WO1997047319A1 - Utilisations d'un recepteur de la chemokine dans l'inhibition de l'infection par le vih-1 - Google Patents

Utilisations d'un recepteur de la chemokine dans l'inhibition de l'infection par le vih-1 Download PDF

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Publication number
WO1997047319A1
WO1997047319A1 PCT/US1997/010619 US9710619W WO9747319A1 WO 1997047319 A1 WO1997047319 A1 WO 1997047319A1 US 9710619 W US9710619 W US 9710619W WO 9747319 A1 WO9747319 A1 WO 9747319A1
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WIPO (PCT)
Prior art keywords
hiv
cells
agent
binding
chemokine
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Application number
PCT/US1997/010619
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English (en)
Inventor
Graham P. Allaway
Tatjana Dragic
Virginia M. Litwin
Paul J. Maddon
John P. Moore
Alexandra Trkola
Original Assignee
Progenics Pharmaceuticals, Inc.
Aaron Diamond Aids Research Centre
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by Progenics Pharmaceuticals, Inc., Aaron Diamond Aids Research Centre filed Critical Progenics Pharmaceuticals, Inc.
Priority to JP50189598A priority Critical patent/JP4183754B2/ja
Priority to EP97930120A priority patent/EP0956044A4/fr
Priority to AU34026/97A priority patent/AU735460B2/en
Publication of WO1997047319A1 publication Critical patent/WO1997047319A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the replication of primary, non-syncytium-inducing (NSI) HIV-1 isolates in CD4 + T-cells is inhibited by the C-C ⁇ -chemokines MlP-l ⁇ , MIP-1S and RANTES (1,2) , but T-cell line-adapted (TCLA) or syncytium-inducing (SI) primary strains are insensitive (2,3) .
  • the ⁇ -chemokines are small (8kDa) , related proteins active on cells of the lymphoid and monocyte lineage (4-8) .
  • Fusin is not known to be a ⁇ -chemokine receptor (7-9) . Summary of the Invention
  • This invention provides a polypeptide having a sequence corresponding to the sequence of a portion of a chemokine receptor capable of inhibiting the fusion of HIV-1 to CD4 * cells and thus infection of the cells.
  • the chemokine receptor is C-C CKR-5.
  • the CCKR-5 is also named as CCR5.
  • the polypeptide comprises amino acids having a sequence of at least one extracellular domain of C-C CKR-5.
  • the portion of a chemokine receptor comprises amino acid sequence MDYQVSSPIYDINYYTSEPCQKINVKQIAAR (SEQ ID NO: 5) .
  • the portion comprises amino acid sequence HYAAAQWDFGNTMCQ (SEQ ID NO: 6) .
  • the portion comprises amino acid sequence RSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIV (SEQ ID NO: 7) .
  • the portion comprises amino acid sequence QEFFGLNNCSSSNRLDQ (SEQ ID NO: 8) .
  • the portion of the chemokine receptor C-C CKR-5 may comprise one or more of the above sequences.
  • the polypeptides may contain part of the above illustrated sequences and still be capable of inhibiting HIV-1 infection. The minimal number of amino acids sufficient to inhibit HIV-1 infection may be determined by the RET or infection assays as described below.
  • This invention also provides a pharmaceutical composition comprising effective amount of one or more of the above polypeptides and a pharmaceutically acceptable carrier.
  • This invention also provides a polypeptide having a sequence corresponding to that of a portion of an HIV-1 glycoprotein capable of specifically binding to the chemokine receptor C- C CKR-5.
  • This invention provides a pharmaceutical composition comprising effective amount of one of more polypeptides having a sequence corresponding to the sequence of a portion of an HIV-l glycoprotein capable of specifically binding to the chemokine receptor C-C CKR-5 and a pharmaceutically acceptable carrier.
  • This invention provides an antibody or a portion of an antibody capable of binding to a chemokine receptor on a CD4 + cell and inhibiting HIV-l infection of the cell.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an antibody capable of binding to a chemokine receptor on a CD4 + cell and inhibiting HIV-l infection of the cell and a pharmaceutically acceptable carrier.
  • This invention provides a method of treating an HIV-l infected subject comprising administering to the subject the above polypeptides, antibody and pharmaceutical compositions.
  • This invention provides a method of reducing the likelihood of a subject from becoming infected by HIV-l comprising administering to the subject the above pharmaceutical compositions .
  • This invention provides a method for inhibiting fusion of HIV-l to CD4 + cells which comprises contacting CD4 cells with a non-chemokine agent capable of binding to the chemokine receptor C-C CKR-5 in an amount and under conditions such that fusion of HIV-l to the CD4 * cells is inhibited.
  • This invention provides a method for inhibiting HIV-l infection of CD4 + cells which comprises contacting CD4 + cells with a non-chemokine agent capable of binding to the chemokine receptor C-C CKR-5 in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited, thereby inhibiting HIV-l infection of the cells.
  • This invention provides a non-chemokine agent capable of binding to the chemokine receptor C-C CKR-5 and inhibiting HIV-l infection.
  • This invention provides a molecule capable of binding to the chemokine receptor C-C CKR-5 and inhibiting HIV-l infection comprising a non-chemokine agent linked to a ligand capable of binding to a cell surface receptor of the CD4 * cells other than the chemokine receptor such that the binding of the non-chemokine agent to the chemokine receptor does not prevent the binding of the ligand to the other receptor.
  • This invention provides a pharmaceutical composition comprising an amount of the above molecules effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • This invention also provides a molecule capable of binding to the chemokine receptor C-C CKR-5 and inhibiting fusion of HIV-l to CD4 + cells comprising a non-chemokine agent linked to a compound capable of increasing the in vivo half-life of the non-chemokine agent.
  • This invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of the molecule capable of binding to the chemokine receptor C-C CKR-5 and inhibiting HIV-l infection comprising a non-chemokine agent linked to a compound capable of increasing the in vivo half-life of the non-chemokine agent effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • This invention provides a method for reducing the likelihood of HIV-l infection in a subject comprising administering the above pharmaceutical compositions to the subject.
  • This invention provides a method for treating HIV-l infection in a subject comprising administering the above pharmaceutical compositions to the subject.
