human CD4 and which could be used
as models for HIV infection of macrophages if the
macrophage-specific factor were known. See, for example, Dunn et
al., Human immunodeficiency virus type I infection of human
CD4-transgenic rabbits, J. Gen. Vir. 76:1327-1336 (1995); Snyder et
al., Development and Tissue-Specific Expression of Human CD4 in
Transgenic Rabbits, Mol. Reprod. & Devel. 40:419-428 (1995);
Killeen et al., Regulated Expression of Human CD4 Rescues Helper
T-Cell Development in Mice Lacking Expression of Endogenous CD4,
EMBO J. 12:1547-1553 (1993); Forte et al., Human CD4 Produced in
Lymphoid Cells of Transgenic Mice Binds HIV gp 120 and Modifies the
Subsets of Mouse T-Cell Populations, Immunogenetics 38:455-459
(1993).
[0009] A goal of research in this field is to find a putative
factor for the macrophage-tropic isolates that could be
co-expressed with CD4 in a small animal. Such co-expression would
provide an animal model to develop efficacious therapies to combat
infection by macrophage-tropic HIV isolates. The discovery of other
essential cofactors would provide new targets for development of
anti-HIV therapies.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the discovery of a new CC
chemokine receptor protein associated with HIV infection (formerly
referred to as "CC CKR5", now more commonly known as "CCR5"). The
invention provides isolated polynucleotides and polypeptides
encoded by CCR5 polynucleotides, as well as antibodies directed
against regions of CCR5 and peptide fragments of CCR5 which block
HIV interaction with the CC CKR5 receptor.
[0011] It is an object of the present invention to provide
therapeutic and preventative medicinal agents effective against HIV
infection and effective in regulating monocyte accumulation and
activation. In accomplishing these and other objects, there has
been provided, in accordance with one aspect of the present
invention a stable, nonhuman cell line, the cells of which contain
DNA encoding CCR5. In accordance with another aspect of the
invention a transgenic non-human mammal is provided comprised of
cells that coexpress human CD4 and CCR5.
[0012] In another aspect of the invention, the invention provides
an antibody which binds to CCR5 and which blocks membrane fusion
between HIV and a target cell. In accordance with another aspect of
the invention, there is provided a cell that expresses a CCR5 gene,
wherein the CCR5 gene is not stably integrated into the genome of
said cell.
[0013] In accordance with yet another aspect of the invention an
isolated and purified peptide fragment of CCR5 is provided that
blocks membrane fusion between HIV and a target cell.
[0014] In yet another aspect, the invention provides a method for
identifying a compound which blocks membrane fusion between HIV and
a CCR5 target cell or between an HIV-infected cell and a CCR5
positive uninfected cell. The method includes the steps of: a)
incubating components comprising the compound and a CDU and CCR5
positive cell under conditions sufficient to allow the components
to interact; b) contacting the components of step a) with HIV or an
HIV-infected cell; and c) measuring the ability of the compound to
block membrane fusion between HIV and the CCR5 positive cell or
between an HIV-infected cell and a CCR5 positive uninfected
cell.
[0015] In accordance with yet another aspect of the invention a
method of inhibiting CCR5 expression in a cell is provided,
comprising introducing into the cell at least one antisense
polynucleotide that causes the inhibition of CCR5 in the cell.
[0016] In accordance with yet another aspect of the invention is
provided a CCR5-binding agent, wherein said agent blocks binding of
a chemokine and HIV to CCR5.
[0017] The antibodies and blocking agents of the invention are also
useful for providing methods for modulating an immune response in
which macrophages are involved. For example, administration of CCR5
agonists or antagonists would be useful for modulating the immune
response.
[0018] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A shows an alignment of amino acid sequences deduced
from cDNAs for CC CKR1 (SEQ ID NO: 9), CC CKR2B (SEQ ID NO: 8), and
for CCR5 (SEQ ID NO: 4). Arabic numbers enumerate a CCR5 amino acid
sequence (SEQ ID NO:4) and a variant with residue changed from
alanine to leucine (SEQ ID NO: 2) that has been deduced from a CCR5
DNA sequence (SEQ ID NO:3 and SEQ ID NO: 1, respectively) and are
left-justified. Putative membrane-spanning segments I-VII are
noted. Vertical bars show identities between adjacent residues and
open boxes show predicted sites for N-linked glycosylation. Dashes
and gaps have been inserted to optimize the alignments.
Extracellular portions of the CCR5 polypeptide are located between
transmembrane domains 2 and 3, transmembrane domains 4 and 5,
transmembrane domains 6 and 7, and in the amino terminal segment
before transmembrane domain 1.
[0020] FIGS. 1B and 1C show the nucleotide and deduced amino acid
sequence (SEQ ID NO:11) for a CCR5 variant where nucleotides
293-296 of the wild-type DNA is changed from CTTG to TGCT resulting
in a change at amino acid residue 127, from Alanine to Leucine. The
translated protein is shown in SEQ ID NO: 2 (and the corresponding
cDNA in SEQ ID NO: 1).
[0021] FIG. 1D shows the nucleotide and deduced amino acid sequence
(SEQ ID NO:3 and 4, respectively) for CCR5.
[0022] FIG. 2 shows CCR5 peptides which inhibit fusion between
cells expressing the HIV-1 Env from the macrophage-tropic Ba-L
isolate and murine cells co-expressing CD4 and CCR5. Peptides were
preincubated with HIV-Env-expressing cells for 1 hour at a
concentration from 0-50 .mu.g/ml before mixing with cells which
express CD4 and CCR5.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0023] The present invention originated from studies on receptor
proteins of chemokines. The inventors cloned, sequenced, and
functionally expressed a human cDNA encoding a novel
macrophage-selective CC chemokine receptor that has been designated
CCR5.
[0024] During their investigation, the inventors discovered that
CCR5 is a necessary cofactor for infection by macrophage-tropic HIV
isolates. More particularly, the inventors found that when they
transgenically expressed human CCR5 in non-human cells which also
transgenically express human CD4, the altered cells could fuse with
cells that express the env envelope protein from macrophage-tropic
strains of HIV. It should be understood that other HIV strains are
"dual-tropic" and have the ability to infect both macrophages and
immortalized T-cell lines and are believed to be able to use more
than one cofactor. Furthermore, the inventors reasoned that
antibodies against CCR5 can inhibit the fusion of cells that
contain CD4 and CCR5, upon contact with cells that express the env
protein from macrophage-tropic strains of HIV. Antibodies which
bind CCR5 can inhibit infection of cells that contain CCR5 and CD4
by macrophage-tropic strains of HIV. The insights of the present
invention enable the development of new tools to study HIV
infection of macrophages and the discovery of new HIV treatment
methodologies based on chemokine receptor biochemistry.
[0025] Chemokine receptors are thought to have seven
transmembrane-domains, are coupled to G-protein and participate in
cellular responses to chemokines. Receptor CCR5 that has been
cloned by the inventors is the fifth human CC chemokine receptor
identified to date. The five receptors bind overlapping but
distinct subsets of CC chemokines. Of the five, only CC chemokine
receptor 5 ("CCR5") displays a CC chemokine specificity profile
that matches the profile for suppression of HIV-1 infection. Cocchi
et al., Science 270, 1811 (1995). RANTES, MIP-1.alpha. and
MIP-1.beta. are potent agonists of CCR5, but MCP-1 and MCP-3 are
not, as summarized by Combadiere et al. in J. Biol. Chem. 270:
16491-4 (1995), J. Biol. Chem. 270: 30235 (1995), and Molec. Biol.
Cell. 6: 224a (1995) and by Samson et al. in Biochemistry 35: 3362
(1996) the disclosures of which are incorporated herein in their
entireties.
Isolation of cDNA Encoding CCR5
[0026] The gene for the chemokine receptor of the present invention
can be cloned from a human cDNA library. Methods used to clone
novel chemokine receptor-like cDNAs from a .lamda.gt11 cDNA library
made from peripheral blood mononuclear cells of a patient with
eosinophilic leukemia have been described by Combadiere et al., DNA
Cell Biol. 14: 673-80 (1995), which is herein incorporated in its
entirety by reference. A cDNA encoding CCR5 also can be isolated by
the procedure described by U.S. provisional patent application
60/010,854 filed on Jan. 30, 1996, which is herein incorporated by
reference.
[0027] The above-described methods can be used to identify DNA
sequences that code for one or more CCR5 polypeptide sequences. A
nucleotide sequence determined by the inventors, herein described
as SEQ ID NO:3 of the present invention, has been deposited with
the Genbank/EMBL data libraries under accession number U57840. But
many other related sequences that code for CCR5 and altered forms
of CCR5 are contemplated in context of the various embodiments
enumerated herein (e.g. SEQ ID NO:1).
[0028] In preferred embodiments fusion between env-expressing
effector cells and CD4-expressing and CCR5-expressing target cells,
prepared by infection with vaccinia virus, induces activation of
Escherichia coli lacZ, causing .beta.-galactosidase production in
fused cells as described by Nussbaum et al., J. Virol. 68: 5411
(1994), which is incorporated in its entirety by reference. The
specificity of cell fusion as measured with this assay is
equivalent to the specificity of infection by HIV-1 virions.
[0029] The invention provides an isolated polynucleotide sequence
encoding a polypeptide having an amino acid sequence as set forth
in SEQ ID NO:4. The term "isolated" as used herein includes
polynucleotides substantially free of other nucleic acids,
proteins, lipids, carbohydrates or other materials with which it is
naturally associated. Polynucleotide sequences of the invention
include DNA, cDNA and RNA sequences which encode CCR5. It is
understood that all polynucleotides encoding all or a portion of
CCR5 are also included herein, as long as they encode a polypeptide
with CCR5 activity (e.g. act as a cofactor for HIV infection). Such
polynucleotides include naturally occurring, synthetic, and
intentionally manipulated polynucleotides. For example, portions of
the mRNA sequence may be altered due to alternate RNA splicing
patterns or the use of alternate promoters for RNA transcription.
As another example, CCR5 polynucleotide may be subjected to
site-directed mutagenesis. The polynucleotide sequence for CCR5
also includes antisense sequences. The polynucleotides of the
invention include sequences that are degenerate as a result of the
genetic code. There are 20 natural amino acids, most of which are
specified by more than one codon. Therefore, all degenerate
nucleotide sequences are included in the invention as long as the
amino acid sequence of CCR5 polypeptide encoded by the nucleotide
sequence is functionally unchanged. Also included are nucleotide
sequences which encode CCR5 polypeptide, such as SEQ ID NO:1. In
addition, the invention also includes a polynucleotide encoding a
polypeptide having the biological activity of an amino acid
sequence of SEQ ID NO:4 and having at least one epitope for an
antibody immunoreactive with CCR5 polypeptide. Assays provided
herein which show association between HIV infection and expression
of CCR5 can be used to detect CCR5 activity.
[0030] The polynucleotide encoding CCR5 includes the nucleotide
sequence in FIG. 1 (SEQ ID NO:1 and 3), as well as nucleic acid
sequences complementary to that sequence. A complementary sequence
may include an antisense nucleotide. When the sequence is RNA, the
deoxyribonucleotides A, G, C, and T of FIG. 1 are replaced by
ribonucleotides A, G, C, and U, respectively. Also included in the
invention are fragments (portions) of the above-described nucleic
acid sequences that are at least 15 bases in length, which is
sufficient to permit the fragment to selectively hybridize to DNA
that encodes the protein of FIG. 1 (e.g. SEQ ID NO: 4). "Selective
hybridization" as used herein refers to hybridization under
moderately stringent or highly stringent physiological conditions
(See, for example, the techniques described in Maniatis et al.,
1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y., incorporated herein by reference), which
distinguishes related from unrelated nucleotide sequences.
[0031] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g. GC v. AT content), and nucleic acid type (e.g. RNA v. DNA) of
the hybridizing regions of the nucleic acids can be considered in
selecting hybridization conditions. An additional consideration is
whether one of the nucleic acids is immobilized, for example, on a
filter.
[0032] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions, e.g.
high stringency conditions, or each of the conditions can be used,
e.g. for 10-15 minutes each, in the order listed above, repeating
any or all of the steps listed. However, as mentioned above,
optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0033] Specifically disclosed herein is a cDNA sequence for CCR5.
SEQ ID NO:3 represents the wild-type sequence and SEQ ID NO:1
represents a cDNA which encodes CCR5 having a conservative
substitution of Leucine for Alanine at amino acid residue 127. The
result of this conservative variation should not affect biological
activity of CCR5 polypeptide or peptides containing the variation
(see Example 5).
[0034] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
or computer-based techniques which are well known in the art. These
include, but are not limited to: 1) hybridization of genomic or
cDNA libraries with probes to detect homologous nucleotide
sequences; 2) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features; 3) polymerase
chain reaction (PCR) on genomic DNA or cDNA using primers capable
of annealing to the DNA sequence of interest; 4) computer searches
of sequence databases for similar sequences; and 5) differential
screening of a subtracted DNA library.
[0035] Preferably the CCR5 polynucleotide of the invention is
derived from a mammalian organism. Screening procedures which rely
on nucleic acid hybridization make it possible to isolate any gene
sequence from any organism, provided the appropriate probe is
available. Oligonucleotide probes, which correspond to a part of
the sequence encoding the protein in question, can be synthesized
chemically. This requires that short, oligopeptide stretches of
amino acid sequence must be known. The DNA sequence encoding the
protein can be deduced from the genetic code, however, the
degeneracy of the code must be taken into account. It is possible
to perform a mixed addition reaction when the sequence is
degenerate. This includes a heterogeneous mixture of denatured
double-stranded DNA. For such screening, hybridization is
preferably performed on either single-stranded DNA or denatured
double-stranded DNA. Hybridization is particularly useful in the
detection of cDNA clones derived from sources where an extremely
low amount of mRNA sequences relating to the polypeptide of
interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it
is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res., 9:879,
1981). Alternatively, a subtractive library, as illustrated herein
is useful for elimination of non-specific cDNA clones.
[0036] When the entire sequence of amino acid residues of the
desired polypeptide is not known, the direct synthesis of DNA
sequences is not possible and the method of choice is the synthesis
of cDNA sequences. Among the standard procedures for isolating cDNA
sequences of interest is the formation of plasmid- or
phage-carrying cDNA libraries which are derived from reverse
transcription of mRNA which is abundant in donor cells that have a
high level of genetic expression. When used in combination with
polymerase chain reaction technology, even rare expression products
can be cloned. In those cases where significant portions of the
amino acid sequence of the polypeptide are known, the production of
labeled single or double-stranded DNA or RNA probe sequences
duplicating a sequence putatively present in the target cDNA may be
employed in DNA/DNA hybridization procedures which are carried out
on cloned copies of the cDNA which have been denatured into a
single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325,
1983).
[0037] A cDNA expression library, such as lambda gt11, can be
screened indirectly for CCR5 peptides having at least one epitope,
using antibodies specific for CCR5. Such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of CCR5 cDNA.
[0038] Alterations in CCR5 nucleic acid include intragenic
mutations (e.g. point mutation, nonsense (stop), missense, splice
site and frameshift) and heterozygous or homozygous deletions.
Detection of such alterations can be done by standard methods known
to those of skill in the art including sequence analysis, Southern
blot analysis, PCR based analyses (e.g. multiplex PCR, sequence
tagged sites (STSs)) and in situ hybridization. Such proteins can
be analyzed by standard SDS-PAGE and/or immunoprecipitation
analysis and/or Western blot analysis, for example.
[0039] DNA sequences encoding CCR5 can be expressed in vitro by DNA
transfer into a suitable host cell. "Host cells" are cells in which
a vector can be propagated and its DNA expressed. The term also
includes any progeny of the subject host cell. It is understood
that all progeny may not be identical to the parental cell since
there may be mutations that occur during replication. However, such
progeny are included when the term "host cell" is used. Methods of
stable transfer, meaning that the foreign DNA is continuously
maintained in the host, are known in the art.