  • This invention provides a method for determining whether a non-chemokine agent is capable of inhibiting the fusion of HIV-l to a CD4 ⁇ C-C CKR-5 cell which comprises: (a) contacting a CD4 + , C-C CKR-5 * cell, which is labeled with a first dye, with a cell expressing an appropriate HIV-l envelope glycoprotein on its surface, which is labeled with a second dye, in the presence of excess of the agent under conditions permitting the fusion of the CD4 * and C- C CKR-5 * cell to the cell expressing the HIV-l envelope glycoprotein on its surface in the absence of an agent known to inhibit fusion of HIV-l to CD4 * , C-C CKR-5 + cell, the first and second dyes being selected so as to allow resonance energy transfer between the dyes; (b) exposing the product of step (a) to conditions which would result in resonance energy transfer if fusion has occurred; and (c) determining whether there is resonance energy transfer, the absence or reduction of transfer indicating that
  • This invention also provides a transgenic nonhuman animal which comprises an isolated DNA molecule encoding the chemokine receptor C-C CKR-5.
  • this transgenic nonhuman animal further comprises an isolated DNA molecule encoding a sufficient portion of the CD4 molecule to permit binding the HIV-l envelope glycoprotein.
  • This invention further provides a transgenic nonhuman animal which comprises an isolated DNA molecule encoding the chemokine receptor C-C CKR-5 and an isolated DNA molecule encoding fusin.
  • this transgenic nonhuman animal further comprises an isolated DNA molecule encoding the full-length or portion of the CD4 molecule sufficient for binding the HIV-l envelope glycoprotein.
  • This invention also provides transformed cells which comprise an isolated nucleic acid molecule encoding the chemokine receptor C-C CKR-5.
  • his invention provides an agent capable of inhibiting HIV-l infection and capable of binding to a chemokine receptor without substantially affecting the said chemokine receptor's capability to bind to chemokines .
  • Figure 1 Specificity, time course and stage of ⁇ -chemokine inhibition of HIV-l replication
  • PM1 cells (1 xlO 6 ) were preincubated with RANTES + MIP- l ⁇ + MIP-l ⁇ (R/M ⁇ /M ⁇ ; lOOng/ml of each) for 24h (-24h) or 2h (-2h) , then washed twice with phosphate buffered saline (PBS) .
  • HIV- 1 (BaL env-complemented) virus 50ng of p24 ; see legend to Table 1) was added for 2h, then the cells were washed and incubated for 48h before measurement of luciferase activity in cell lysates as described previously (10,11) .
  • virus and R/M ⁇ /M ⁇ were added simultaneously to cells, and at the indicated time points (lh, 3h, etc) the cells were washed twice in PBS, resuspended in culture medium and incubated for 48h prior to luciferase assay.
  • Time 0 represents the positive control, to which no ⁇ -chemokines were added.
  • +2h represents the mixture of virus with cells for 2h prior to washing twice in PBS, addition of R/M ⁇ /M ⁇ and continuation of the culture for a further 48h before luciferase assay.
  • Lanes 2 and 6) are negative controls (no virus) .
  • Lanes 3 and 7 are negative controls (no virus) . All viral stocks used for the PCR assay were treated with DNAse for 30 min at 37°C, and tested for DNA contamination before use. After 2h, the cells were washed and resuspended in medium containing the same ⁇ -chemokines for a further 8h. DNA was then extracted from infected cells using a DNA/RNA isolation kit (US Biochemicals) . First round nested PCR was performed with primers : U3 + , 5' -CAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGG-3 ' (SEQ
  • FIG. 2 HIV-l env-mediated membrane fusion of cells transiently expressing C-C CKR-5 Membrane fusion mediated by ⁇ -chemokine receptors expressed in HeLa cells was demonstrated as follows: Cells were transfected with control plasmid pcDNA3.1 or plasmid pcDNA3.1 -CKR constructs using lipofectin (Gibco BRL) . The pcDNA3.1 plasmid carries a T7-polymerase promoter and transient expression of ⁇ -chemokine receptors was boosted by infecting cells with lxlO 7 pfu of vaccinia encoding the T7-polymerase (vFT7.3) 4h post-lipofection (9) .
  • U87MG-CD4 cells were transiently lipofected with CCR5 mutants, then infected with NLluc/ADA (dark hatched bars) , NLluc/JR-FL (light hatched bars) or NLluc/DH123 (white bars) luciferase-expressing chimeric viruses (1,2 of the Third Series of
  • Luciferase activity due c.p.s. was measured 72h post-infection(1, 2) and standardized for lipofection efficiency and receptor expression levels.
  • the co-receptor activity of each mutant designated on the x-axis is expressed as a percentage of the wild-type co-receptor activity (100%) , and is the mean ⁇ s.d. of three independent experiments each performed in quadruplicate. (*) indicates that the amino acid is also different in murine CCR5.
  • JR-FL env gene black bars
  • the vFT7pol system was used to enhance co-receptor expression (hatched bars) (1,4, 5,13 of the Third Series of Experiments) .
  • the extent of cell-cell fusion was determined using the RET assay(1.18 of the Third Series of Experiments) .
  • The. % RET values shown are the means ⁇ s.d. of three independent experiments, each performed in duplicate.
  • 2D7-PE binding is presented as [1- (m.f.i. with gpl20/m.f.i. without gpl20)] x 100%, and is the mean +.s.d. of three independent experiments.
  • Figure 8 Flow cvtometric analysis of the binding of sCD4- gp!20 complexes to (B)CCR5 ⁇ and (B)CCR5 LI .2 cells, a murine pre-B lymphoma line
  • the inhibitors were the CC chemokines MIP-l/S or RANTES at the concentrations indicated on the x axis.
  • the inhibition was measured using the RET assay, with the cell combinations indicated.
  • This invention provides a polypeptide having a sequence corresponding to the sequence of a portion of a chemokine receptor capable of inhibiting the fusion of HIV-l to CD4 4 cells and thus infection of the cell.
  • the chemokine receptor is C-C CKR-5 (CCR5) .
  • the fragment comprises at least one extracellular domain of the chemokine receptor C-C CKR-5.
  • the extracellular domain is the second extracellular loop of CCR5.
  • the chemokine receptor is CCR3 or CKR-2b(31,32) .
  • the sequence of a portion of the chemokine receptor includes the original amino acids or modified amino acids from the receptor, their derivatives and analogues. Such sequence should retain the ability to inhibit HIV-l infection. Sequences of fusin are also included.
  • the portion of a chemokine receptor comprises amino acid sequence MDYQVSSPIYDINYYTSEPCQKINVKQIAAR (SEQ ID NO: 5) .
  • the portion comprises amino acid sequence HYAAAQWDFGNTMCQ (SEQ ID NO: 6) .
  • the portion comprises amino acid sequence RSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIV (SEQ ID NO: 7) .
  • the portion comprises amino acid sequence QEFFGLNNCSSSNRLDQ (SEQ ID NO: 8) .
  • the portion of the chemokine receptor C-C CKR-5 may comprise one or more of the above sequences.
  • the polypeptides may contain part of the above illustrated sequences and still be capable of inhibiting HIV-l infection. The minimal number of amino acids sufficient to inhibit HIV-l infection may be determined by the RET or infection assays as described below .
  • polypeptides described above may be fusion molecules such that the fragments are linked to other molecules .
  • the molecule is a CD4-based molecule.
  • CD4- based molecules are known in the art and described in Allaway et al. (1996), Patent Cooperation Treaty Application No. PCT/US95/08805, publication no. WO 96/02575, the content of which is incorporated by reference into this application.
  • the polypeptide contains multiple units of one or more portions of a chemokine receptor.
  • the polypeptide contains sequences corresponding to multiple units of one or more extracellular domains of the chemokine receptor C-C CKR-5.
  • This invention also provides a pharmaceutical composition comprising effective amount of the above polypeptide and a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • This invention also provides a polypeptide having a sequence corresponding to that of a portion of an HIV-l envelope glycoprotein capable of specifically binding to the chemoreceptor C-C CKR-5.
  • a sequence may be identified by routine experiments. For example, overlapping synthetic peptides representing fragments of gpl20 or gp41 can be tested in the RET assay for their ability to inhibit fusion between cells expressing the envelope glycoprotein of HIV-l clinical isolates and cells expressing CD4 and C-C CKR-5. Peptides inhibiting fusion in this assay are also screened in the RET assay for ability to inhibit fusion mediated by the envelope glycoprotein of HIV-l laboratory-adapted- strains and peptides which are inhibitory in this later assay are discarded. As an alternative method, the peptides can be tested for their ability to compete with chemokines for binding to cell expressing C-C CKR-5.
  • This invention provides a pharmaceutical composition comprising effective amount of the polypeptide comprising a fragment of HIV-l glycoprotein capable of specifically binding to the chemokine receptor C-C CKR-5 and a pharmaceutically acceptable carrier.
  • This invention provides an antibody or a portion of an antibody thereof capable of binding to a chemokine receptor on a CD4 + cell and inhibiting HIV-l infection of the cell.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising effective amount of antibody capable of binding to a chemokine receptor and inhibiting HIV-l infection and a pharmaceutically acceptable carrier.
  • This invention provides a method of treating an HIV-l infected subject comprising administering to the subject the above pharmaceutical compositions.
  • This invention provides a method of reducing the likelihood of a subject from becoming infected by HIV-l comprising administering to the subject the above pharmaceutical compositions.
  • This invention provides a method for inhibiting fusion of
  • HIV-l to CD4 + cells which comprises contacting CD4 + cells with a non-chemokine agent capable of binding to the chemokine receptor C-C CKR-5 in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited.
  • This invention provides a method for inhibiting HIV-l infection of CD4 * cells which comprises contacting CD4 * cells with a non-chemokine agent capable of binding to the chemokine receptor C-C CKR-5 in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited, thereby inhibiting HIV-l infection.
  • the non-chemokine agents of this invention are capable of binding to chemokine receptors and inhibiting fusion of HIV- 1 to CD4 * cells.
  • the non-chemokine agents include, but are not limited to, chemokine fragments and chemokine derivatives and analogues, but do not include naturally occurring chemokines.
  • the non-chemokine agent is an oligopeptide. In another embodiment, the non-chemokine agent is a polypeptide. In still another embodiment, the non-chemokine agent is a nonpeptidyl agent.
  • This invention provides a non-chemokine agent capable of binding to the chemokine receptor C-C CKR-5 and inhibiting fusion of HIV-l to CD4 + cells.
  • This invention provides a molecule capable of binding to the chemokine receptor C-C CKR-5 and inhibiting fusion of HIV-l to CD4 + cells comprising a non-chemokine agent linked to a ligand capable of binding to a cell surface receptor of the CD4 + cells other than the chemokine receptor such that the binding of the non-chemokine agent to the chemokine receptor does not prevent the binding of the ligand to the other receptor.
  • the cell surface receptor is CD4.
  • the ligand comprises an antibody or a portion of an antibody.
  • This invention provides a pharmaceutical composition comprising an amount of the above molecule effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • This invention also provides a molecule capable of binding to the chemokine receptor C-C CKR-5 and inhibiting fusion of HIV-l to CD4 * cells comprising a non-chemokine agent linked to a compound capable of increasing the in vivo half-life of the non-chemokine agent.
  • the compound is polyethylene glycol.
  • This invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of the molecule capable of binding to the chemokine receptor C-C CKR-5 and inhibiting fusion of HIV-l to CD4 * cells
  • This invention provides a method for reducing the likelihood of HIV-l infection in a subject comprising administering the above pharmaceutical compositions to the subject.
  • This invention provides a method for treating HIV-l infection in a subject comprising administering the above pharmaceutical compositions to the subject.
  • This invention provides a method for determining whether a non-chemokine agent is capable of inhibiting the fusion of HIV-l to a CD4 * , C-C CKR-5 cell which comprises: (a) contacting a CD4 ⁇ C-C CKR-5 + cell, which is labeled with a first dye, with a cell expressing an appropriate HIV-l envelope glycoprotein on its surface, which is labeled with a second dye, in the presence of excess of the agent under conditions permitting the fusion of the CD4 + and C-C CKR-5' cell to the cell expressing the HIV-l envelope glycoprotein on its surface in the absence of an agent known to inhibit fusion of HIV-l to CD4 ⁇ C-C CKR-5 cell, the first and second dyes being selected so as to allow resonance energy transfer between the dyes; (b) exposing the product of step (a) to conditions which would result in resonance energy transfer if fusion has occurred; and (c) determining whether there is resonance energy transfer, the absence or reduction of transfer indicating that the agent is
  • the agent is an oligopeptide. In another embodiment, the agent is a polypeptide. In still another embodiment, the agent is a nonpeptidyl agent. In a further embodiment, the CD4 + cell is a PM1 cell. In a separate embodiment, the cell expressing the HIV-l envelope glycoprotein is a HeLa cell expressing HIV-l JR _ FL ⁇ gpl20/gp41.