[0040] In the present invention, the CCR5 polynucleotide sequences
may be inserted into a recombinant expression vector. The term
"recombinant expression vector" refers to a plasmid, virus or other
vehicle known in the art that has been manipulated by insertion or
incorporation of the CCR5 genetic sequences. Such expression
vectors contain a promoter sequence which facilitates the efficient
transcription of the inserted genetic sequence of the host. The
expression vector typically contains an origin of replication, a
promoter, as well as specific genes which allow phenotypic
selection of the transformed cells. Vectors suitable for use in the
present invention include, but are not limited to the T7-based
expression vector for expression in bacteria (Rosenberg, et al.,
Gene, 56: 125, 1987), the pMSXND expression vector for expression
in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521,
1988) and baculovirus-derived vectors for expression in insect
cells. The DNA segment can be present in the vector operably linked
to regulatory elements, for example, a promoter (e.g. T7,
metallothionein I, or polyhedrin promoters).
[0041] Polynucleotide sequences encoding CCR5 can be expressed in
either prokaryotes or eukaryotes. Hosts can include microbial,
yeast, insect and mammalian organisms. However, since mature CCR5
is glycosylated, the choice of host cells depends on whether or not
the glycosylated or non-glycosylated form of CCR5 is desired.
Methods of expressing DNA sequences having eukaryotic or viral
sequences in prokaryotes are well known in the art. Biologically
functional viral and plasmid DNA vectors capable of expression and
replication in a host are known in the art. Such vectors are used
to incorporate DNA sequences of the invention.
[0042] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the CCR5 coding
sequence and appropriate transcriptional/-translational control
signals. These methods include in vitro recombinant DNA techniques,
synthetic techniques, and in vivo recombination/genetic techniques.
(See, for example, the techniques described in Maniatis et al.,
1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y.)
[0043] A variety of host-expression vector systems may be utilized
to express the CCR5 coding sequence. These include but are not
limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing the CCR5 coding sequence; yeast transformed with
recombinant yeast expression vectors containing the CCR5 coding
sequence; plant cell systems infected with recombinant virus
expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g. Ti plasmid) containing the CCR5 coding
sequence; insect cell systems infected with recombinant virus
expression vectors (e.g. baculovirus) containing the CCR5 coding
sequence; or animal cell systems infected with recombinant virus
expression vectors (e.g. retroviruses, adenovirus, vaccinia virus)
containing the CCR5 coding sequence, or transformed animal cell
systems engineered for stable expression. Since CCR5 has not been
confirmed to contain carbohydrates, both bacterial expression
systems as well as those that provide for translational and
post-translational modifications may be used; e.g. mammalian,
insect, yeast or plant expression systems.
[0044] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, etc. may be used in
the expression vector (see e.g. Bitter et al., 1987, Methods in
Enzymology 153:516-544). For example, when cloning in bacterial
systems, inducible promoters such as pL of bacteriophage .gamma.,
plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be
used. When cloning in mammalian cell systems, promoters derived
from the genome of mammalian cells (e.g. metallothionein promoter)
or from mammalian viruses (e.g. the retrovirus long terminal
repeat; the adenovirus late promoter; the vaccinia virus 7.5K
promoter) may be used. Promoters produced by recombinant DNA or
synthetic techniques may also be used to provide for transcription
of the inserted CCR5 coding sequence.
[0045] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al.,
Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et
al., 1987, Expression and Secretion Vectors for Yeast, in Methods
in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y.,
Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL
Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene
Expression in Yeast, Methods in Enzymology, Eds. Berger &
Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular
Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al.,
Cold Spring Harbor Press, Vols. I and II. A constitutive yeast
promoter such as ADH or LEU2 or an inducible promoter such as GAL
may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning
Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press,
Wash., D.C.). Alternatively, vectors may be used which promote
integration of foreign DNA sequences into the yeast chromosome.
[0046] Eukaryotic systems, and preferably mammalian expression
systems, allow for proper post-translational modifications of
expressed mammalian proteins to occur. Eukaryotic cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, phosphorylation, and advantageously,
plasma membrane insertion of the gene product may be used as host
cells for the expression of CCR5.
[0047] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the CCR5 coding sequence
may be ligated to an adenovirus transcription/translation control
complex, e.g. the late promoter and tripartite leader sequence.
Alternatively, the vaccinia virus 7.5K promoter may be used. (e.g.
see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:
7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et
al., 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931). Of particular
interest are vectors based on bovine papilloma virus which have the
ability to replicate as extrachromosomal elements (Sarver, et al.,
1981, Mol. Cell. Biol. 1: 486). Shortly after entry of this DNA
into mouse cells, the plasmid replicates to about 100 to 200 copies
per cell. Transcription of the inserted cDNA does not require
integration of the plasmid into the host's chromosome, thereby
yielding a high level of expression. These vectors can be used for
stable expression by including a selectable marker in the plasmid,
such as, for example, the neo gene. Alternatively, the retroviral
genome can be modified for use as a vector capable of introducing
and directing the expression of the CCR5 gene in host cells (Cone
& Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349-6353).
High level expression may also be achieved using inducible
promoters, including, but not limited to, the metallothionine IIA
promoter and heat shock promoters.
[0048] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with the CCR5 cDNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. The selectable marker in the recombinant
plasmid confers resistance to the selection and allows cells to
stably integrate the plasmid into their chromosomes and grow to
form foci which in turn can be cloned and expanded into cell lines.
For example, following the introduction of foreign DNA, engineered
cells may be allowed to grow for 1-2 days in an enriched media, and
then are switched to a selective media. A number of selection
systems may be used, including but not limited to the herpes
simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska &
Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes
can be employed in tk-, hgprt.sup.- or aprt.sup.- cells
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:
3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527);
gpt, which confers resistance to mycophenolic acid (Mulligan &
Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo, which
confers resistance to the aminoglycoside G-418 (Colberre-Garapin et
al., 1981, J. Mol. Biol. 150:1); and hygro, which confers
resistance to hygromycin (Santerre et al., 1984, Gene 30: 147)
genes. Recently, additional selectable genes have been described,
namely trpB, which allows cells to utilize indole in place of
tryptophan; hisD, which allows cells to utilize histinol in place
of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci.
USA 85: 8047); and ODC (ornithine decarboxylase) which confers
resistance to the ornithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In:
Current Communications in Molecular Biology, Cold Spring Harbor
Laboratory ed.).
[0049] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransformed with DNA
sequences encoding the CCR5 of the invention, and a second foreign
DNA molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
[0050] Cell Lines
[0051] In one embodiment, the present invention relates to stable
recombinant cell lines, the cells of which express CCR5 polypeptide
or coexpress human CD4 and CCR5 and contain DNA that encodes CCR5.
Suitable cell types include but are not limited to cells of the
following types: NIH 3T3 (Murine), Mv 1 lu (Mink), BS-C-1 (African
Green Monkey) and human embryonic kidney (HEK) 293 cells. Such
cells are described, for example, in the Cell Line Catalog of the
American Type Culture Collection (ATCC). These cells can be stably
transformed by a method known to the skilled artisan. See, for
example, Ausubel et al., Introduction of DNA Into Mammalian Cells,
in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, sections 9.5.1-9.5.6
(John Wiley & Sons, Inc. 1995). "Stable" transformation in the
context of the invention means that the cells are immortal to the
extent of having gone through at least 50 divisions.
[0052] CCR5 can be expressed using inducible or constitutive
regulatory elements for such expression. Commonly used constitutive
or inducible promoters, for example, are known in the art. The
desired protein encoding sequence and an operably linked promoter
may be introduced into a recipient cell either as a non-replicating
DNA (or RNA) molecule, which may either be a linear molecule or,
more preferably, a closed covalent circular molecule. Since such
molecules are incapable of autonomous replication, the expression
of the desired molecule may occur through the transient expression
of the introduced sequence. Alternatively, permanent expression may
occur through the integration of the introduced sequence into the
host chromosome. Therefore the cells can be transformed stably or
transiently.
[0053] An example of a vector that may be employed is one which is
capable of integrating the desired gene sequences into the host
cell chromosome. Cells which have stably integrated the introduced
DNA into their chromosomes can be selected by also introducing one
or more markers which allow for selection of host cells which
contain the expression vector. The marker may complement an
auxotrophy in the host (such as leu2, or ura3, which are common
yeast auxotrophic markers), biocide resistance, e.g., antibiotics,
or heavy metals, such as copper, or the like. The selectable marker
gene can either be directly linked to the DNA gene sequences to be
expressed, or introduced into the same cell by co-transfection.
[0054] In a preferred embodiment, the introduced sequence will be
incorporated into a plasmid or viral vector capable of autonomous
replication in the recipient host. Any of a wide variety of vectors
may be employed for this purpose. Factors of importance in
selecting a particular plasmid or viral vector include: the ease
with which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not
contain the vector; the number of copies of the vector which are
desired in a particular host; and whether it is desirable to be
able to "shuttle" the vector between host cells of different
species.
[0055] For a mammalian host, several possible vector systems are
available for expression. One class of vectors utilize DNA elements
which provide autonomously replicating extra-chromosomal plasmids,
derived from animal viruses such as bovine papilloma virus, polyoma
virus, adenovirus, or SV40 virus. A second class of vectors include
vaccinia virus expression vectors. A third class of vectors relies
upon the integration of the desired gene sequences into the host
chromosome. Cells which have stably integrated the introduced DNA
into their chromosomes may be selected by also introducing one or
more markers (e.g. an exogenous gene) which allow selection of host
cells which contain the expression vector. The marker may provide
for prototropy to an auxotrophic host, biocide resistance, e.g.
antibiotics, or heavy metals, such as copper or the like. The
selectable marker gene can either be directly linked to the DNA
sequences to be expressed, or introduced into the same cell by
co-transformation. Additional elements may also be needed for
optimal synthesis of mRNA. These elements may include splice
signals, as well as transcription promoters, enhancers, and
termination signals. The cDNA expression vectors incorporating such
elements include those described by Okayama, H., Mol. Cell. Biol.,
3:280 (1983), and others.
[0056] Once the vector or DNA sequence containing the construct has
been prepared for expression, the DNA construct may be introduced
(transformed) into an appropriate host. Various techniques may be
employed, such as protoplast fusion, calcium phosphate
precipitation, electroporation or other conventional
techniques.
[0057] Transgenic Animals
[0058] In another embodiment, the present invention relates to
transgenic animals having cells that coexpress human CD4 and CCR5.
Such transgenic animals represent a model system for the study of
HIV infection and the development of more effective anti-HIV
therapeutics.
[0059] The term "animal" here denotes all mammalian species except
human. It also includes an individual animal in all stages of
development, including embryonic and fetal stages. Farm animals
(pigs, goats, sheep, cows, horses, rabbits and the like), rodents
(such as mice), and domestic pets (for example, cats and dogs) are
included within the scope of the present invention.
[0060] A "transgenic" animal is any animal containing cells that
bear genetic information received, directly or indirectly, by
deliberate genetic manipulation at the subcellular level, such as
by microinjection or infection with recombinant virus. "Transgenic"
in the present context does not encompass classical crossbreeding
or in vitro fertilization, but rather denotes animals in which one
or more cells receive a recombinant DNA molecule. Although it is
highly preferred that this molecule be integrated within the
animal's chromosomes, the present invention also contemplates the
use of extrachromosomally replicating DNA sequences, such as might
be engineered into yeast artificial chromosomes.
[0061] The term "transgenic animal" also includes a "germ cell
line" transgenic animal. A germ cell line transgenic animal is a
transgenic animal in which the genetic information has been taken
up and incorporated into a germ line cell, therefore conferring the
ability to transfer the information to offspring. If such offspring
in fact possess some or all of that information, then they, too,
are transgenic animals.
[0062] It is highly preferred that the transgenic animals of the
present invention be produced by introducing into single cell
embryos DNA encoding CCR5 and DNA encoding human CD4, in a manner
such that these polynucleotides are stably integrated into the DNA
of germ line cells of the mature animal and inherited in normal
mendelian fashion. Advances in technologies for embryo
micromanipulation now permit introduction of heterologous DNA into
fertilized mammalian ova. For instance, totipotent or pluripotent
stem cells can be transformed by microinjection, calcium phosphate
mediated precipitation, liposome fusion, retroviral infection or
other means, the transformed cells are then introduced into the
embryo, and the embryo then develops into a transgenic animal. In a
preferred method, developing embryos are infected with a retrovirus
containing the desired DNA, and transgenic animals produced from
the infected embryo.
[0063] In a most preferred method the appropriate DNAs are
coinjected into the pronucleus or cytoplasm of embryos, preferably
at the single cell stage, and the embryos allowed to develop into
mature transgenic animals. These techniques are well known. For
instance, reviews of standard laboratory procedures for
microinjection of heterologous DNAs into mammalian (mouse, pig,
rabbit, sheep, goat, cow) fertilized ova include: Hogan et al.,
MANIPULATING THE MOUSE EMBRYO (Cold Spring Harbor Press 1986);
Krimpenfort et al., Bio/Technology 9:86 (1991); Palmiter et al.,
Cell 41:343 (1985); Kraemer et al., GENETIC MANIPULATION OF THE
EARLY MAMMALIAN EMBRYO (Cold Spring Harbor Laboratory Press 1985);
Hammer et al., Nature, 315:680 (1985); Purcel et al., Science,
244:1281 (1986); Wagner et al., U.S. Pat. No. 5,175,385;
Krimpenfort et al., U.S. Pat. No. 5,175,384, the respective
contents of which are incorporated by reference.
[0064] The cDNA that encodes CCR5 can be fused in proper reading
frame under the transcriptional and translational control of a
vector to produce a genetic construct that is then amplified, for
example, by preparation in a bacterial vector, according to
conventional methods. See, for example, the standard work: Sambrook
et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor
Press 1989), the contents of which are incorporated by reference.
The amplified construct is thereafter excised from the vector and
purified for use in producing transgenic animals.
[0065] Production of transgenic animals containing the gene for
human CD4 have been described. See Snyder et al., supra; Dunn et
al., supra, the contents of which are incorporated by
reference.
[0066] The term "transgenic" as used herein additionally includes
any organism whose genome has been altered by in vitro manipulation
of the early embryo or fertilized egg or by any transgenic
technology to induce a specific gene knockout. The term "gene
knockout" as used herein, refers to the targeted disruption of a
gene in vivo with complete loss of function that has been achieved
by any transgenic technology familiar to those in the art. In one
embodiment, transgenic animals having gene knockouts are those in
which the target gene has been rendered nonfunctional by an
insertion targeted to the gene to be rendered non-functional by
homologous recombination. As used herein, the term "transgenic"
includes any transgenic technology familiar to those in the art
which can produce an organism carrying an introduced transgene or
one in which an endogenous gene has been rendered non-functional or
"knocked out."
[0067] The transgene to be used in the practice of the subject
invention is a DNA sequence comprising a modified CCR5 coding
sequence. In a preferred embodiment, the CCR5 gene is disrupted by
homologous targeting in embryonic stem cells. For example, the
entire mature C-terminal region of the CCR5 gene may be deleted as
described in the examples below. Optionally, the CCR5 disruption or
deletion may be accompanied by insertion of or replacement with
other DNA sequences, such as a non-functional CCR5 sequence. In
other embodiments, the transgene comprises DNA antisense to the
coding sequence for CCR5. In another embodiment, the transgene
comprises DNA encoding an antibody or receptor peptide sequence
which is able to bind to CCR5. Where appropriate, DNA sequences
that encode proteins having CCR5 activity but differ in nucleic
acid sequence due to the degeneracy of the genetic code may also be
used herein, as may truncated forms, allelic variants and
interspecies homologues.