  • This invention also provides a transgenic nonhuman animal which comprises an isolated DNA molecule encoding the chemokine receptor C-C CKR-5.
  • this transgenic nonhuman animal further comprises an isolated DNA molecule encoding a sufficient portion of the CD4 molecule to permit binding the HIV-l envelope glycoprotein.
  • This invention further provides a transgenic nonhuman animal which comprises an isolated DNA molecule encoding the chemokine receptor C-C CKR-5 and an isolated DNA molecule encoding fusin.
  • this transgenic nonhuman animal further comprises an isolated DNA molecule encoding a sufficient portion of the CD4 molecule to permit binding the HIV-l envelope glycoprotein.
  • One means available for producing a transgenic animal is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium (Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)) .
  • DNA or cDNA encoding the C-C CKR-5 chemokine receptor or CD4 is purified from a vector by methods well known in the art.
  • Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans-gene.
  • tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene.
  • the DNA in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a pipet puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term.
  • pseudopregnant mouse a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant
  • microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.
  • This invention provides transformed cells which comprise an isolated nucleic acid molecule encoding the chemokine receptor C-C CKR-5.
  • This invention also provides an agent capable of inhibiting HIV-l infection and capable of binding to a chemokine receptor without substantially affecting the said chemokine receptor's capability to bind to chemokines.
  • the term "without substantially affecting" mean that after the binding of the agent to the chemokine receptor, the chemokine receptor should still be able to bind to chemokines .
  • a higher concentration of the chemokine is required to achieve the degree of binding observed if the chemokine receptor had not been bound to the agent .
  • the chemokine concentration required to achieve the same binding is two fold. In another embodiment, the chemokine concentration is ten fold.
  • the chemokine receptor is CCR5. In another embodiment, the chemokine receptor is CXCR4 , CCR3 or CCR-2b.
  • This agent may be an oligopeptide, a nonpeptidyl agent or a polypeptide.
  • this agent can be an antibody or a portion of an antibody.
  • This invention further provides a pharmaceutical composition comprising an amount of the above agent effective to inhibit fusion of HIV-l to CD4 * cells and a pharmaceutically acceptable carrier.
  • This invention provides a method for inhibiting HIV-l infection of CD4 + cells which comprises contacting such CD4 * cells with an agent capable of inhibiting HIV-l infection and capable of binding to a chemokine receptor without substantially affecting the said chemokine receptor's capability to bind to chemokines.
  • This invention also provides a molecule capable of binding to the chemokine receptor CCR5 and inhibiting fusion of HIV- 1 to CD4 * cells without substantially affecting the said chemokine receptor's capability to bind to chemokines linked to a compound capable of increasing the in vivo half-life of the non-chemokine agent.
  • the compound is polyethylene glycol .
  • This invention provides a pharmaceutical composition comprising an amount of the above molecule effective to inhibit HIV-l infection and a pharmaceutically acceptable carrier.
  • This invention provides a method for reducing the likelihood of HIV-l infection in a subject comprising administering the above pharmaceutical composition to the subject.
  • This invention provides a method for treating HIV-l infection in a subject comprising administering the above pharmaceutical composition to the subject.
  • This invention provides a method for determining whether an agent is capable of inhibiting HIV-l infection comprising steps of: (a) fixing a chemokine receptor on a solid matrix;
  • step (b) contacting the agent with the fixed chemokine receptor under conditions permitting the binding of the agent to the chemokine receptor; (c) removing the unbound agent; (d) contacting the fixed chemokine receptor resulting in step
  • step (c) with a gpl20 in the presence of CD4 under conditions permitting the binding of the gpl20/CD4 complex and the chemokine receptor in the absence of the agent; (e) measuring the amount of bound gpl20/CD4 complex; and (f) comparing the amount determined in step (d) with the amount determined in the absence of the agent, a decrease of the amount indicating that the agent is capable of inhibiting HIV-l infection.
  • This invention also provides a method for determining whether an agent is capable of inhibiting HIV-l infection comprising steps of: (a) fixing a chemokine receptor on a solid matrix; (b) contacting the agent with the fixed chemokine receptor; (c) contacting the mixture in step (b) with a gpl20 in the presence of CD4 under conditions permitting the binding of the gpl20/CD4 complex and the chemokine receptor in the absence of the agent; (d) measuring the amount of bound gpl20/CD4 complex; and (e) comparing the amount determined in step (d) with the amount determined in the absence of the agent, a decrease of the amount indicating that the agent is capable of inhibiting HIV-l infection.
  • This invention also provides a method for determining whether an agent is capable of inhibiting HIV-l infection comprising steps of: (a) fixing a gpl20/CD4 complex on a solid matrix; (b) contacting the agent with the fixed gpl20/CD4 complex under conditions permitting the binding of the agent to the gpl20/CD4 complex; (c) removing unbound agent,- (d) contacting the fixed gpl20/CD4 complex resulting from step (c) with a chemokine receptor under conditions permitting the binding of the gpl20/CD4 complex and the chemokine receptor in the absence of the agent; (e) measuring the amount of bound chemokine receptor; and (f) comparing the amount determined in step (e) with the amount determined in the absence of the agent, a decrease of the amount indicating that the agent is capable of inhibiting HIV-l infection.
  • This invention provides a method for determining whether an agent is capable of inhibiting HIV-l infection comprising steps of: (a) fixing a gpl20/CD4 on a solid matrix; (b) contacting the agent with the fixed gpl20/CD4 complex; (c) contacting the mixture in step (b) with a chemokine receptor under conditions permitting the binding of the gpl20/CD4 complex and the chemokine receptor in the absence of the agent; (d) measuring the amount of bound chemokine receptor; (e) comparing the amount determined in step (d) with the amount determined in the absence of the agent, a decrease of the amount indicating that the agent is capable of inhibiting HIV-l infection.
  • CD4 include soluble CD4, fragment of CD4 or polypeptides incorporating the gpl20 binding site of CD4 capable of binding gpl20 and enabling the binding of gpl20 to the appropriate chemokine receptor.