[0068] Antibodies which Bind to CCR5 Inhibit Fusion
[0069] In another embodiment, the present invention relates to
antibodies that bind CCR5 that block env-mediated membrane fusion
(i) associated with HIV entry into a human CD4-positive target cell
or (ii) between an HIV-infected cell and an uninfected human
CD4-positive target cell. The invention also includes antibodies
that bind to CCR5 and inhibit chemokine binding. For example, such
antibodies may be useful for ameliorating immune response disorders
associated with macrophages. Antibodies of the invention may also
inhibit gp120 binding to CCR5. Such antibodies could represent
research and diagnostic tools in the study of HIV infection and the
development of more effective anti-HIV therapeutics. In addition,
pharmaceutical compositions comprising antibodies against CCR5 may
represent effective anti-HIV therapeutics.
[0070] An antibody suitable for blocking env-mediated membrane
fusion, inhibiting chemokine binding, or blocking gp120 binding to
CCR5, is specific for at least one portion of an extracellular
region of the CCR5 polypeptide, as shown in FIG. 1 (SEQ ID NO:2 and
4). For example, one of skill in the art can use the peptides in
SEQ ID NO:5-7 or other extracellular amino acids of CCR5 to
generate appropriate antibodies of the invention. Alternatively,
one of skill in the art can use whole cells expressing CCR5 as an
immunogen for generation of anti-CCR5 antibodies which either block
env-mediated membrane fusion, inhibit chemokine binding or block
gp120 binding to CCR5. Anti-CCR5 antibodies of the invention may
have any or all of these functions.
[0071] A target cell includes but is not limited to a cell of the
following types: Mv 1 lu, NIH 3T3, BS-C-1, HEK293 cells and primary
human T-cells and macrophages. Antibodies of the invention include
polyclonal antibodies, monoclonal antibodies, and fragments of
polyclonal and monoclonal antibodies.
[0072] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, for example, Green et al.,
Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS
(Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al.,
Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992),
which are hereby incorporated by reference.
[0073] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature
256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor
Pub. 1988), which are hereby incorporated by reference. Briefly,
monoclonal antibodies can be obtained by injecting mice with a
composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the B lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures. Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, e.g. Coligan et al., sections 2.7.1-2.7.12 and sections
2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG),
in METHODS 1N MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana
Press 1992).
[0074] Methods of in vitro and in vivo multiplication of monoclonal
antibodies is well-known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g. osyngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane (tetramethylpentadecane) prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0075] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention may also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg et
al., International Patent Publication WO 91/11465 (1991) and Losman
et al., Int. J. Cancer 46:310 (1990), which are hereby incorporated
by reference.
[0076] Alternatively, a therapeutically useful anti-CCR5 antibody
may be derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in its
entirety by reference. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988);
Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc.
Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech.
12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1993),
which are hereby incorporated by reference.
[0077] Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., METHODS: A COMPANION TO
METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12: 433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
[0078] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet. 7:13 (1994); Lonberg et
al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579
(1994), which are hereby incorporated by reference.
[0079] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of the antibody or by expression in E.
coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5 S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, by Goldenberg, U.S. Pat. Nos.
4,036,945 and 4,331,647, and references contained therein. These
patents are hereby incorporated in their entireties by reference.
See also Nisonhoff et al., Arch. Biochem. Biophys's. 89:230 (1960);
Porter, Biochem. J. 73:119 (1959); Edelman et al., METHODS IN
ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et
al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0080] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0081] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g. Sandhu, supra. Preferably, the Fv
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by Whitlow et al.,
METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97
(1991); Bird et al., Science 242:423-426 (1988); Ladner et al.,
U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77
(1993); and Sandhu, supra.
[0082] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., METHODS: A COMPANION TO
METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).
[0083] Antibodies that bind to CXCR4 chemokine receptor, another
HIV fusion cofactor receptor, have been shown to block fusion of
HIV strains that use CXCR4 receptor for infection (Feng, et al.,
Science 272:872, 1996; Endres, et al., Cell 87:745, 1996).
[0084] Variants of CCR5
[0085] The term "CCR5 variant" as used herein means a molecule that
simulates at least part of the structure of CCR5 and interferes
with the fusion of cells that express env with cells that express
CD4 and CCR5. The env protein of certain HIV isolates may
participate in HIV infectivity by binding to CCR5 at the surface of
certain cells. While not wishing to be bound by a particular theory
of the invention, the inventors believe that CCR5 variants may
interfere in HIV infectivity by competing with the binding of CCR5
to env. CCR5 variants may also be useful in preventing chemokine
binding, thereby ameliorating symptoms of macrophage associated
immune disorders.
[0086] In one embodiment, the present invention relates to peptides
and peptide derivatives that have fewer amino acid residues than
CCR5 and that block membrane fusion between HIV and a target cell.
Such peptides and peptide derivatives could represent research and
diagnostic tools in the study of HIV infection and the development
of more effective anti-HIV therapeutics. The preferred peptide
fragments of CCR5 according to the invention include those which
correspond to the regions of CCR5 that are exposed on the cell
surface (e.g. SEQ ID NO:5, 6 or 7).
[0087] The invention relates not only to peptides and peptide
derivatives of naturally-occurring CCR5, but also to CCR5 mutants
and chemically synthesized derivatives of CCR5 that block membrane
fusion between HIV and a target cell. For example, changes in the
amino acid sequence of CCR5 are contemplated in the present
invention. CCR5 can be altered by changing the DNA encoding the
protein (e.g. SEQ ID NO:1 &2). Preferably, only conservative
amino acid alterations are undertaken, using amino acids that have
the same or similar properties. Illustrative amino acid
substitutions include the changes of: alanine to serine; arginine
to lysine; asparagine to glutamine or histidine; aspartate to
glutamate; cysteine to serine; glutamine to asparagine; glutamate
to aspartate; glycine to proline; histidine to asparagine or
glutamine; isoleucine to leucine or valine; leucine to valine or
isoleucine; lysine to arginine, glutamine, or glutamate; methionine
to leucine or isoleucine; phenylalanine to tyrosine, leucine or
methionine; serine to threonine; threonine to serine; tryptophan to
tyrosine; tyrosine to tryptophan or phenylalanine; valine to
isoleucine or leucine.
[0088] Variants useful for the present invention comprise analogs,
homologs, muteins and mimetics of CCR5 that retain the ability to
block membrane fusion. Peptides of the CCR5 refer to portions of
the amino acid sequence of CCR5 that also retain this ability. The
variants can be generated directly from CCR5 itself by chemical
modification, by proteolytic enzyme digestion, or by combinations
thereof. Additionally, genetic engineering techniques, as well as
methods of synthesizing polypeptides directly from amino acid
residues, can be employed.
[0089] Peptides of the invention include the following which
correspond to extracellular loops of CCR5 (amino acid designations
are according to the single letter code):
TABLE-US-00001 (SEQ ID NO: 5) extracellular loop-1 (el-1):
A/LAAQWDFGNTMC (SEQ ID NO: 6) extracellular loop-2 (el-2):
RSQKEGLHYTCSSHFPYSQYQFWK (SEQ ID NO: 7) extracellular loop-3
(el-3): QEFFGLNNCSSSNRLD
FIG. 2 shows the ability of SEQ ID NO: 5, 6, and 7 to inhibit
fusion between cells expressing the HIV-1 env (from the macrophage
tropic Ba-L isolate) and murine cells co-expressing CD4 and
CCR5.
[0090] Peptides of the invention can be synthesized by such
commonly used methods as t-BOC or FMOC protection of alpha-amino
groups. Both methods involve stepwise syntheses whereby a single
amino acid is added at each step starting from the C terminus of
the peptide (See, Coligan et al., Current Protocols in Immunology,
Wiley Interscience, 1991, Unit 9). Peptides of the invention can
also be synthesized by the well known solid phase peptide synthesis
methods described by Merrifield (J. Am. Chem. Soc., 85:2149, 1962),
and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman,
San Francisco, 1969, pp. 27-62), using a
copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g
polymer. On completion of chemical synthesis, the peptides can be
deprotected and cleaved from the polymer by treatment with liquid
HF-10% anisole for about 1/4-1 hours at 0.degree. C. After
evaporation of the reagents, the peptides are extracted from the
polymer with 1% acetic acid solution which is then lyophilized to
yield the crude material. This can normally be purified by such
techniques as gel filtration on Sephadex G-15 using 5% acetic acid
as a solvent. Lyophilization of appropriate fractions of the column
will yield the homogeneous peptide or peptide derivatives, which
can then be characterized by such standard techniques as amino acid
analysis, thin layer chromatography, high performance liquid
chromatography, ultraviolet absorption spectroscopy, molar
rotation, solubility, and quantitated by the solid phase Edman
degradation.
[0091] Alternatively, peptides can be produced by recombinant
methods as described below.
[0092] The term "substantially purified" as used herein refers to a
molecule, such as a peptide that is substantially free of other
proteins, lipids, carbohydrates, nucleic acids, and other
biological materials with which it is naturally associated. For
example, a substantially pure molecule, such as a polypeptide, can
be at least 60%, by dry weight, the molecule of interest. One
skilled in the art can purify CCR5 peptides using standard protein
purification methods and the purity of the polypeptides can be
determined using standard methods including, e.g. polyacrylamide
gel electrophoresis (e.g. SDS-PAGE), column chromatography (e.g.
high performance liquid chromatography (HPLC)), and amino-terminal
amino acid sequence analysis.
[0093] Non-peptide compounds that mimic the binding and function of
CCR5 ("mimetics") can be produced by the approach outlined in
Saragovi et al., Science 253: 792-95 (1991). Mimetics are molecules
which mimic elements of protein secondary structure. See, for
example, Johnson et al., "Peptide Turn Mimetics," in BIOTECHNOLOGY
AND PHARMACY, Pezzuto et al., Eds., (Chapman and Hall, New York
1993). The underlying rationale behind the use of peptide mimetics
is that the peptide backbone of proteins exists chiefly to orient
amino acid side chains in such a way as to facilitate molecular
interactions. For the purposes of the present invention,
appropriate mimetics can be considered to be the equivalent of CCR5
itself.
[0094] Longer peptides can be produced by the "native chemical"
ligation technique which links together peptides (Dawson, et al.,
Science, 266:776, 1994). Variants can be created by recombinant
techniques employing genomic or cDNA cloning methods. Site-specific
and region-directed mutagenesis techniques can be employed. See
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY vol. 1, ch. 8 (Ausubel et
al. eds., J. Wiley & Sons 1989 & Supp. 1990-93); PROTEIN
ENGINEERING (Oxender & Fox eds., A. Liss, Inc. 1987). In
addition, linker-scanning and PCR-mediated techniques can be
employed for mutagenesis. See PCR TECHNOLOGY (Erlich ed., Stockton
Press 1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 &
2, supra. Protein sequencing, structure and modeling approaches for
use with any of the above techniques are disclosed in PROTEIN
ENGINEERING, loc. cit., and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
vols. 1 & 2, supra.
[0095] If the compounds described above are employed, the skilled
artisan can routinely insure that such compounds are amenable for
use with the present invention in view of the vaccinia cell fusion
system described herein. If a compound blocks env-mediated membrane
fusion (i) involved in HIV entry into a human CD4-positive target
cell or (ii) between an HIV-infected cell and an uninfected human
CD4-positive target cell, the compound is suitable according to the
invention.
[0096] CCR5-Binding and Blocking Agents
[0097] In yet another embodiment, the present invention relates to
CCR5-binding agents that block membrane fusion between HIV and a
target cell. Such agents could represent research and diagnostic
tools in the study of HIV infection and the development of more
effective anti-HIV therapeutics. In addition, pharmaceutical
compositions comprising CCR5-binding agents may represent effective
anti-HIV therapeutics. In the context of HIV infection, the phrase
"CCR5-binding agent" denotes a naturally occurring ligand of CCR5
such as, for example: RANTES, MIP-1.alpha. or MIP-1.beta.; a
synthetic ligand of CCR5, or appropriate derivatives of the natural
or synthetic ligands. The determination and isolation of ligands is
well described in the art. See, e.g. Lerner, Trends NeuroSci.
17:142-146 (1994) which is hereby incorporated in its entirety by
reference. A CCR5-binding agent that blocks env-mediated membrane
fusion (i) involved in HIV entry into a human CD4-positive target
cell or (ii) between an HIV-infected cell and an uninfected human
CD4-positive target cell is suitable according to the invention.
Further, a CCR5 blocking or binding agent includes an agent which
inhibits gp120 binding to CCR5 or chemokine binding to CCR5.
[0098] In yet another embodiment, the present invention relates to
CCR5-binding agents that interfere with binding between CCR5 and a
chemokine. Such binding agents may interfere by competitive
inhibition, by non-competitive inhibition or by uncompetitive
inhibition.
[0099] Interference with normal binding between CCR5 and one or
more chemokines can result in a useful pharmacological effect
related to inflammation because CCR5 binds chemokines that regulate
monocyte accumulation and activation in inflamed tissue sites.
Nevertheless, while monocyte chemotaxis is the most widely shared
and perhaps best described function for MIP-1.alpha., MIP-1.beta.
and RANTES, apparently each of the CC CKRs that bind one or more of
these chemokines connect specifically and differentially to
additional monocyte functions such as T-cell costimulation.
[0100] Monocytes are long-lived cells capable of further
differentiation as they move from the blood to establish residence
in the tissues as macrophages. The functional properties of tissue
macrophages differ in different organs, and in the same organ
depending on the presence of priming agents, i.e., agents that can
change the behavior of monocytes and make them more sensitive to
chemoattractants. CCR5-binding or blocking agents can interfere
with the normal functioning of this system to reduce inflammation
and are contemplated by the present invention. Anti-CCR5 antibodies
of the invention are also useful in this context.
[0101] Screen for CCCKR5 Binding and Blocking Compositions
[0102] In another embodiment, the invention provides a method for
identifying a composition which binds to CCR5 or blocks HIV
env-mediated membrane fusion. The method includes incubating
components comprising the composition and CCR5 under conditions
sufficient to allow the components to interact and measuring the
binding of the composition to CCR5. Compositions that bind to CCR5
include peptides, peptidomimetics, polypeptides, chemical compounds
and biologic agents as described above. In addition to inhibition
of cell fusion, one of skill in the art could screen for inhibition
of gp120 binding or inhibition of CCR5 binding to a chemokine to
determine if a compound or composition was a CCR5 binding or
blocking agent.
[0103] Incubating includes conditions which allow contact between
the test composition and CCR5. Contacting includes in solution and
in solid phase. The test ligand(s)/composition may optionally be a
combinatorial library for screening a plurality of compositions.
Compositions identified in the method of the invention can be
further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
method usually applied to the detection of a specific DNA sequence
such as PCR, oligomer restriction (Saiki, et al., Bio/Technology,
3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe
analysis (Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278,
1983), oligonucleotide ligation assays (OLAs) (Landegren, et al.,
Science, 241: 1077, 1988), and the like. Molecular techniques for
DNA analysis have been reviewed (Landegren, et al., Science,
242:229-237, 1988).
[0104] To determine if a composition can functionally complex with
the receptor protein, induction of the exogenous gene is monitored
by monitoring changes in the protein levels of the protein encoded
for by the exogenous gene, for example. When a composition(s) is
found that can induce transcription of the exogenous gene, it is
concluded that this composition(s) can bind to the receptor protein
coded for by the nucleic acid encoding the initial sample test
composition(s).
[0105] Expression of the exogenous gene can be monitored by a
functional assay or assay for a protein product, for example. The
exogenous gene is therefore a gene which will provide an
assayable/measurable expression product in order to allow detection
of expression of the exogenous gene. Such exogenous genes include,
but are not limited to, reporter genes such as chloramphenicol
acetyltransferase gene, an alkaline phosphatase gene,
beta-galactosidase, a luciferase gene, a green fluorescent protein
gene, guanine xanthine phosphoribosyltransferase, alkaline
phosphatase, and antibiotic resistance genes (e.g. neomycin
phosphotransferase).
[0106] Expression of the exogenous gene is indicative of
composition-receptor binding, thus, the binding or blocking
composition can be identified and isolated. The compositions of the
present invention can be extracted and purified from the culture
media or a cell by using known protein purification techniques
commonly employed, such as extraction, precipitation, ion exchange
chromatography, affinity chromatography, gel filtration and the
like. Compositions can be isolated by affinity chromatography using
the modified receptor protein extracellular domain bound to a
column matrix or by heparin chromatography.
[0107] Also included in the screening method of the invention is
combinatorial chemistry methods for identifying chemical compounds
that bind to CCR5. Ligands/compositions that bind to CCR5 can be
assayed in standard cell:cell fusion assays, such as the vaccinia
assay described herein to determine whether the composition
inhibits or blocks env-mediated membrane fusion (i) involved in HIV
entry into a human CD4-positive target cell or (ii) between an
HIV-infected cell and an uninfected human CD4-positive target cell.
Screening methods include inhibition of chemokine binding to CCR5
(e.g. use radiolabeled chemokine) or inhibition of labeled gp120.
For example, a derivative of RANTES was shown to act as a CCR5
receptor antagonist (RANTES 9-68; Arenzana-Selsdedos et al., Nature
383:400, 1996, incorporated by reference). AOP-RANTES and
Met-RANTES were shown to bind with high affinity yet failed to
induce chemotaxis signalling, thereby acting as an antagonist
(Simmons et al., Science 276:276, 1997). Thus, the screening method
is also useful for identifying variants, binding or blocking
agents, etc., which functionally, if not physically (e.g.
sterically) act as antagonists or agonists, as desired.
[0108] Pharmaceutical Compositions
[0109] The invention also includes various pharmaceutical
compositions that block membrane fusion between HIV and a target
cell. The pharmaceutical compositions according to the invention
are prepared by bringing an antibody against CCR5, a peptide or
peptide derivative of CCR5, a CCR5 mimetic, or a CCR5-binding agent
according to the present invention into a form suitable for
administration to a subject using carriers, excipients and
additives or auxiliaries. Frequently used carriers or auxiliaries
include magnesium carbonate, titanium dioxide, lactose, mannitol
and other sugars, talc, milk protein, gelatin, starch, vitamins,
cellulose and its derivatives, animal and vegetable oils,
polyethylene glycols and solvents, such as sterile water, alcohols,
glycerol and polyhydric alcohols. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial, anti-oxidants, chelating agents and inert gases.
Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co.,
1405-1412, 1461-1487 (1975) and The National Formulary XIV., 14th
ed. Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's The Pharmacological Basis for Therapeutics
(7th ed.).
[0110] In another embodiment, the invention relates to a method of
blocking the membrane fusion between HIV and a target cell. This
method involves administering to a subject a therapeutically
effective dose of a pharmaceutical composition containing the
compounds of the present invention and a pharmaceutically
acceptable carrier. "Administering" the pharmaceutical composition
of the present invention may be accomplished by any means known to
the skilled artisan. By "subject" is meant any mammal, preferably a
human.
[0111] The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units are tablets, capsules
and suppositories. For treatment of a patient, depending on
activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the patient,
different daily doses are necessary. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or else
several smaller dose units and also by multiple administration of
subdivided doses at specific intervals.
[0112] The pharmaceutical compositions according to the invention
are in general administered topically, intravenously, orally or
parenterally or as implants, but even rectal use is possible in
principle. Suitable solid or liquid pharmaceutical preparation
forms are, for example, granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, aerosols, drops or injectable solution in ampule form and
also preparations with protracted release of active compounds, in
whose preparation excipients and additives and/or auxiliaries such
as disintegrants, binders, coating agents, swelling agents,
lubricants, flavorings, sweeteners or solubilizers are customarily
used as described above. The pharmaceutical compositions are
suitable for use in a variety of drug delivery systems. For a brief
review of present methods for drug delivery, see Langer, Science,
249: 1527-1533 (1990), which is incorporated herein by
reference.
[0113] The pharmaceutical compositions according to the invention
may be administered locally or systemically. By "therapeutically
effective dose" is meant the quantity of a compound according to
the invention necessary to prevent, to cure or at least partially
arrest the symptoms of the disease and its complications. Amounts
effective for this use will, of course, depend on the severity of
the disease and the weight and general state of the patient.
Typically, dosages used in vitro may provide useful guidance in the
amounts useful for in situ administration of the pharmaceutical
composition, and animal models may be used to determine effective
dosages for treatment of particular disorders. Various
considerations are described, e.g., in Gilman et al. (eds.) (1990)
GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS,
8th ed., Pergamon Press; and REMINGTON'S PHARMACEUTICAL SCIENCES,
17th ed. (1990), Mack Publishing Co., Easton, Pa., each of which is
herein incorporated by reference.
[0114] Testing for New Pharmaceutical Compositions
[0115] In a preferred embodiment, the invention is a method for
screening a compound ("test substance") for anti-HIV
pharmacological activity. In this embodiment, the CCR5 and CD4
genes are expressed in one type of eukaryotic cell and incubated
with a second type of eukaryotic cell that expresses an HIV
envelope protein ("env"). Fusion between at least one cell of each
type with the other type is then monitored. The test substance is
added to the incubation solution before or after mixing of the
cells and its effect on the fusion rate of cells is determined by
any of a number of means. One means to monitor fusion is to include
a system that results in the production of an active
.beta.-galactosidase upon cell fusion as described in Nussbaum et
al., 1994, supra. If the test molecule inhibits HIV infectivity
then the presence of the molecule will decrease the cell fusion
response. In the case where the test substance binds a naturally
occurring molecule present in the human that is necessary for HIV
infectivity, then addition of the test molecule may decrease cell
fusion.
[0116] The cell fusion assay can be used to determine the
functional ability of CCR5 to confer env-mediated fusion competence
to a diverse range of CD4-positive (e.g. either recombinantly
produced or naturally occurring) cell types: e.g. NIH 3T3 (murine);
BS-C-1 (African green monkey); HEK293 (human); and Mv 1 Lu (mink).
In addition, unusual, fusion-incompetent, CD4-positive human cell
types can be employed (U-87 MG glioblastoma; and SCL1).
[0117] Variations of drug screening methods are known to the
artisan of average skill in this field. Consequently, the cell
fusion assay can be used in a wide variety of formats to exploit
the properties of the CCR5 receptor to screen for drugs that are
effective against HIV.
[0118] Antisense or Ribozyme Inhibition of CCR5 for HIV Therapy
[0119] Antisense technology offers a very specific and potent means
of inhibiting HIV infection of cells that contain CCR5, for
example, by decreasing the amount of CCR5 expression in a cell.
Antisense polynucleotides in context of the present invention
includes both short sequences of DNA known as oligonucleotides of
usually 10-50 bases in length as well as longer sequences of DNA
that may exceed the length of the CCR5 gene sequence itself
Antisense polynucleotides useful for the present invention are
complementary to specific regions of a corresponding target mRNA.
Hybridization of antisense polynucleotides to their target
transcripts can be highly specific as a result of complementary
base pairing. The capability of antisense polynucleotides to
hybridize is affected by such parameters as length, chemical
modification and secondary structure of the transcript which can
influence polynucleotide access to the target site. See Stein et
al, Cancer Research 48:2659 (1988). An antisense polynucleotide can
be introduced to a cell by introducing a DNA segment that codes for
the polynucleotide into the cell such that the polynucleotide is
made inside the cell. An antisense polynucleotide can also be
introduced to a cell by adding the polynucleotide to the
environment of the cell such that the cell can take up the
polynucleotide directly. The latter route is preferred for the
shorter polynucleotides of up to about 20 bases in length.
[0120] In selecting the preferred length for a given
polynucleotide, a balance must be struck to gain the most favorable
characteristics. Shorter polynucleotides such as 10- to 15-mers,
while offering higher cell penetration, have lower gene
specificity. In contrast, while longer polynucleotides of 20-30
bases offer better specificity, they show decreased uptake kinetics
into cells. See Stein et al., PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDE
ANALOGUES in "Oligodeoxynucleotides--Antisense Inhibitors of Gene
Expression" Cohen, ed. McMillan Press, London (1988). Accessibility
to mRNA target sequences also is of importance and, therefore,
loop-forming regions in targeted mRNAs offer promising targets.
[0121] In this disclosure the term "polynucleotide" encompasses
both oligomeric nucleic acid moieties of the type found in nature,
such as the deoxyribonucleotide and ribonucleotide structures of
DNA and RNA, and man-made analogues which are capable of binding to
nucleic acids found in nature. The polynucleotides of the present
invention can be based upon ribonucleotide or deoxyribonucleotide
monomers linked by phosphodiester bonds, or by analogues linked by
methyl phosphonate, phosphorothioate, or other bonds. They may also
comprise monomer moieties which have altered base structures or
other modifications, but which still retain the ability to bind to
naturally occurring DNA and RNA structures. Such polynucleotides
may be prepared by methods well-known in the art, for instance
using commercially available machines and reagents available from
Perkin-Elmer/Applied Biosystems (Foster City, Calif.).
[0122] Phosphodiester-linked polynucleotides are particularly
susceptible to the action of nucleases in serum or inside cells,
and therefore in a preferred embodiment the polynucleotides of the
present invention are phosphorothioate or methyl phosphonate-linked
analogues, which have been shown to be nuclease-resistant. Persons
of ordinary skill in this art will be able to select other linkages
for use in the invention. These modifications also may be designed
to improve the cellular uptake and stability of the
polynucleotides.
[0123] In another embodiment of the invention, the antisense
polynucleotide is an RNA molecule produced by introducing an
expression construct into the target cell. The RNA molecule thus
produced is chosen to have the capability to hybridize to CCR5
mRNA. Such molecules that have this capability can inhibit
translation of the CCR5 mRNA and thereby inhibit the ability of HIV
to infect cells that contain the RNA molecule.
[0124] The polynucleotides which have the capability to hybridize
with mRNA targets can inhibit expression of corresponding gene
products by multiple mechanisms. In "translation arrest," the
interaction of polynucleotides with target mRNA blocks the action
of the ribosomal complex and, hence, prevents translation of the
messenger RNA into protein. Haeuptle et al., Nucl. Acids. Res.
14:1427 (1986). In the case of phosphodiester or phosphorothioate
DNA polynucleotides, intracellular RNase H can digest the targeted
RNA sequence once it has hybridized to the DNA oligomer. Walder and
Walder, Proc. Natl. Acad. Sci. USA 85:5011 (1988). As a further
mechanism of action, in "transcription arrest" it appears that some
polynucleotides can form "triplex," or triple-helical structures
with double stranded genomic DNA containing the gene of interest,
thus interfering with transcription by RNA polymerase.
Giovannangeli et al., Proc. Natl. Acad. Sci. 90:10013 (1993);
Ebbinghaus et al. J. Clin. Invest. 92:2433 (1993).
[0125] In one preferred embodiment, CCR5 polynucleotides are
synthesized according to standard methodology. Phosphorothioate
modified DNA polynucleotides typically are synthesized on automated
DNA synthesizers available from a variety of manufacturers. These
instruments are capable of synthesizing nanomole amounts of
polynucleotides as long as 100 nucleotides. Shorter polynucleotides
synthesized by modern instruments are often suitable for use
without further purification. If necessary, polynucleotides may be
purified by polyacrylamide gel electrophoresis or reverse phase
chromatography. See Sambrook et al., MOLECULAR CLONING: A
Laboratory Manual, Vol. 2, Chapter 11, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989).
[0126] Alternatively, a CCR5 polynucleotide in the form of
antisense RNA may be introduced to a cell by its expression within
the cell from a standard DNA expression vector. CCR5 DNA antisense
sequences can be cloned from standard plasmids into expression
vectors, which expression vectors have characteristics permitting
higher levels of, or more efficient expression of the resident
polynucleotides. At a minimum, these constructs require a
prokaryotic or eukaryotic promoter sequence which initiates
transcription of the inserted DNA sequences. A preferred expression
vector is one where the expression is inducible to high levels.
This is accomplished by the addition of a regulatory region which
provides increased transcription of downstream sequences in the
appropriate host cell. See Sambrook et al., Vol. 3, Chapter 16
(1989).
[0127] For example, CCR5 antisense expression vectors can be
constructed using the polymerase chain reaction (PCR) to amplify
appropriate fragments from single-stranded cDNA of a plasmid such
as pRc in which CCR5 cDNA has been incorporated. Fang et al., J.
Biol. Chem. 267:25889-25897 (1992). Polynucleotide synthesis and
purification techniques are described in Sambrook et al. and
Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
(Wiley Interscience 1987) (hereafter "Ausubel"), respectively. The
PCR procedure is performed via well-known methodology. See, for
example, Ausubel, and Bangham, "The Polymerase Chain Reaction:
Getting Started," in PROTOCOLS IN HUMAN MOLECULAR GENETICS (Humana
Press 1991). Moreover, PCR kits can be purchased from companies
such as Stratagene Cloning Systems (La Jolla, Calif.) and
Invitrogen (San Diego, Calif.).
[0128] The products of PCR are subcloned into cloning vectors. In
this context, a "cloning vector" is a DNA molecule, such as a
plasmid, cosmid or bacteriophage, that can replicate autonomously
in a host prokaryotic cell. Cloning vectors typically contain one
or a small number of restriction endonuclease recognition sites at
which foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function of the
vector, as well as a marker gene that is suitable for use in the
identification and selection of cells transformed with the cloning
vector. Suitable cloning vectors are described by Sambrook et al.,
Ausubel, and Brown (ed.), MOLECULAR BIOLOGY LABFAX (Academic Press
1991). Cloning vectors can be obtained, for example, from GIBCO/BRL
(Gaithersburg, Md.), Clontech Laboratories, Inc. (Palo Alto,
Calif.), Promega Corporation (Madison, Wis.), Stratagene Cloning
Systems (La Jolla, Calif.), Invitrogen (San Diego, Calif.), and the
American Type Culture Collection (Rockville, Md.).
[0129] Preferably, the PCR products are ligated into a "TA" cloning
vector. Methods for generating PCR products with a thymidine or
adenine overhang are well-known to those of skill in the art. See,
for example, Ausubel at pages 15.7.1-15.7.6. Moreover, kits for
performing TA cloning can be purchased from companies such as
Invitrogen (San Diego, Calif.).
[0130] Cloned antisense fragments are amplified by transforming
competent bacterial cells with a cloning vector and growing the
bacterial host cells in the presence of the appropriate antibiotic.
See, for example, Sambrook et al., and Ausubel. PCR is then used to
screen bacterial host cells for CCR5 antisense orientation clones.
The use of PCR for bacterial host cells is described, for example,
by Hofmann et al., "Sequencing DNA Amplified Directly from a
Bacterial Colony," in PCR PROTOCOLS: METHODS AND APPLICATIONS,
White (ed.), pages 205-210 (Humana Press 1993), and by Cooper et
al., "PCR-Based Full-Length cDNA Cloning Utilizing the
Universal-Adaptor/Specific DOS Primer-Pair Strategy," Id. at pages
305-316.
[0131] Cloned antisense fragments are cleaved from the cloning
vector and inserted into an expression vector. For example, HindIII
and XbaI can be used to cleave the antisense fragment from TA
cloning vector pCR.TM.-II (Invitrogen; San Diego, Calif.). Suitable
expression vectors typically contain (1) prokaryotic DNA elements
coding for a bacterial origin of replication and an antibiotic
resistance marker to provide for the amplification and selection of
the expression vector in a bacterial host; (2) DNA elements that
control initiation of transcription, such as a promoter; and (3)
DNA elements that control the processing of transcripts, such as a
transcription termination/polyadenylation sequence.