  • gpl20 is the gpl20 from an appropriate strain of HIV-l.
  • gpl20 from the macrophage tropic clinical isolate will bind to the chemokine receptor CCR5
  • gpl20 from the laboratory adapted T-tropic strain HIV-l ⁇ will bind to the chemokine receptor CXCR4.
  • the CD4 is a soluble CD4.
  • the chemokine receptor which may be used in the above assay includes CCR5, CXCR4, CCR3 and CCR-2b.
  • the chemokine receptor is expressed on a cell.
  • the cell is a LI .2 cell.
  • the chemokine receptor is purified and reconstituted in liposomes. Such chemokine receptor embedded in the lipid bilayer of liposomes retains the gpl20 binding activity of the receptor.
  • the gpl20, CD4 or both may be labelled with a detectable marker in the above assays. Markers including radioisotope or enzymes such as horse radish peroxidase may be used in this invention.
  • the gpl20 or CD4 or the chemokine receptor is labelled with biotin.
  • the biotmylated gpl20, or CD4 or the chemokine receptor is detected by: (i) incubating with streptavidin-phycoerythrin, (ii) washing the incubated mixture resulting from step (i) , and (iii) measuring the amount of bound gpl20 using a plate reader, exciting at 530nm, reading emission at 590nm.
  • This invention also provides an agent determined to be capable of inhibiting HIV-l infection by the above methods, which is previously unknown.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the agent determined to be capable of inhibiting HIV-l infection by the above methods and a pharmaceutically acceptable carrier.
  • the agent is an oligopeptide.
  • the agent is a polypeptide.
  • the agent is a nonpeptidyl agent.
  • This invention also provides a molecule capable of binding to the chemokine receptor CCR5 and inhibiting fusion of HIV- 1 to CD4 + cells comprising the above determined agent linked to a compound capable of increasing the in vivo half-life of the non-chemokine agent.
  • the compound is polyethylene glycol.
  • This invention also provides a pharmaceutical composition comprising an amount of the above molecule effective to inhibit HIV-l infection and a pharmaceutically acceptable carrier.
  • This invention provides a method for reducing the likelihood of HIV-l infection in a subject comprising administering the above pharmaceutical compositions to the subject.
  • This invention provides a method for treating HIV-l infection in a subject comprising administering the above pharmaceutical composition to the subject.
  • ⁇ -chemokines inhibit HIV-l replication
  • NL4/3 ⁇ env which also carries the luciferase reporter gene
  • envelope glycoproteins expressed in trans 10,11
  • Various env-complemented viruses were tested in PM1 cells, a variant of HUT-78 that has the unique ability to support replication of primary and TCLA HIV-l strains, allowing comparison of envelope glycoprotein functions against a common cellular background (2,12) .
  • MlP-l ⁇ , MIP-l ⁇ and RANTES are most active against HIV-l in combination (2,3) , and strongly inhibited infection of PM1 cells by complemented viruses whose envelopes are derived from the NSI primary strains ADA and
  • PM1 cells were cultured as described by Lusso et al (12) .
  • Ficoll/hypaque- isolated PBMC from laboratory workers (LW) stimulated with PHA for 72h before depletion of CD8+ Lymphocytes with anti-CD8 immunomagnetic beads (DYNAL, Great Neck, NY).
  • CD4+ Lymphocytes were maintained in culture medium containing interleukin-2 (lOOU/ml; Hofmann LaRoche, Nutley, NJ) , as described previously (3).
  • Target cells (1- 2x10 s ) were infected with supernatants (10-50ng of HIV-l p24) from 293 -cells co- transfected with an
  • NL4 / 3 henv- luciferase vector and a HIV-l env -expressing vector (10,11).
  • ⁇ -Chemokines R & D Systems, Minneapolis
  • the ⁇ - chemokine concentration range was selected based on prior studies (2,3). After 2h, the cells were washed twice with PBS, resuspended in ⁇ - chemokine- containing media and maintained for 48-96h. Luciferase activity in cell lysates was measured as described previously (10,11).
  • luciferase activity /ng p24/mg protein, expressed relative to that in virus-control cultures lacking ⁇ -chemokines (100%), and are the means of duplicate or sextuplicate determinations, nd, not done.
  • RANTES and MIP-l ⁇ were strongly active when added individually, while other ⁇ -chemokines - MlP-l ⁇ , MCP-1, MCP-2 and MCP-3 (refs. 13-15) - were weaker inhibitors (Table la) .
  • MlP-l ⁇ , MIP-l ⁇ and RANTES did not inhibit infection of PM1 cells by the TCLA strains NL4/3 and HxB2, or by the amphotropic murine leukemia virus (MuLV-Ampho) pseudotype (Table la) .
  • MuLV-Ampho amphotropic murine leukemia virus
  • the env-complementation assay was used to assess HIV-l entry into CD4+ T-cells from two control individuals (LW4 and LW5) .
  • MlP-l ⁇ , MIP-l ⁇ and RANTES strongly inhibited infection by the NSI primary strain JR-FL infection of LW4' s and LW5's CD4 + T-cells, and weakly reduced HxB2 infection of LW cells (Table lb) , suggesting that there may be some overlap in receptor usage on activated CD4 + T-cells by different virus strains.
  • BaL env-mediated replication in normal PBL was also inhibited by MlP-l ⁇ , MIP-l ⁇ and RANTES, albeit with significant inter-donor variation in sensitivity (data not shown) .
  • ⁇ -chemokines inhibited HIV-l replication by showing that complete inhibition of infection of PM1 cells required the continuous presence of ⁇ -chemokines for up to 5h after addition of ADA or BaL env-complemented virus (Fig.la) .
  • Pre-treatment of the cells with ⁇ -chemokines for 2h or 24h prior to infection had no inhibitory effect if the cells were subsequently washed before virus addition.
  • adding ⁇ -chemokines 2h after virus only minimally affected virus entry (Fig. la) .
  • a PCR-based assay was next used to detect HIV-l early DNA reverse transcripts in PM1 cells after lOh of infection; reverse transcription of ADA, but not of NL4/3, could not be detected in the presence of MIP-l ⁇ and RANTES (Fig. lb) .