[0132] For a mammalian host, the transcriptional and translational
regulatory signals preferably are derived from viral sources, such
as adenovirus, bovine papilloma virus, simian virus, or the like,
in which the regulatory signals are associated with a particular
gene which has a high level of expression. Suitable transcriptional
and translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0133] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1: 273
(1982)); the TK promoter of Herpes virus (McKnight, Cell 31: 355
(1982)); the SV40 early promoter (Benoist et al., Nature 290: 304
(1981)); the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l Acad. Sci. USA 79: 6777 (1982)); and the cytomegalovirus
promoter (Foecking et al., Gene 45: 101 (1980)).
[0134] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
fusion gene expression if the prokaryotic promoter is regulated by
a eukaryotic promoter. Zhou et al., Mol. Cell. Biol. 10: 4529
(1990); Kaufman et al., Nucl. Acids Res. 19: 4485 (1991).
[0135] A vector for introducing at least one antisense
polynucleotide into a cell by expression from a DNA is the vector
pRc/CMV (Invitrogen, San Diego, Calif.), which provides a high
level of constitutive transcription from mammalian
enhancer-promoter sequences. Cloned CCR5 antisense vectors are
amplified in bacterial host cells, isolated from the cells, and
analyzed as described above.
[0136] Another possible method by which antisense sequences may be
exploited is via gene therapy. Virus-like vectors, usually derived
from retroviruses, may prove useful as vehicles for the importation
and expression of antisense constructs in human cells. Generally,
such vectors are non-replicative in vivo, precluding any unintended
infection of non-target cells. In such cases, helper cell lines are
provided which supply the missing replicative functions in vitro,
thereby permitting amplification and packaging of the antisense
vector. A further precaution against accidental infection of
non-target cells involves the use of target cell-specific
regulatory sequences. When under the control of such sequences,
antisense constructs would not be expressed in normal tissues.
[0137] Two prior studies have explored the feasibility of using
antisense polynucleotides to inhibit the expression of a heparin
binding growth factor. Kouhara et al., Oncogene 9: 455-462 (1994);
Morrison, J. Biol. Chem. 266: 728 (1991). Kouhara et al. showed
that androgen-dependent growth of mouse mammary carcinoma cells
(SC-3) is mediated through induction of androgen-induced, heparin
binding growth factor (AIGF). An antisense 15-mer corresponding to
the translation initiation site of AIGF was measured for its
ability to interfere with androgen-induction of SC-3 cells. At
concentrations of 5 .mu.M, the antisense polynucleotide effectively
inhibited androgen-induced DNA synthesis. Morrison showed that
antisense polynucleotides targeted against basic fibroblast growth
factor can inhibit growth of astrocytes in culture. Thus, the
general feasibility of targeting an individual gene product in a
mammalian cell has been established.
[0138] Antisense polynucleotides according to the present invention
are derived from any portion of the open reading frame of the CCR5
cDNA. Preferably, mRNA sequences (i) surrounding the translation
initiation site and (ii) forming loop structures are targeted.
Based upon the size of the human genome, statistical studies show
that a DNA segment approximately 14-15 base pairs long will have a
unique sequence in the genome. To ensure specificity of targeting
CCR5 RNA, therefore, it is preferred that the antisense
polynucleotides are at least 15 nucleotides in length. Thus, the
shortest polynucleotides contemplated by the present invention
encompass nucleotides corresponding to positions 1-14, 1-15, 1-16,
1-17, 1-18, 1-19, 2-16, 3-17, etc. of the CCR5 cDNA sequence.
Position 1 refers to the first nucleotide of the CCR5 coding
region.
[0139] Not every antisense polynucleotide will provide a sufficient
degree of inhibition or a sufficient level of specificity for the
CCR5 target. Thus, it will be necessary to screen polynucleotides
to determine which have the proper antisense characteristics. A
preferred method to assay for a useful antisense polynucleotide is
the inhibition of cell fusion between: (1) cells that contain CD4
and CCR5; and (2) cells that contain env.
[0140] Administration of an antisense polynucleotide to a subject,
either as a naked, synthetic polynucleotide or as part of an
expression vector, can be effected via any common route (oral,
nasal, buccal, rectal, vaginal, or topical), or by subcutaneous,
intramuscular, intraperitoneal, or intravenous injection.
Pharmaceutical compositions of the present invention, however, are
advantageously administered in the form of injectable compositions.
A typical composition for such purpose comprises a pharmaceutically
acceptable solvent or diluent and other suitable, physiologic
compounds. For instance, the composition may contain polynucleotide
and about 10 mg of human serum albumin per milliliter of a
phosphate buffer containing NaCl.
[0141] As much as 700 milligrams of antisense polynucleotide has
been administered intravenously to a patient over a course of 10
days (i.e., 0.05 mg/kg/hour) without signs of toxicity. Sterling,
"Systemic Antisense Treatment Reported," Genetic Engineering News
12: 1, 28 (1992).
[0142] Other pharmaceutically acceptable excipients include
non-aqueous or aqueous solutions and non-toxic compositions
including salts, preservatives, buffers and the like. Examples of
non-aqueous solutions are propylene glycol, polyethylene glycol,
vegetable oil and injectable organic esters such as ethyloleate.
Aqueous solutions include water, alcoholic/aqueous solutions,
saline solutions, parenteral vehicles such as sodium chloride,
Ringer's dextrose, etc. Intravenous vehicles include fluid and
nutrient replenishers. Preservatives include antimicrobial,
anti-oxidants, chelating agents and inert gases. The pH and exact
concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art. A
preferred pharmaceutical composition for topical administration is
a dermal cream or transdermal patch.
[0143] Antisense polynucleotides or their expression vectors may be
administered by injection as an oily suspension. Suitable
lipophilic solvents or vehicles include fatty oils, such as sesame
oil, or synthetic fatty acid esters, such as ethyloleate or
triglycerides. Moreover, antisense polynucleotides or vectors may
be combined with a lipophilic carrier such as any one of a number
of sterols including cholesterol, cholate and deoxycholic acid. A
preferred sterol is cholesterol. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension
including, for example, sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension also contains
stabilizers.
[0144] An alternative formulation for the administration of
antisense CCR5 polynucleotides involves liposomes. Liposome
encapsulation provides an alternative formulation for the
administration of antisense CCR5 polynucleotides and expression
vectors. Liposomes are microscopic vesicles that consist of one or
more lipid bilayers surrounding aqueous compartments. See,
generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.
Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46: 618
(1993). Liposomes are similar in composition to cellular membranes
and as a result, liposomes can be administered safely and are
biodegradable. Depending on the method of preparation, liposomes
may be unilamellar or multilamellar, and liposomes can vary in size
with diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s). See, for example, Machy et al.,
LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46: 1576 (1989). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0145] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents. Scherphof et al.,
Ann. N.Y. Acad. Sci. 446: 368 (1985).
[0146] After intravenous administration, conventional liposomes are
preferentially phagocytosed into the reticuloendothelial system.
However, the reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means. Claassen et al., Biochim. Biophys. Acta 802: 428 (1984). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatised phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system. Allen et al., Biochim. Biophys. Acta
1068: 133 (1991); Allen et al., Biochim. Biophys. Acta 1150: 9
(1993). These Stealth.RTM. liposomes have an increased circulation
time and an improved targeting to tumors in animals. Woodle et al.,
Proc. Amer. Assoc. Cancer Res. 33: 2672 (1992). Human clinical
trials are in progress, including Phase III clinical trials against
Kaposi's sarcoma. Gregoriadis et al., Drugs 45: 15 (1993).
[0147] Antisense polynucleotides and expression vectors can be
encapsulated within liposomes using standard techniques. A variety
of different liposome compositions and methods for synthesis are
known to those of skill in the art. See, for example, U.S. Pat. No.
4,844,904, U.S. Pat. No. 5,000,959, U.S. Pat. No. 4,863,740, and
U.S. Pat. No. 4,975,282, all of which are hereby incorporated by
reference.
[0148] Liposomes can be prepared for targeting to particular cells
or organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For instance, antibodies
specific to tumor associated antigens may be incorporated into
liposomes, together with antisense polynucleotides or expression
vectors, to target the liposome more effectively to the tumor
cells. See, for example, Zelphati et al., Antisense Research and
Development 3: 323-338 (1993), describing the use "immunoliposomes"
containing antisense polynucleotides for human therapy.
[0149] In general, the dosage of administered liposome-encapsulated
antisense polynucleotides and vectors will vary depending upon such
factors as the patient's age, weight, height, sex, general medical
condition and previous medical history. Dose ranges for particular
formulations can be determined by using a suitable animal
model.
[0150] The above approaches can also be used not only with
antisense nucleic acid, but also with ribozymes, or triplex agents
to block transcription or translation of a specific CCR5 mRNA,
either by masking that mRNA with an antisense nucleic acid or
triplex agent, or by cleaving it with a ribozyme.
[0151] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991;
Helene, C., Anticancer Drug Design, 6(6):569, 1991).
[0152] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0153] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0154] Homozygous and Heterozygous Mutations in CCR5
[0155] It is known that in some cases, a homozygous or heterozygous
mutation in a polypeptide or a regulatory region of a gene confers
a molecular basis for a difference in function. Bertina, et al. and
Greengard, et al. (Bertina, et al., Nature, 369:64, 1994;
Greengard, et al., Lancet, 343:1361, 1994), first identified the
molecular basis for the FV abnormality. The phenotype of APC
resistance was shown to be associated with heterozygosity or
homozygosity for a single point mutation in the FV gene that
resulted in the substitution of arginine at amino acid residue 506
with glutamine (FV R506Q). This R506Q mutation prevents APC from
cleaving a peptide bond at Arg-506 in FV that is required to
inactivate factor Va (Bertina, supra; Sun, et al., Blood, 83:3120,
1994).
[0156] Similarly, the present invention envisions diagnostic and
prognostic, and in addition, therapeutic approaches to treatment of
HIV-associated syndromes based on homozygosity or heterozygosity of
CCR5 mutants. For example, while not wanting to be bound by a
particular theory, it is believed that a subject having a
homozygous mutant of CCR5 may be HIV resistant or exhibit a slower
rate of disease progression. Along the same lines, a subject having
a heterozygous mutation in CCR5 may exhibit a slower rate of
disease progression than a patient having a wild type CCR5.
Mutations included in the CCR5 coding region may also result in
inactivating mutations. In addition, a mutation in the regulatory
region of CCR5 gene may prevent or inhibit expression of CCR5,
thereby providing resistance to some degree from HIV infection.
[0157] Once an individual having a homozygous or heterozygous
mutant in CCR5 is identified, it is envisioned that cells from that
individual, once matched for histocompatibility, can be
transplanted to an HIV positive individual, or to an "at risk"
individual.
[0158] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLE 1
Cloning and Sequence Analysis of the CCR5 Gene
[0159] Complementary DNAs were obtained from a .lamda.gt11 cDNA
library (Combadiere et al., DNA Cell Biol. cit op.). One of the
cDNAs obtained, designated clone 63-2, had a novel sequence highly
related to CC CKR2B, but it extended only from bp 105 to 813 of the
CC CKR2B open reading frame. The 63-2 cDNA was used as a
hybridization probe to screen under low stringency conditions
(final wash in 5.times.SSPE: described by Maniatis et al.,
Molecular Cloning a Laboratory Manual, Cold Spring Harbor
Laboratory, at 55.degree. C. for 30 min) a .lamda.pCEV9 cDNA
library prepared from endotoxin-stimulated human peripheral blood
monocytes as described previously (Combadiere et al., J. Biol.
Chem. 270: 29671-5 and J. Biol. Chem. 270: 16491-4 (1995)). One of
the isolated clones, designated clone 8.5, matched and extended the
sequence of clone 63-2. A 1.4 kb fragment of the clone 8.5 cDNA was
excised from the vector DNA by Bam HI and Bst XI double digestion,
blunt-ended with Pfu DNA polymerase, subcloned into the Eco RV site
of pBluescript II KS (Stratagene, La Jolla, Calif.), and sequenced
completely on both strands. The cDNA insert was then subcloned
between the Bam HI and Hind III sites of the mammalian expression
vector pREP9 (Invitrogen, San Diego, Calif.).
[0160] The clone 8.5 cDNA is 1.4 kb in length. The 5'- and
3'-untranslated regions and open reading frame are 26,288 and 1056
bp, respectively. The 3'-untranslated region is not polyadenylated
and lacks a polyadenylation consensus sequence. The ATG codon
proposed to initiate translation is flanked by sequence that
conforms favorably with established consensus rules. M. Kozak,
Nucleic Acids Res. 15:8125-48 (1987). The open reading frame
contains 352 codons.
[0161] Seven segments of the deduced amino acid sequence from SEQ
ID NO: 1 have a high content of hydrophobic amino acids consistent
with membrane-spanning domains as well as multiple amino acids
conserved in analogous positions of the known
seven-transmembrane-domain receptor rhodopsin. These considerations
clearly indicate that CCR5 is ancestrally related to rhodopsin-like
receptors, and strongly suggest that it functions as a
seven-transmembrane-domain G protein-coupled receptor. A database
search revealed that the highest sequence identity occurs with
chemokine receptors. In particular, the amino acid sequence of CCR5
is 57, 70, 75, 51 and 48% identical to CC CKR1, CC CKR2A, CC CKR2B,
CC CKR3 and CC CKR4, respectively, with lower identity
(approximately 30%) to the CXC chemokine receptors, IL-8 receptors
A and B. An alignment of the amino acid sequence of CCR5 with those
of CC CKR1 and CC CKR2B is shown in FIG. 1.
[0162] The CC CKR2B sequence is eight amino acids longer than CCR5
due to a substantially longer N-terminal segment. The two sequences
are otherwise colinear with the exception of a four amino acid gap
in the putative second extracellular loop of CC CKR2B relative to
that of CCR5.
[0163] Residues of CCR5 that differ from CC CKR2B in the alignment
are found mostly in the putative extracellular domains (the N
terminal segment and the sequence between putative transmembrane
domains 2 and 3, 4 and 5, and 6 and 7) and adjacent portions of the
transmembrane domains, and in the C-terminal segment which is
predicted to lie in the cytoplasm (FIG. 2). Like other
seven-transmembrane-domain receptors, the C-terminal tail has a
high content of serine and threonine residues that may be sites for
receptor phosphorylation as they are in rhodopsin and the .beta.
adrenergic receptor. CCR5 also contains cysteine residues that by
analogy with other seven-transmembrane domain receptors could be a
site for palmitoylation, tethering this domain to the plasma
membrane.
[0164] The net charge of the N-terminal extracellular segment of
CCR5 is -1. The corresponding domain of CC CKR2B has a net charge
of zero, whereas for other known chemokine receptors this domain is
highly acidic. Like all other known chemokine receptors, CCR5 has
conserved cysteine residues in the N-terminal segment and the third
predicted extracellular loop that could form a disulfide bond.
Cysteine residues are less frequently found in this location in
other seven-transmembrane-domain receptors. CCR5 lacks a consensus
sequence for N-linked glycosylation, whereas one or more are found
in other CC CKR's.
[0165] The CCR5 Gene. When total human genomic DNA was digested
with Pst I, Eco RI, Hind III and Xba I and hybridized with a CCR5
cDNA probe extending from bp 69 to 789 relative to the start of the
open reading frame, 2-4 bands that hybridized with different
intensity were detected in each lane. The probe used contains a
recognition site for Pst I at bp 532 of the CCR5 open reading frame
but not for the other restriction enzymes used. The pattern is most
consistent with multiple small cross-hybridizing genes. In fact,
restriction fragments of genomic clones isolated for CC CKR2, CC
CKR3 and CCR5 can account for all of the bands seen. The CC CKR2B,
CC CKR3 and CCR5 open reading frames lack intervening
sequences.
EXAMPLE 2
Chemokine Binding to CCR5
[0166] Human embryonic kidney (HEK) 293 cells (a total of 10.sup.7)
grown to log phase in DMEM and 10% fetal bovine serum were
electroporated with 20 .mu.g of plasmid DNA, and G418-resistant
colonies were picked and expanded as described previously
(Combadiere et al., op cit.). The cell lines studied contained
large amounts of the recombinant CCR5 mRNA, but lacked CC CKR3 mRNA
and native 8.5 mRNA, as assessed by Northern blot analysis of total
RNA using full-length cDNA probes. The methods used to create HEK
293 cell lines stably expressing CC CKR1 and CC CKR2B have been
described previously (Combadiere et al., J. Biol. Chem. 270:
29671-5).
[0167] Transfected HEK 293 cells (a total of 10.sup.6) were
incubated in duplicate with 0.2 nM .sup.125I-labeled RANTES, MCP-1,
MIP-1.alpha., MIP-1.beta. or MCP-3 (specific activity -2200
Ci/mmol, Du Pont/NEN, Boston, Mass.) and varying concentrations of
unlabeled recombinant human chemokines (Peprotech, Rocky Hill,
N.J.) in 200 .mu.l of binding medium (RPMI 1640 with 1 mg/ml BSA
and 25 mM HEPES, pH 7.4). After incubation for 1 h at 4.degree. C.
or 37.degree. C., unbound chemokines were separated from cells by
pelleting through a 10% sucrose/PBS cushion, and the
cell-associated counts were determined. Specific binding was
determined by the difference in counts in the presence and absence
of 1250-fold molar excess of unlabeled chemokine.
[0168] Receptor activation from chemokine binding was assessed by
real time measurement of [Ca.sup.+2].sub.i changes using 2 million
transfected HEK 293 cells loaded with FURA-2. Ratio fluorescence of
cells was measured as described previously. Combadiere et al., J.
Biol. Chem. 270: 16491-4 (1995). J. Van Damme (Rega Institute,
Leuven) provided chemically synthesized human MCP-2 protein
according to a procedure described in Proost et al., Cytokine 7:
97-104 (1995). O. Yoshie (Shionogi Institute, Osaka) provided
recombinant human eotaxin. Where indicated, cells loaded with
FURA-2 were incubated in holotoxin of B. pertussis (List, Campbell,
Calif.) 250 ng/ml for 2 h at 37.degree. C., then washed twice in
PBS and resuspended in HBSS. Cell viability was .about.80% by
trypan blue exclusion after pertussis toxin treatment. ATP was
purchased from Sigma Co. (St. Louis, Mo.).
Agonists for CCR5. Given the high sequence similarity of CCR5 with
other CC chemokine receptors, the inventors predicted that CCR5
would be specific for CC chemokines. To test this, the inventors
transfected HEK 293 cells, which normally are unresponsive to
stimulation with chemokines, with the clone 8.5 cDNA and measured
the calcium flux responses induced by a panel of chemokines. The
calcium flux response is strongly associated with chemotaxis,
degranulation and other higher order leukocyte responses to
chemokines, and is a sensitive and specific measure of receptor
activation. Neither untransfected nor mock-transfected and selected
HEK 293 cells responded to any of the chemokines tested. In
contrast, six independent HEK 293 cell lines stably transfected
with the clone 8.5 cDNA, three each from two separate
transfections, exhibited [Ca.sup.2+].sub.i transients in response
to MIP-1.alpha., RANTES and MIP-1.beta., but not in response to
MCP-1, MCP-2, MCP-3, eotaxin, IL-8 or .gamma.IP-10 all tested at
100 nM. MIP-1.alpha., MIP-1.beta. and RANTES were similar in
potency and efficacy, the concentrations for half-maximal and
maximal responses ranging from 5-40 and 25-50 nM, respectively.
These results indicate that CCR5 is a CC chemokine receptor
selective for MIP-1.alpha., MIP-1.beta. and RANTES. Desensitization
of CCR5. After activation, chemokine receptors have altered
sensitivity to repeated stimulation with the activating agonist and
other agonists. When the same chemokine was added in succession to
CCR5 transfectants, cells responded to the first addition but not
the second, indicating that the receptor underwent homologous
desensitization to all three of its agonists. When different
agonists were added in succession, MIP-1.alpha. or RANTES given
first blocked the response to MIP-1.beta. given second, and
MIP-1.beta. or RANTES given first blocked the response to
MIP-1.alpha. given second. But MIP-1.alpha. or MIP-1.beta. given
first reduced, but did not eliminate, the response to RANTES given
second. MCP-1 had no effect on the responses to MIP-1.alpha.,
MIP-1.beta. or RANTES. These data show a functional interaction of
MIP-1.alpha., MIP-1.beta. and RANTES with the same receptor, CCR5.
G Protein Coupling to CCR5. Known chemokine receptors couple to
G.sub.i-type G proteins, which unlike other classes of G proteins
are functionally sensitive to pertussis toxin. Treatment of CCR5
transfectants with pertussis toxin completely abolished the calcium
flux response to MIP-1.alpha., MIP-1.beta. and RANTES. In contrast,
the calcium flux response to ATP was largely unaffected. These data
indicate that CCR5 in HEK 293 cells is coupled to G proteins of the
G.sub.i class.
[0169] Binding of CC Chemokines to CCR5. The calcium flux results
show that cells expressing CCR5 have acquired the capacity to
respond to the presence of MIP-1.alpha., MIP-1.beta. and RANTES.
The mechanism of this effect appears to be related to specific
binding to CCR5 on the cell surfaces as judged by radioligand
binding assays with intact HEK 293 cells that were stably
transfected with CCR5 and by comparison with HEK 293 cells stably
transfected with CC CKR1 and CC CKR2B as positive and negative
controls, respectively. The results for CCR5 are quite complex, and
the results for the positive and negative controls are described
first.
[0170] The total amounts of .sup.125I-MIP-1.alpha.,
.sup.125I-MIP-1.beta. and .sup.125I-RANTES that bound to CC CKR2B
were similar in magnitude to untransfected HEK 293 cells, and were
completely non-specific in both cases, whether the assays were
carried out at 4.degree. C. or 37.degree. C. In contrast, CC CKR2B
transfectants specifically bound both .sup.125I-MCP-1 and
.sup.125I-MCP-3 at both 4.degree. C. and 37.degree. C. These
results are consistent with the known agonists for CC CKR2B.
[0171] In the case of CC CKR1, specific binding of
.sup.125I-MIP-1.alpha. was 5-10-fold greater than non-specific
binding at both 4.degree. C. and 37.degree. C., whereas specific
binding of .sup.125I-MCP-1 was not detectable. The K.sub.i for
homologous competition binding of .sup.125I-MIP-1.alpha. at
4.degree. C. was 10 nM. These results are similar to previously
published results, are consistent with MIP-1.alpha.'s agonist
activity for CC CKR1, and represent a positive control for
MIP-1.alpha. binding. Each of the six CCR5 transfectants were
tested at 4.degree. C. The total binding of .sup.125I-MIP-1.alpha.,
.sup.125I-MIP-1.beta. and .sup.125I-RANTES was equal to the
background levels established for the CC CKR2B transfectant, and
was completely non-specific even when radioligand concentrations as
high as 5 nM were tested. Yet, all six cell lines exhibited clear
and robust calcium flux responses to MIP-1.alpha., MIP-1.beta. and
RANTES.
[0172] The G418 concentration in the media of the CCR5 transfectant
that exhibited the strongest calcium flux response was increased
from 1 to 3 mg/ml for one week. A cell line was derived, named
CCR5.1 that exhibited calcium flux responses to MIP-1.alpha.,
MIP-1.beta. and RANTES that were consistently double those of the
parental cell line. This cell line exhibited specific binding at
4.degree. C. for MIP-1.alpha., MIP-1.beta. and RANTES.
[0173] To increase the sensitivity of the binding assay,
non-equilibrium conditions at 37.degree. C. were used to increase
the ratio of specific to non-specific binding for
.sup.125I-MIP-1.alpha. .sup.125I-MIP-1.beta. and .sup.125I-RANTES
by a factor of 2-4 for both CC CKR1 and CCR5.1 cell lines, compared
to results obtained at 4.degree. C. .sup.125I-MCP-1 did not bind
specifically to either CC CKR1 or CCR5.1 cells at 37.degree. C.,
whereas .sup.125I-MCP-3 bound specifically to CC CKR1 but not to
CCR5.1 cells.
[0174] The .sup.125I-MIP-1.alpha. and .sup.125I-MIP-1.beta. binding
sites on CC CKR1 and CCR5.1 cells were easily distinguished in two
ways. First, .sup.125I-MIP-1.alpha. and .sup.125I-MIP-1.beta.
binding to CC CKR1, but not to CCR5.1, was competed effectively by
unlabeled MCP-3. Second, unlabeled MIP-1.alpha. competed 20-fold
more effectively for .sup.125I-MIP-1.alpha. binding to CC CKR1 than
to CCR5.1 (half-maximal inhibitory
concentrations[IC.sub.50].about.5 and 100 nM, respectively), and
unlabeled MIP-1.beta. competed-2-fold more effectively for
.sup.125I-MIP-.beta. binding to CCR5.1 than to CC CKR1
(IC.sub.50S.about.100 and 200 nM, respectively).
[0175] At 37.degree. C., .sup.125I-RANTES bound to both CC CKR1 and
CCR5.1 cells at low but significantly increased levels compared to
the negative control CC CKR2B cells. Excess unlabeled RANTES
reduced 125I-RANTES binding to CCR5.1 cells (IC.sub.50.about.80
nM). The .sup.125I-RANTES binding sites on CC CKR1 and CCR5.1 could
be distinguished by heterologous competition with excess unlabeled
MIP-1.alpha. (IC.sub.50-20 and 100 nM for CC CKR1 and CCR5.1
respectively).
Distribution of CCR5 RNA. Compared to other CC CKRs, CCR5 is most
like CC CKR2A and CC CKR2B not only in its primary sequence but
also in its RNA distribution. Full-length CC CKR2B and CCR5 open
reading frame probes recognized a 3.5 kb RNA band by Northern blot
hybridization in total RNA made from adherent monocytes. Neither
probe recognized RNA in neutrophil or eosinophil samples. To
determine whether the CCR5 probe was merely cross-hybridizing to
the monocyte CC CKR2 mRNA, a 30-mer antisense oligonucleotide
specific for CCR5 was designed. This probe also detected the 3.5 kb
monocyte mRNA species. A similar analysis using specific
oligonucleotide probes has indicated that both CC CKR2A and CC
CKR2B RNA is present in adherent monocytes.
EXAMPLE 3
Cell Fusion Assay Suitable for Drug Screening
[0176] A vaccinia cell fusion system is used to assay the
functional ability of CCR5 to confer env-mediated fusion competence
to CD4-positive nonhuman cells. This assay is carried out as
described in Nussbaum et al., 1994, supra. In the assay murine NIH
3T3 cells or human HeLa cells are first transfected with the
plasmid pSC59.CCR5 and then co-infected with various vaccinia
viruses: vTF7-3 (containing the T7 RNA polymerase gene); vCB3
(containing the human CD4 gene); and vaccinia WR (a negative
control). A different cell population is co-infected with various
vaccinia viruses: vCB-21R (containing the E. coli lacZ gene under
the transcriptional control of a T7 promoter (P.sub.T7-lacZ)) along
with either a vaccinia virus that encodes a Ba-L env gene from a
macrophage-tropic isolate or vCB-16 (a negative control, containing
a mutant env gene encoding an uncleavable, nonfusogenic
unc/env).
[0177] The cell populations described above are incubated overnight
at 31.degree. C. to allow expression of the vaccinia-encoded
proteins. The cells are washed and mixtures of each combination are
prepared in 96-well microtiter plates. Each well contains equal
numbers of T7 RNA polymerase-containing cells and lacZ
gene-containing cells. Replicate plates are incubated for 2-4 hours
at 37.degree. C. to allow fusion. Samples on one plate are treated
with NP-40 and aliquots are assayed for .beta.-galactosidase
activity using a 96-well absorbance reader.
[0178] The .beta.-galactosidase assay results from this experiment
will show that NIH 3T3 cells coexpressing human CD4 and CCR5 are
highly competent for fusion with cells expressing env from the
macrophage-tropic isolate (Ba-L) but not from a T-cell line-tropic
isolate (LAV). In contrast, the data will indicate that NIH 3T3
cells coexpressing human CD4 alone or CCR5 alone are incompetent
for fusion with cells expressing env. Furthermore, the low
background levels of .beta.-galactosidase produced will indicate
that NIH 3T3 cells coexpressing human CD4 and CCR5 do not fuse with
cells expressing mutant unc/env.
[0179] In a related experiment, several colonies of stable,
transformed mink cells that coexpress human CD4 and CCR5 would be
tested for susceptibility to HIV-1 infection by macrophage-tropic
or dual-tropic HIV-1 strains (e.g. strains that use CCR5).
Transformants containing the human CD4 gene and an irrelevant
control gene are used as negative controls. Direct measurements of
p24 (HIV core antigen) production will indicate that HIV-1
infection is productive with cells that coexpress human CD4 and
CCR5, but not with the negative controls. Moreover, the efficiency
of HIV-1 infection of transformed, CD4-positive, CCR5-positive,
nonhuman cells is high enough to be detected directly.
EXAMPLE 4
Anti-CCR5 Antibody Blocks env-Mediated Membrane Fusion
[0180] Based on the known topology of 7-transmembrane segment
proteins, four regions of CCR5 are predicted to be exposed at the
cell surface. Natural or synthetic peptides are produced or
synthesized by methods well-known in the art that correspond to
each of these 4 regions. Rabbit antisera is raised by immunization
with peptide-KLH (keyhole limpet hemocyanin) conjugates. Total
immunoglobulin is purified from the preimmune and the immune sera
by chromatographic separation with Protein-A Sepharose.
Alternatively, whole cells expressing CCR5 can be used to generate
anti-CCR5 antibodies.
[0181] Antibodies raised against an 28 amino acid N-terminal
portion of CCR5 or against the extracellular loops (e.g. el-1), can
block membrane fusion between macrophage-tropic strains Ba-L,
SF162, JR-FL and ADA of HIV and human macrophages, in other words
strains that use CCR5. (See, for example, Feng et al., 1996, supra;
Endres et al., 1996, supra).
EXAMPLE 5
Specificity of CCR5 for env from Macrophage-Tropic Isolates
[0182] The sensitivity of fusion mediated by env from different HIV
isolates is tested with antibodies prepared against the N-terminal
portion of CCR5. The anti-CCR5 antibodies inhibit fusion mediated
by the prototypic macrophage-tropic Ba-L env, but will not inhibit
fusion mediated by the prototypic T-cell line-tropic LAV env. The
fusion inhibition with anti-CCR5 antibodies is not due to
nonspecific inhibitory effects on the cells. Coexpression of CCR5
enhances fusion more with env from macrophage-tropic strains (Ba-L,
SF162, JR-FL, and ADA) than with env from T-cell line-tropic
isolates (IIIB, LAV, and RF).
EXAMPLE 6
CCR5 Peptides Block env-Mediated Membrane Fusion
[0183] Synthetic peptides that correspond to the predicted
extracellular loops of CCR5 were prepared and tested for inhibition
of env-mediated membrane fusion. Peptides were as follows:
TABLE-US-00002 (SEQ ID NO: 5) extracellular loop-1: LAAQWDFGNTMC
(SEQ ID NO: 6) extracellular loop-2: RSQKEGLHYTCSSHFPYSQYQFWK (SEQ
ID NO: 7) extracellular loop-3: QEFFGLNNCSSSNRLD
[0184] The peptides were tested using vaccinia-based expression and
reporter gene assay system (see Example 3 above). Cell fusion was
quantitated by determining the level of .beta.-galactosidase in
detergent cell lysates.
[0185] FIG. 2 shows that each peptide (0-50 .mu.g/ml) was able to
inhibit fusion between cells expressing the HIV-1 Env from the
macrophage-tropic Ba-L isolate and murine cells co-expressing CD4
and CCR5.