  • inhibition by ⁇ -chemokines requires their presence during at least one of the early stages of HIV-l replication: virus attachment, fusion and early reverse transcription. These sites of action were discriminated, first by testing whether ⁇ -chemokines inhibited binding of JR-FL or BRU gpl20 to soluble CD4, or of tetrameric CD4-IgG2 binding to HeLa-JR-FL cells expressing oligomeric envelope glycoproteins (17) .
  • ⁇ -chemokines inhibit a step after CD4 binding, when conformational changes in the envelope glycoproteins lead to fusion of the viral and cellular membranes (18) .
  • Cell-cell membrane fusion is also induced by the gpl20-CD4 interaction, and can be monitored directly by resonance energy transfer (RET) between fluorescent dyes incorporated into cell membranes (17) .
  • RET resonance energy transfer
  • OKT4a completely inhibits membrane fusion of PM1 cells with HeLa cells expressing the envelope glycoproteins of either JR-FL (HeLa-JR-FL) or BRU (HeLa-BRU) , confirming the specificity of the process (17) .
  • CD4 * target cells (mi togen- activated CD4* lymphocytes or PM1 cells) were labeled wi th octadecyl rhodamine (Molecular Probes, Eugene, OR) , and HeLa -JR-FL cells, HeLa -BRU cells (or con trol HeLa cells, not shown) were labeled wi th octadecyl fluorescein (Molecular Probes) , overnight a t 37°C.
  • Equal numbers of labeled target cells and env- expressing cells were mixed in 96-well pla tes and ⁇ - chemokines (or CD4 MAb OKT4a) were added at the final concentra tions (ng/ml ) indicated in parentheses in the first column . Fluorescence emission values were determined 4h after cell mixing (17) . If cell fusion occurs, the dyes are closely associated in the conjoined membrane such tha t exci ta tion of fluorescein a t 450nm resul ts in resonance energy transfer (RET) and emission by rhodamine at 590nm.
  • RET resonance energy transfer
  • the %RET value is defined by a calculation described el sewhere (17) , and each is the mean of triplica te determinations . These values were, for HeLa -JR-FL and HeLa -BRU cells respectively: PM1 cells 11 . 5%, 10 . 5%; LW5 CD4 + cells, 6. 0%, 10 . 5%; R/M ⁇ /M ⁇ , RANTES + MlP-l ⁇ + MlP-l ⁇ .
  • C-C CKR-5 in HeLa-CD4 (human) , COS-CD4 (simian) and 3T3-CD4 (murine) cells rendered each of them readily infectible by the primary, NSI strains ADA and BaL in the env-complementation assay of HIV-l entry (Table 3) .
  • C-C CKR-4 and C-C CKR-5 have no introns (4-8,15,22) and were isolated by PCR performed directly on a human genomic DNA pool derived from the PBMC of seven healthy donors.
  • Oligonucleotides overlapping the ATG and the stop codons and containing BamHI and Xhol restriction sites for directional cloning into the pcDNA3.1 expression vector (Invitrogen
  • LESTR also known as fusin or HUMSTR (4,9,24) was cloned by PCR performed directly on cDNA derived from PMl cells, using sequences derived from the NIH database. Listed below are the 5' and 3' primer pairs used in first (5-1 and 3-1) and second (5-2 and 3-2) round PCR amplification of the CKR genes directly from human genomic DNA, and of LESTR from PMl cDNA. Only a single set of primers was used to amplify CKR-5.
  • L/5-1 AAG CTT GGA GAA CCA GCG GTT ACC ATG GAG GGG
  • ATC (SEQ ID NO: 9) ;
  • L/5-2 GTC TGA GTC TGA GTC AAG CTT GGA GAA CCA (SEQ ID NO: 10);
  • L/3-1 CTC GAG CAT CTG TGT TAG CTG GAG TGA AAA CTT GAA GAC
  • CKR-1.-C1/5-1 AAG CTT CAG AGA GAA GCC GGG ATG GAA ACT CC
  • Cl/3-1 CTC GAG CTG AGT CAG AAC CCA GCA GAG AGT TC (SEQ ID NO: 15);
  • CKR-2a:C2/5-l AAG CTT CAG TAC ATC CAC AAC ATG CTG TCC AC (SEQ ID NO: 17) ;
  • C2/5-2 GTC TGA GTC TGA GTC AAG CTT CAG TAC ATC (SEQ ID NO:
  • C2/3-1 CTC GAG CCT CGT TTT ATA AAC CAG CCG AGA C (SEQ ID NO:
  • C2/3-2 GTC TGA GTC TGA GTC CTC GAG CCT CGT TTT (SEQ ID NO: 19);
  • C3/3-1 CTC GAG CAG ACC TAA AAC ACA ATA GAG AGT TCC (SEQ ID NO: 1
  • C4/3-1 CTC GAG CCA TTT CAT TTT TCT ACA GGA CAG CAT C (SEQ ID NO: 27) ;
  • CKR- 5: C5/5-12 GTC TGA GTC TGA GTC AAG CTT AAC AAG ATG GAT
  • TAT CAA (SEQ ID NO: 29) ;
  • C5/3-12 GTC TGA GTC TGA GTC CTC GAG TCC GTG TCA CAA GCC
  • CAC (SEQ ID NO: 30) .
  • the human CD4- expressing cell lines HeLa-CD4 (P42) , 3T3-CD4 (sc6) and COS-CD4 (Z28T1) (23) were transfected with the different pcDNA3.1-CKR constructs by the calcium phosphate method, then infected 48h later with different reporter viruses (200ng of HIV-l p24/l0 6 cells) in the presence or absence of ⁇ -chemokines (400ng/ml each of RANTES, MlP-l ⁇ and
  • C-C CKR-5 The second receptor function of C-C CKR-5 was confirmed in assays of env-mediated membrane fusion.
  • C-C CKR-5 was transiently expressed in COS and HeLa cell lines that permanently expressed human CD4, both cell lines fused strongly with HeLa cells expressing the JR-FL envelope glycoproteins, whereas no fusion occurred when control plasmids were used (data not shown) .
  • Expression of LESTR instead of C-C CKR-5 did not permit either C0S-CD4 or HeLa- CD4 cells to fuse with HeLa-JR-FL cells, but did allow fusion between C0S-CD4 cells and HeLa-BRU cells (data not shown) .
  • the fusion capacity of ⁇ -chemokine receptors was also tested in the RET assay.