EXAMPLE 7
Cell Lines Expressing CCR5
[0186] Human HeLa, human embryonic kidney (HEK) 293, and murine NIH
3T3 cell lines (American Type Culture Collection, Rockville, Md.)
were cultured in DMEM-10 (Dulbecco's modified Eagle's medium
[Quality Biologicals, Gaithersburg, Md.] containing 10% fetal
bovine serum [FBS, HyClone, Logan, Utah], 2 mM L-glutamine, 100
U/ml penicillin and 100 .mu.g/ml streptomycin). The human PM1 T
cell line (Lusso et al., 1995) was obtained from the NIH AIDS
Research and Reference Reagent Program (Rockville, Md.) and was
grown in RPMI-10 (RPMI 1640 medium [Quality Biologicals] containing
10% FBS, 10 mM HEPES, 2 mM glutamine, and antibiotics). Recombinant
vaccinia virus stocks were prepared by standard procedures (Earl et
al., 1991). Pertussis toxin was obtained from List (Campbell,
Calif.). Recombinant chemokines were purchased from Peprotech
(Rocky Hill, N.J.). Fura 2-AM and propidium iodide were obtained
from Molecular Probes (Eugene, Oreg.). Sodium azide and ATP were
from Sigma (St. Louis, Mo.).
[0187] CCR5 Constructs. Epitope-tagged variants of CCR5 were
created to enable detection by the M5 monoclonal antibody (Kodak,
Rochester, N.Y.). The CCR5 open reading frame was amplified by PCR
using the following primers: 1) for full-length CCR5 (designated
CCR5): a 3'-oligonucleotide containing (from 3' to 5') 27 bases
complementary to the last 9 codons of CCR5, 3 bases for the stop
codon, 6 bases for an Xho I restriction site and 8 miscellaneous
bases; 2) for CCR5 lacking most of the cytoplasmic C-terminus
(designated CCR5.sub.306): a 3'-oligonucleotide containing (from 3'
to 5') 27 bases complementary to codons 298-306 of CCR5, 3 bases
for a stop codon, 6 bases for an Xho I restriction site and 8
miscellaneous bases; and 3) for both constructs: a
5'-oligonucleotide containing (from 5' to 3') 8 miscellaneous
bases, 6 bases for a Hind III site, 3 bases for the start codon, 24
bases encoding the flag epitope DYKDDDDK (SEQ ID NO: 10) and 27
bases complementary to CCR5 codons 2 to 10. The resulting two PCR
products were digested and subcloned between the Hind III and Xho I
sites of the changes using a MSIII fluorimeter (Photon Technology
International, S. Brunswick, N.J.) in HEK 293 cell lines expressing
receptor constructs as previously described. Fuerst, T. R., Niles,
E. G., Studier, F. W., and Moss, B. (1986). Briefly, cells were
loaded with 2 .mu.M FURA-2 AM at 37.degree. C. for 45 min, washed
twice and resuspended at 10.sup.6 cells/ml in HBSS, pH 7.4. Two ml
of the cell suspension were placed in a stirred, water-jacketed
cuvette at 37.degree. C. and excited sequentially at 340 and 380
nm. Fluorescence emission was monitored at 510 nm before and after
addition of agonists. For some experiments, cells were incubated
with 250 ng/ml pertussis toxin for 3 h prior to functional
assay.
Cell Fusion Assay. Fusion between effector cells expressing HIV-1
Env and target cells expressing CD4 was quantitated by a
vaccinia-based reporter gene assay in which .beta.-galactosidase is
produced selectively in fused cells (Nussbaum et al., 1994). As
effector cells, HeLa cells were coinfected with vCB-21R, which
encodes the E. Coli LacZ gene under control of the bacteriophage T7
promoter (J. Virol. 70, 5487-5494.), and a recombinant vaccinia
virus encoding one of the following HIV-1 Envs (PNAS. 92,
9004-9008.): M-tropic Envs Ba-L (vCB-43; note this is a correction
of the nomenclature used for this virus in Broder and Berger,
1995), ADA (vCB-39), SF-162 (vCB-32), and JR-FL (vCB-28); and Unc,
an uncleavable mutant of IIIB (vCB-16). In one protocol, the target
cells were HEK 293 cell transfectants stably expressing the
indicated CCR5 constructs. These cells were coinfected with vTF7-3
encoding T7 RN polymerase (Fuerst et al., 1993) and vCB-3 encoding
human CD4 (Cell 85, 1149-12158.). In both viruses the foreign genes
are linked to vaccinia early/late promoters; the multiplicity of
infection was 10 pfu/cell for each virus. In another protocol, the
target cells were NIH 3T3 cells transfected with pcDNA3-based
plasmids encoding CCR5 or CCR5.sub.306 using DOTAP lipofection
(Boehringer Mannheim, Indianapolis, Ind.); control cells were
transfected with pcDNA3 vector alone. After 4 h incubation in DOTAP
at 37.degree. C., cells were coinfected with vTF7-3 and vCB-3;
expression of the CCR5 constructs was driven by the T7 promoter.
Cell cultures were incubated at 31.degree. C. overnight.
[0188] Cell surface expression of CCR5 and CCR5.sub.306 was
analyzed by flow cytometry using as the probe either a rabbit
polyclonal antiserum generated against a synthetic peptide
representing the predicted extracellular amino terminal domain of
CCR5 (amino acids 1-28), or a mAb recognizing the Flag epitope.
Specific cell surface staining at comparable intensity was obtained
when HEK293 cells stably transfected with either CCR5 or
CCR5.sub.306 were incubated with the anti-CCR5 antiserum. In
contrast, cells stably transfected with the closely related
receptor CCR2b (75% amino acid identity) gave only background
fluorescence equivalent to that observed with the signaling is
required for the HIV-1 coreceptor activity of CCR5, using a
quantitative vaccinia-based reporter gene assay of HIV-1
Env-mediated cell fusion HEK 293 cell transfectants expressing CCR5
or CCR5.sub.306 (along with vaccinia-encoded CD4) were tested for
their ability to fuse with HeLa cells expressing vaccinia-encoded
Envs from several M-tropic strains. Comparable levels of fusion
occurred with CCR5 and CCR5.sub.306 for each Env tested. Similar
results were obtained in an alternative protocol whereby CCR5 and
CCR5.sub.306 were expressed on NIH 3T3 cells using a transient
vaccinia expression system. Thus, the C-terminal truncation that
abolished the G protein signal transduction activity of CCR5 had no
effect on fusion coreceptor activity.
[0189] Pertussis toxin provided an alternative means to test the
requirement for G protein signal transduction in the fusion
coreceptor activity of CCR5. In the cell fusion assay using the
M-tropic Ba-L Env, high concentration of the toxin (500 ng/ml) had
no effect on the fusion coreceptor activity of CCR5 expressed
transiently in NIH 3T3 cells.
[0190] The effects of pertussis toxin were also tested on
productive HIV-1 infection, using the Jurkat-derived T cell line
PM1 as the target. PM1 cells express CD4 and are highly susceptible
to M-tropic HIV-1 strains. Moreover, CCR5 mRNA is expressed in
these cells and infection by M-tropic HIV-1 on PM1 cells is CCR5.
In the continuous presence of pertussis toxin (500 ng/ml), robust
infection by the M-tropic Ba-L isolate in PM1 cells was observed.
Consistent with this, when PM1 cells were used as target cells in
the cell fusion assay with effector HeLa cells expressing the Ba-L
Env, high levels of fusion activity were observed and this was
completely resistant to pertussis toxin. Thus, pertussis toxin at
concentrations that potently block G protein-mediated signal
transduction had minimal effect on either Env-mediated cell fusion
or productive infection. These results parallel earlier reports
that pertussis toxin did not process known as receptor
sequestration or downmodulation. This process is thought to explain
in part the phenomenon of receptor desensitization and could be
important either for HIV-1 Env-dependent membrane fusion, and/or
chemokine inhibition of fusion. CCR5 was strongly downmodulated by
chemokine ligands, whereas the truncated CCR5.sub.306 receptor was
unaffected. This indicates that, in addition to containing critical
determinants of signaling, the C-terminal domain of CCR5 also
contains critical determinants for chemokine-mediated
down-modulation.
[0191] It will be apparent to those skilled in the art that various
modifications and variations can be made to the compositions and
processes of this invention. Thus, it is intended that the present
invention cover such modifications and variations, provided they
come within the scope of the appended claims and their equivalents.
Sequence CWU 1
1
1111225DNAHomo sapiensCDS(27)..(1082) 1aagaaactct ccccgggtgg aacaag
atg gat tat caa gtg tca agt cca atc 53Met Asp Tyr Gln Val Ser Ser
Pro Ile1 5tat gac atc aat tat tat aca tcg gag ccc tgc caa aaa atc
aat gtg 101Tyr Asp Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Gln Lys Ile
Asn Val10 15 20 25aag caa atc gca gcc cgc ctc ctg cct ccg ctc tac
tca ctg gtg ttc 149Lys Gln Ile Ala Ala Arg Leu Leu Pro Pro Leu Tyr
Ser Leu Val Phe30 35 40atc ttt ggt ttt gtg ggc aac atg ctg gtc atc
ctc atc ctg ata aac 197Ile Phe Gly Phe Val Gly Asn Met Leu Val Ile
Leu Ile Leu Ile Asn45 50 55tgc aaa agg ctg aag agc atg act gac atc
tac ctg ctc aac ctg gcc 245Cys Lys Arg Leu Lys Ser Met Thr Asp Ile
Tyr Leu Leu Asn Leu Ala60 65 70atc tct gac ctg ttt ttc ctt ctt act
gtc ccc ttc tgg gct cac tac 293Ile Ser Asp Leu Phe Phe Leu Leu Thr
Val Pro Phe Trp Ala His Tyr75 80 85ttg gcc gcc cag tgg gac ttt gga
aat aca atg tgt caa ctc ttg aca 341Leu Ala Ala Gln Trp Asp Phe Gly
Asn Thr Met Cys Gln Leu Leu Thr90 95 100 105ggg ctc tat ttt ata ggc
ttc ttc tct gga atc ttc ttc atc atc ctc 389Gly Leu Tyr Phe Ile Gly
Phe Phe Ser Gly Ile Phe Phe Ile Ile Leu110 115 120ctg aca atc gat
agg tac ctg gct gtc gtc cat gct gtg ttt gct tta 437Leu Thr Ile Asp
Arg Tyr Leu Ala Val Val His Ala Val Phe Ala Leu125 130 135aaa gcc
agg acg gtc acc ttt ggg gtg gtg aca agt gtg atc act tgg 485Lys Ala
Arg Thr Val Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp140 145
150gtg gtg gct gtg ttt gcg tct ctc cca gga atc atc ttt acc aga tct
533Val Val Ala Val Phe Ala Ser Leu Pro Gly Ile Ile Phe Thr Arg
Ser155 160 165caa aaa gaa ggt ctt cat tac acc tgc agc tct cat ttt
cca tac agt 581Gln Lys Glu Gly Leu His Tyr Thr Cys Ser Ser His Phe
Pro Tyr Ser170 175 180 185cag tat caa ttc tgg aag aat ttc cag aca
tta aag ata gtc atc ttg 629Gln Tyr Gln Phe Trp Lys Asn Phe Gln Thr
Leu Lys Ile Val Ile Leu190 195 200ggg ctg gtc ctg ccg ctg ctt gtc
atg gtc atc tgc tac tcg gga atc 677Gly Leu Val Leu Pro Leu Leu Val
Met Val Ile Cys Tyr Ser Gly Ile205 210 215cta aaa act ctg ctt cgg
tgt cga aat gag aag aag agg cac agg gct 725Leu Lys Thr Leu Leu Arg
Cys Arg Asn Glu Lys Lys Arg His Arg Ala220 225 230gtg agg ctt atc
ttc acc atc atg att gtt tat ttt ctc ttc tgg gct 773Val Arg Leu Ile
Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp Ala235 240 245ccc tac
aac att gtc ctt ctc ctg aac acc ttc cag gaa ttc ttt ggc 821Pro Tyr
Asn Ile Val Leu Leu Leu Asn Thr Phe Gln Glu Phe Phe Gly250 255 260
265ctg aat aat tgc agt agc tct aac agg ttg gac caa gct atg cag gtg
869Leu Asn Asn Cys Ser Ser Ser Asn Arg Leu Asp Gln Ala Met Gln
Val270 275 280aca gag act ctt ggg atg acg cac tgc tgc atc aac ccc
atc atc tat 917Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro
Ile Ile Tyr285 290 295gcc ttt gtc ggg gag aag ttc aga aac tac ctc
tta gtc ttc ttc caa 965Ala Phe Val Gly Glu Lys Phe Arg Asn Tyr Leu
Leu Val Phe Phe Gln300 305 310aag cac att gcc aaa cgc ttc tgc aaa
tgc tgt tct att ttc cag caa 1013Lys His Ile Ala Lys Arg Phe Cys Lys
Cys Cys Ser Ile Phe Gln Gln315 320 325gag gct ccc gag cga gca agc
tca gtt tac acc cga tcc act ggg gag 1061Glu Ala Pro Glu Arg Ala Ser
Ser Val Tyr Thr Arg Ser Thr Gly Glu330 335 340 345cag gaa ata tct
gtg ggc ttg tgacacggac tcaagtgggc tggtgaccca 1112Gln Glu Ile Ser
Val Gly Leu350gtcagagttg tgcacatggc ttagttttca tacacagcct
gggctggggg tggggtggga 1172gaggtctttt ttaaaaggaa gttactgtta
tagagggtct aagattcatc cat 12252352PRTHomo sapiens 2Met Asp Tyr Gln
Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr1 5 10 15Ser Glu Pro
Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu20 25 30Leu Pro
Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn35 40 45Met
Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met50 55
60Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu65
70 75 80Leu Thr Val Pro Phe Trp Ala His Tyr Leu Ala Ala Gln Trp Asp
Phe85 90 95Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile
Gly Phe100 105 110Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile
Asp Arg Tyr Leu115 120 125Ala Val Val His Ala Val Phe Ala Leu Lys
Ala Arg Thr Val Thr Phe130 135 140Gly Val Val Thr Ser Val Ile Thr
Trp Val Val Ala Val Phe Ala Ser145 150 155 160Leu Pro Gly Ile Ile
Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr165 170 175Thr Cys Ser
Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn180 185 190Phe
Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu195 200
205Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg
Cys210 215 220Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile
Phe Thr Ile225 230 235 240Met Ile Val Tyr Phe Leu Phe Trp Ala Pro
Tyr Asn Ile Val Leu Leu245 250 255Leu Asn Thr Phe Gln Glu Phe Phe
Gly Leu Asn Asn Cys Ser Ser Ser260 265 270Asn Arg Leu Asp Gln Ala
Met Gln Val Thr Glu Thr Leu Gly Met Thr275 280 285His Cys Cys Ile
Asn Pro Ile Ile Tyr Ala Phe Val Gly Glu Lys Phe290 295 300Arg Asn
Tyr Leu Leu Val Phe Phe Gln Lys His Ile Ala Lys Arg Phe305 310 315
320Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala
Ser325 330 335Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser
Val Gly Leu340 345 35031225DNAHomo sapiensCDS(27)..(1082)
3aagaaactct ccccgggtgg aacaag atg gat tat caa gtg tca agt cca atc
53Met Asp Tyr Gln Val Ser Ser Pro Ile1 5tat gac atc aat tat tat aca
tcg gag ccc tgc caa aaa atc aat gtg 101Tyr Asp Ile Asn Tyr Tyr Thr
Ser Glu Pro Cys Gln Lys Ile Asn Val10 15 20 25aag caa atc gca gcc
cgc ctc ctg cct ccg ctc tac tca ctg gtg ttc 149Lys Gln Ile Ala Ala
Arg Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe30 35 40atc ttt ggt ttt
gtg ggc aac atg ctg gtc atc ctc atc ctg ata aac 197Ile Phe Gly Phe
Val Gly Asn Met Leu Val Ile Leu Ile Leu Ile Asn45 50 55tgc aaa agg
ctg aag agc atg act gac atc tac ctg ctc aac ctg gcc 245Cys Lys Arg
Leu Lys Ser Met Thr Asp Ile Tyr Leu Leu Asn Leu Ala60 65 70atc tct
gac ctg ttt ttc ctt ctt act gtc ccc ttc tgg gct cac tat 293Ile Ser
Asp Leu Phe Phe Leu Leu Thr Val Pro Phe Trp Ala His Tyr75 80 85gct
gcc gcc cag tgg gac ttt gga aat aca atg tgt caa ctc ttg aca 341Ala
Ala Ala Gln Trp Asp Phe Gly Asn Thr Met Cys Gln Leu Leu Thr90 95
100 105ggg ctc tat ttt ata ggc ttc ttc tct gga atc ttc ttc atc atc
ctc 389Gly Leu Tyr Phe Ile Gly Phe Phe Ser Gly Ile Phe Phe Ile Ile
Leu110 115 120ctg aca atc gat agg tac ctg gct gtc gtc cat gct gtg
ttt gct tta 437Leu Thr Ile Asp Arg Tyr Leu Ala Val Val His Ala Val
Phe Ala Leu125 130 135aaa gcc agg acg gtc acc ttt ggg gtg gtg aca
agt gtg atc act tgg 485Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr
Ser Val Ile Thr Trp140 145 150gtg gtg gct gtg ttt gcg tct ctc cca
gga atc atc ttt acc aga tct 533Val Val Ala Val Phe Ala Ser Leu Pro
Gly Ile Ile Phe Thr Arg Ser155 160 165caa aaa gaa ggt ctt cat tac
acc tgc agc tct cat ttt cca tac agt 581Gln Lys Glu Gly Leu His Tyr
Thr Cys Ser Ser His Phe Pro Tyr Ser170 175 180 185cag tat caa ttc
tgg aag aat ttc cag aca tta aag ata gtc atc ttg 629Gln Tyr Gln Phe
Trp Lys Asn Phe Gln Thr Leu Lys Ile Val Ile Leu190 195 200ggg ctg
gtc ctg ccg ctg ctt gtc atg gtc atc tgc tac tcg gga atc 677Gly Leu
Val Leu Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile205 210
215cta aaa act ctg ctt cgg tgt cga aat gag aag aag agg cac agg gct
725Leu Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg
Ala220 225 230gtg agg ctt atc ttc acc atc atg att gtt tat ttt ctc
ttc tgg gct 773Val Arg Leu Ile Phe Thr Ile Met Ile Val Tyr Phe Leu
Phe Trp Ala235 240 245ccc tac aac att gtc ctt ctc ctg aac acc ttc
cag gaa ttc ttt ggc 821Pro Tyr Asn Ile Val Leu Leu Leu Asn Thr Phe
Gln Glu Phe Phe Gly250 255 260 265ctg aat aat tgc agt agc tct aac
agg ttg gac caa gct atg cag gtg 869Leu Asn Asn Cys Ser Ser Ser Asn
Arg Leu Asp Gln Ala Met Gln Val270 275 280aca gag act ctt ggg atg
acg cac tgc tgc atc aac ccc atc atc tat 917Thr Glu Thr Leu Gly Met
Thr His Cys Cys Ile Asn Pro Ile Ile Tyr285 290 295gcc ttt gtc ggg
gag aag ttc aga aac tac ctc tta gtc ttc ttc caa 965Ala Phe Val Gly
Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln300 305 310aag cac
att gcc aaa cgc ttc tgc aaa tgc tgt tct att ttc cag caa 1013Lys His
Ile Ala Lys Arg Phe Cys Lys Cys Cys Ser Ile Phe Gln Gln315 320
325gag gct ccc gag cga gca agc tca gtt tac acc cga tcc act ggg gag
1061Glu Ala Pro Glu Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly
Glu330 335 340 345cag gaa ata tct gtg ggc ttg tgacacggac tcaagtgggc
tggtgaccca 1112Gln Glu Ile Ser Val Gly Leu350gtcagagttg tgcacatggc
ttagttttca tacacagcct gggctggggg tggggtggga 1172gaggtctttt
ttaaaaggaa gttactgtta tagagggtct aagattcatc cat 12254352PRTHomo
sapiens 4Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr
Tyr Thr1 5 10 15Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala
Ala Arg Leu20 25 30Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly
Phe Val Gly Asn35 40 45Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys
Arg Leu Lys Ser Met50 55 60Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile
Ser Asp Leu Phe Phe Leu65 70 75 80Leu Thr Val Pro Phe Trp Ala His
Tyr Ala Ala Ala Gln Trp Asp Phe85 90 95Gly Asn Thr Met Cys Gln Leu
Leu Thr Gly Leu Tyr Phe Ile Gly Phe100 105 110Phe Ser Gly Ile Phe
Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu115 120 125Ala Val Val
His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe130 135 140Gly
Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser145 150
155 160Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His
Tyr165 170 175Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe
Trp Lys Asn180 185 190Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu
Val Leu Pro Leu Leu195 200 205Val Met Val Ile Cys Tyr Ser Gly Ile
Leu Lys Thr Leu Leu Arg Cys210 215 220Arg Asn Glu Lys Lys Arg His
Arg Ala Val Arg Leu Ile Phe Thr Ile225 230 235 240Met Ile Val Tyr
Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu245 250 255Leu Asn
Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser260 265
270Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu Gly Met
Thr275 280 285His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly
Glu Lys Phe290 295 300Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His
Ile Ala Lys Arg Phe305 310 315 320Cys Lys Cys Cys Ser Ile Phe Gln
Gln Glu Ala Pro Glu Arg Ala Ser325 330 335Ser Val Tyr Thr Arg Ser
Thr Gly Glu Gln Glu Ile Ser Val Gly Leu340 345 350512PRTHomo
sapiensMISC_FEATURE(1)..(1)Xaa at position 1 is Ala or Leu 5Xaa Ala
Ala Gln Trp Asp Phe Gly Asn Thr Met Cys1 5 10624PRTHomo sapiens
6Arg Ser Gln Lys Glu Gly Leu His Tyr Thr Cys Ser Ser His Phe Pro1 5
10 15Tyr Ser Gln Tyr Gln Phe Trp Lys20716PRTHomo sapiens 7Gln Glu
Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser Asn Arg Leu Asp1 5 10
158360PRTHomo sapiens 8Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg
Asn Thr Asn Glu Ser1 5 10 15Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr
Asp Tyr Gly Ala Pro Cys20 25 30His Lys Phe Asp Val Lys Gln Ile Gly
Ala Gln Leu Leu Pro Pro Leu35 40 45Tyr Ser Leu Val Phe Ile Phe Gly
Phe Val Gly Asn Met Leu Val Val50 55 60Leu Ile Leu Ile Asn Cys Lys
Lys Leu Lys Cys Leu Thr Asp Ile Tyr65 70 75 80Leu Leu Asn Leu Ala
Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro85 90 95Leu Trp Ala His
Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met100 105 110Cys Lys
Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile115 120
125Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val
His130 135 140Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly
Val Val Thr145 150 155 160Ser Val Ile Thr Trp Leu Val Ala Val Phe
Ala Ser Val Pro Gly Ile165 170 175Ile Phe Thr Lys Cys Gln Lys Glu
Asp Ser Val Tyr Val Cys Gly Pro180 185 190Tyr Phe Pro Arg Gly Trp
Asn Asn Phe His Thr Ile Met Arg Asn Ile195 200 205Leu Gly Leu Val
Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly210 215 220Ile Leu
Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg225 230 235
240Ala Val Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe
Trp245 250 255Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln
Glu Phe Phe260 265 270Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu
Asp Gln Ala Thr Gln275 280 285Val Thr Glu Thr Leu Gly Met Thr His
Cys Cys Ile Asn Pro Ile Ile290 295 300Tyr Ala Phe Val Gly Glu Lys
Phe Arg Arg Tyr Leu Ser Val Phe Phe305 310 315 320Arg Lys His Ile
Thr Lys Arg Phe Cys Lys Gln Cys Pro Val Phe Tyr325 330 335Arg Glu
Thr Val Asp Gly Val Thr Ser Thr Asn Thr Pro Ser Thr Gly340 345
350Glu Gln Glu Val Ser Ala Gly Leu355 3609355PRTHomo sapiens 9Met
Glu Thr Pro Asn Thr Thr Glu Asp Tyr Asp Thr Thr Thr Glu Phe1 5 10
15Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys Val Asn Glu Arg Ala Phe20
25 30Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe Val Ile
Gly35 40 45Leu Val Gly Asn Ile Leu Val Val Leu Val Leu Val Gln Tyr
Lys Arg50 55 60Leu Lys Asn Met Thr Ser Ile Tyr Leu Leu Asn Leu Ala
Ile Ser Asp65 70 75 80Leu Leu Phe Leu Phe Thr Leu Pro Phe Trp Ile
Asp Tyr Lys Leu Lys85 90 95Asp Asp Trp Val Phe Gly Asp Ala Met Cys
Lys Ile Leu Ser Gly Phe100 105 110Tyr Tyr Thr Gly Leu Tyr Ser Glu
Ile Phe Phe Ile Ile Leu Leu Thr115 120 125Ile Asp Arg Tyr Leu Ala
Ile Val His Ala Val Phe Ala Leu Arg Ala130 135 140Arg Thr Val Thr
Phe Gly Val Ile Thr Ser Ile Ile Ile Trp Ala Leu145 150 155 160Ala
Ile Leu Ala Ser Met Pro Gly Leu Tyr Phe Ser Lys Thr Gln Trp165 170
175Glu Phe Thr His His Thr Cys Ser Leu His Phe Pro His Glu Ser
Leu180 185 190Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys Leu Asn Leu
Phe Gly Leu195 200 205Val Leu Pro Leu Leu Val Met Ile Ile Cys Tyr
Thr Gly Ile Ile Lys210 215 220Ile Leu Leu Arg Arg Pro Asn Glu Lys
Lys Ser Lys Ala Val Arg Leu225 230 235 240Ile Phe Val Ile Met Ile
Ile Phe Phe Leu Phe Trp Thr Pro Tyr Asn245 250 255Leu Thr Ile Leu
Ile Ser Val Phe Gln Asp Phe Leu Phe Thr His Glu260 265 270Cys Glu
Gln Ser
Arg His Leu Asp Leu Ala Val Gln Val Thr Glu Val275 280 285Ile Ala
Tyr Thr His Cys Cys Val Asn Pro Val Ile Tyr Ala Phe Val290 295
300Gly Glu Arg Phe Arg Lys Tyr Leu Arg Gln Leu Phe His Arg Arg
Val305 310 315 320Ala Val His Leu Val Lys Trp Leu Pro Phe Leu Ser
Val Asp Arg Leu325 330 335Glu Arg Val Ser Ser Thr Ser Pro Ser Thr
Gly Glu His Glu Leu Ser340 345 350Ala Gly
Phe355108PRTArtificialFLAG epitope 10Asp Tyr Lys Asp Asp Asp Asp
Lys1 5111256DNAHomo sapiensCDS(1)..(1254) 11gat ccg tcg acc gcc att
atg gat gga tgg caa gaa act ctc ccc ggg 48Asp Pro Ser Thr Ala Ile
Met Asp Gly Trp Gln Glu Thr Leu Pro Gly1 5 10 15tgg aac aag atg gat
tat caa gtg tca agt cca atc tat gac atc aat 96Trp Asn Lys Met Asp
Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn20 25 30tat tat aca tcg
gag ccc tgc caa aaa atc aat gtg aag caa atc gca 144Tyr Tyr Thr Ser
Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala35 40 45gcc cgc ctc
ctg cct ccg ctc tac tca ctg gtg ttc atc ttt ggt ttt 192Ala Arg Leu
Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe50 55 60gtg ggc
aac atg ctg gtc atc ctc atc ctg ata aac tgc aaa agg ctg 240Val Gly
Asn Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu65 70 75
80aag agc atg act gac atc tac ctg ctc aac ctg gcc atc tct gac ctg
288Lys Ser Met Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp
Leu85 90 95ttt ttc ctt ctt act gtc ccc ttc tgg gct cac tac ttg gcc
gcc cag 336Phe Phe Leu Leu Thr Val Pro Phe Trp Ala His Tyr Leu Ala
Ala Gln100 105 110tgg gac ttt gga aat aca atg tgt caa ctc ttg aca
ggg ctc tat ttt 384Trp Asp Phe Gly Asn Thr Met Cys Gln Leu Leu Thr
Gly Leu Tyr Phe115 120 125ata ggc ttc ttc tct gga atc ttc ttc atc
atc ctc ctg aca atc gat 432Ile Gly Phe Phe Ser Gly Ile Phe Phe Ile
Ile Leu Leu Thr Ile Asp130 135 140agg tac ctg gct gtc gtc cat gct
gtg ttt gct tta aaa gcc agg acg 480Arg Tyr Leu Ala Val Val His Ala
Val Phe Ala Leu Lys Ala Arg Thr145 150 155 160gtc acc ttt ggg gtg
gtg aca agt gtg atc act tgg gtg gtg gct gtg 528Val Thr Phe Gly Val
Val Thr Ser Val Ile Thr Trp Val Val Ala Val165 170 175ttt gcg tct
ctc cca gga atc atc ttt acc aga tct caa aaa gaa ggt 576Phe Ala Ser
Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly180 185 190ctt
cat tac acc tgc agc tct cat ttt cca tac agt cag tat caa ttc 624Leu
His Tyr Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe195 200
205tgg aag aat ttc cag aca tta aag ata gtc atc ttg ggg ctg gtc ctg
672Trp Lys Asn Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val
Leu210 215 220ccg ctg ctt gtc atg gtc atc tgc tac tcg gga atc cta
aaa act ctg 720Pro Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu
Lys Thr Leu225 230 235 240ctt cgg tgt cga aat gag aag aag agg cac
agg gct gtg agg ctt atc 768Leu Arg Cys Arg Asn Glu Lys Lys Arg His
Arg Ala Val Arg Leu Ile245 250 255ttc acc atc atg att gtt tat ttt
ctc ttc tgg gct ccc tac aac att 816Phe Thr Ile Met Ile Val Tyr Phe
Leu Phe Trp Ala Pro Tyr Asn Ile260 265 270gtc ctt ctc ctg aac acc
ttc cag gaa ttc ttt ggc ctg aat aat tgc 864Val Leu Leu Leu Asn Thr
Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys275 280 285agt agc tct aac
agg ttg gac caa gct atg cag gtg aca gag act ctt 912Ser Ser Ser Asn
Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu290 295 300ggg atg
acg cac tgc tgc atc aac ccc atc atc tat gcc ttt gtc ggg 960Gly Met
Thr His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly305 310 315
320gag aag ttc aga aac tac ctc tta gtc ttc ttc caa aag cac att gcc
1008Glu Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile
Ala325 330 335aaa cgc ttc tgc aaa tgc tgt tct att ttc cag caa gag
gct ccc gag 1056Lys Arg Phe Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu
Ala Pro Glu340 345 350cga gca agc tca gtt tac acc cga tcc act ggg
gag cag gaa ata tct 1104Arg Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly
Glu Gln Glu Ile Ser355 360 365gtg ggc ttg tga cac gga ctc aag tgg
gct ggt gac cca gtc aga gtt 1152Val Gly Leu His Gly Leu Lys Trp Ala
Gly Asp Pro Val Arg Val370 375 380gtg cac atg gct tag ttt tca tac
aca gcc tgg gct ggg ggt ggg gtg 1200Val His Met Ala Phe Ser Tyr Thr
Ala Trp Ala Gly Gly Gly Val385 390 395gga gag gtc ttt ttt aaa agg
aag tta ctg tta tag agg gtc taa gat 1248Gly Glu Val Phe Phe Lys Arg
Lys Leu Leu Leu Arg Val Asp400 405 410tca tcc at 1256Ser Ser
* * * * *