  • the extent of fusion between HeLa-JR-FL cells and C-C CKR-5-expressing HeLa-CD4 cells was greater than the constitutive level of fusion between HeLa-BRU cells and HeLa-CD4 cells (Fig.2) .
  • the fusion-conferring function of C-C CKR-5 for primary, NSI HIV-l strains has therefore been confirmed in two independent fusion assays .
  • MlP-l ⁇ , MIP-l ⁇ and RANTES inhibit HIV-l infection at the entry stage, by interfering with the virus-cell fusion reaction subsequent to CD4 binding. It was also shown that C-C CKR-5 can serve as a second receptor for entry of primary NSI strains of HIV-l into CD4+ T-cells, and that the interaction of ⁇ -chemokines with C-C CKR-5 inhibits the HIV-l fusion reaction.
  • Recombinant gp41 (IIIB) ectodomain was from Viral Therapeutics Inc., and V3 peptides were obtained either from Repligen or the UK MRC AIDS Reagent Repository.
  • Recombinant MN gpl20 (Genentech) , SF-2 gpl20 (Chiron) and CM243 gpl20 were provided by the NIAID AIDS Reagents Repository, and W61D gpl20 (SmithKline Beecham, Belgium) was from the UK MRC AIDS Reagent Repository.
  • the expression of the gene is under the control of the cytomegalovirus immediate-early promoter.
  • the V3 loops were excised by the splicing-by-overlap- extension technique, such that the cysteines defining the loop were retained and spanned by the peptide sequence TGAGH. All constructs were sequenced to verify that no mutations were introduced during the cloning manipulations.
  • the proteins were expressed in stably transfected Chinese hamster ovary cells (DXB-11) , selected in nucleoside-free medium and amplified using methotrexate, following previously described methods (17) .
  • the secreted proteins were purified to >95% homogeneity in a non-denaturing process comprising an ion exchange, Galanthus nivalus lectin affinity and gel filtration chromatography.
  • the purified proteins bound sCD4 with nanomolar affinity(18) .
  • SF162 gpl60 For expression of SF162 gpl60, a 3.5-kb EcoRl-BamHl fragment containing the env gene was excised from the SV40-based vector pSM and subcloned into the Rl/Bgllll sites of the ⁇ - actin-based expression vector pCAGGS.
  • SF170 gpl60 For expression of SF170 gpl60, a 3.8-kb fragment containing the env gene was excised from the pBSKS* plasmid, blunted by treatment with T4 DNA polymerase, and subcloned into the RV/Xhol sites of pCAGGS.
  • the expression plasmids were transfected into 293 T cells by calcium phosphate co-precipitation. Soluble gpl20 in the culture supernatant was collected after three days, filtered through 0.2- ⁇ m filters and concentrated over an Amicon 1000 membrane.
  • the preparation of soluble, oligomeric forms of the JR-FL and 94RW020 envelope glycoproteins (and also monomeric gpl20 from 92TH014) was as follows.
  • the JR-FL env gene was provided by I. Chen (UCLA) and the env genes of 92TH014 and 92RW020 were obtained from the NIAID AIDS Reagent Repository(30) .
  • the monomeric gpl20 or oligomeric gpl20/gp41 concentrations in unpurified culture supernatants were estimated by denaturing the proteins (19) , then dot- blotting onto nitrocellulose membranes and detecting the gpl20 with a cocktail of murine monoclonal antibodies to continuous epitopes (19) , followed by an anti-mouse IgG-HRP conjugate and the ECL chemiluminescence system (Amersham) .
  • Purified, monomeric JR-FL gpl20 was used as a concentration standard(17) .
  • the concentration of oligomeric gpl20/gp41 complexes was defined as the total concentration of monomeric gpl20 subunits in the preparation.
  • High-affinity CD4 binding of the gpl20s was confirmed by enzyme-linked immunosorbent assay (ELISA) (19) .
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • CD4 + T cells were purified from the activated PBMCs by positive selection using anti-CD4 immunomagnetic beads (Dynal Inc.) .
  • the purified lymphocytes were cultured for at least 3 days at 2 x IO 6 / ml in medium containing IL- 2 (200 U ml "1 ) before being used in the 125 I-MIP-l/3 binding assay.
  • the cells were screened for CCR-5-defective alleles (14) , and only cells from wild-type donors were used (except when specified) .
  • 293 cells were transfected with pcDNA3.l-ckr-5 (ref. 1) using the calcium phosphate method, and resistant clones were selected in culture medium containing 1 ⁇ g ml "1 neomycin (G418; Sigma) . Resistant cells were subcloned and tested for CCR-5 expression in a binding assay using 125 I-MIP-l/3.
  • T cells were washed twice in ice-cold binding buffer (RPMI).
  • the /3-chemokine receptor CCR-5 is an essential co-factor for fusion of HIV-l strains of the non-syncytium-inducing (NSI) phenotype with CD4 + T-cells (1-5) .
  • the primary binding site for human immunodeficiency virus (HIV) -1 is the CD4 molecule, and the interaction is mediated by the viral surface glycoprotein gpl20 (6, 7) .
  • the mechanism of CCR-5 function during HIV-l entry has not been defined, but we have shown previously that its /S-chemokine ligands prevent HIV-l from fusing with the cell(l) .
  • CCR-5 acts as a second binding site for HIV-l simultaneously with or subsequent to the interaction between gpl20 and CD4.
  • CD4 binding although not absolutely necessary for the gpl20-CCR-5 interaction, greatly increases its efficiency.
  • MIP-l / S is the most specific ligand for CCR-5 (8-10) because MlP-l ⁇ and RANTES also bind with high affinity to other members of the /3-chemokine receptor family on lymphoid cells (8-11) .
  • MIP-1/3 as the CCR-5 ligand in the competition assays.
  • CCR-5 is a mitogen-response gene. Its expression in quiescent, purified CD4* T-cells is usually minimal, but 3 days after activation of the cells by phytohaemagglutinin and interleukin (IL)-2, we observed strong increases in CCR-5 messenger RNA and 125 I-labelled-
  • 125 I-MIP-lj ⁇ (0.1 nM) binding to cells from three such individuals was 92 ⁇ 12 c.p.m. per 2 x 10 6 cells (mean ⁇ s.d.) .
  • mean binding to cells from 21 control individuals was 1,044 ⁇ 1,073 c.p.m. per 2 x 10 6 cells (range, 222-4,846 c.p.m.) .
  • Most of the 125 I-MIP-10 reactivity with activated CD4 + T cells therefore derives from binding to CCR-5.
  • Recombinant proteins were titrated in the presence of 0.1 nM 125 I-MIP-l/3 and added to activated CD4 + T cells. Percentage inhibitions of 125 I-MIP-l/3 binding at each gpl20 concentration are shown, and are the means ⁇ s.d. of 2-4 independent experiments. No value indicates that the gpl20 molecule was not tested at that concentration (several molecules were not available at concentrations > l ⁇ g ml "1 ) . * An oligomeric gpl20/gp41 complex.
  • peptides (15-residue if not specified) from the V3 loops of the following strains were also inactive: JR-FL (32-residue) , RA, VS, Case-B (each NSI) ; HxB2, MN, SF- 2 (each TCLA) (peptides were added at l ⁇ g ml ": , the approximate molar equivalent of 60 ⁇ g ml "1 gpl20) .
  • HIV-l strains from genetic subtypes A, B, C and E can use CCR-5 for entry(3) , and we have found that MIP-1/3 inhibits the replication of most primary, NSI HIV-l strains from subtypes A to E. This breadth of reactivity of HIV-l with CCR-5 extend to the 125 I-MIP-l/3 competition assay. Recombinant gpl20s from the following NSI primary strains were competitive, with half-maximal inhibition of MIP-1/3 binding occurring at concentrations around 0.1-0.5 ⁇ g ml "1 :
  • JR-FL (subtype B) , SF162 (B) , W61D (B) , 92TH014 (B) , SF170
  • JR-FL gpl20 (2ug ml "1 ) inhibition of 25 I-MIP-1/3 binding to activated CD4 + T cells was tested in the presence or absence of sCD4 (50 ⁇ g ml "1 ) or monoclonal antibodies to CD4 (50 ⁇ g ml "1 ) or antibodies to gpl20 (20 ⁇ g ml ) .
  • the level of specific 125 I-MIP-l/3 binding (c.p.m.) Recorded in the presence of gpl20 but the absence of antibody was set at 0%, and the level recorded in the absence of both gpl20 and antibody was set at 100%. A negative percentage reversal indicates that the competitive effect of gpl20 on 125 I-MIP-l/3 binding was increased in the presence of the antibody. Also listed are the approximate positions of the antibody epitopes on gpl20, as defined(19,20) . References to the origin of the antibodies are described elsewhere (19,20) or listed in the Methods section.
  • JR-FL gpl20 and CCR-5 requires at least 100-fold higher gpl20 concentrations on CD4 " cells than on CD4 * cells.
  • binding of gpl20 to CD4 on the cell surface increases the probability of a gpl20-CCR-5 interaction; either a gpl20-CD4-CCR-5 ternary complex forms, or there are sequential interactions of gpl20 with CD4, then CCR-5.
  • One possibility is that the high-affinity association of gpl20 with CD4 increases the probability of a lower-affinity interaction of gpl20 with
  • CCR-5 (a proximity effect) .
  • sCD4 does not substitute for cell-surface CD4 , at least with JR-FL gpl20.
  • binding to CD4 may be necessary to (better) expose a CCR-5 binding site on gpl20. This may be especially important in the context of virions, where some regions of the oligomeric envelope glycoproteins (including the V3 loop) that are accessible on monomeric gpl20 are not optimally exposed before CD4 binding (18) .
  • the monoclonal antibodies 2G12, 17b, 447-D, 48d, IgGlbl2, G3-508 and 697-D were also tested against the oligomeric JR-FL gpl20/gp41 protein, and all except 697-D inhibited the interaction of this protein with CCR-5 (not shown) .
  • simian immunodeficiency virus (SIV) strains can also use human CCR- 5 as a second receptor (Z.W. Chen and P. Marx, personal communication) , but the V3 regions of HIV-l and SIV have almost no primary sequence homology. Can all these sequences each form a binding site for the same, conserved cellular protein, when similar V3 sequences from TCLA HIV-l strains cannot (Table 4)? Twin-site models of the interaction of ligands with chemokine receptors (8) leave open the possibility that a relatively conserved section of the V3 loop could be one component of a multi-point binding site for CCR-5 on gpl20.
  • the CCR-5 binding site must include a region of gpl20 that is strongly conserved across the primate immunodeficiency viruses, not just across the HIV-l genetic subtypes. Whether this is also the case for HIV-l interactions with CXCR-4 remains to be determined.
  • the structure of the V3 loop may influence the nature of a complex binding site for CCR-5 on gpl20.
  • a region of gpl20 overlapped by (but not necessarily identical to) the CD4- induced epitopes of monoclonal antibodies 48d and 17b is a good candidate for such a site.
  • These antibodies recognize similar conformationally sensitive structures that are probably located around the bases of the VI, V2 and V3 loops (22, 23) .
  • V3 loop from both HxBc2 gpl20 and JR-FL gpl20 destroys the 48d and 17b epitopes (22 , 23) (unpublished data) , which may be relevant to the inability of the ⁇ -V3 JR-FL gpl20 to interact with CCR-5 (Fig. 5A) , and single amino-acid changes in the V3 and C4 regions of HIV-1 ⁇ ! also have major effects on the structure of these epitopes (24) .
  • sCD4 inhibited the interaction between JR-FL gpl20 and CCR-5 on CD4 * cells, for some strains of HIV-l and (especially) HIV-2 and SIV, sCD4 might enhance the efficiency of second- receptor interactions, and thereby facilitate the entry of these primate immunodeficiency viruses into CD4 or CD4 * cells(25,26) .
  • the CC-chemokine receptor CCR5 is required for the efficient fusion of M-tropic HIV-l strains with the plasma membrane of CD4+ cells (1-5) , and interacts directly with the viral surface glycoprotein gpl20(6,7) .
  • receptor chimera studies have provided useful information(8-10) , the domains of CCR5 that function for HIV-l entry, including the site of gpl20 interaction, have not been unambiguously identified.
  • the wt and mutant CCR5 proteins were transiently expressed in both U87MG-CD4 and SCL-1-CD4 cells and their abilities to support entry mediated by HIV-l envelope glycoproteins were determined using an env-complementation assay with a luciferase readout (Fig. 5) (1,2) .
  • Fig. 5 a luciferase readout
  • These non-lymphoid human cell lines were chosen because they lack the CCR5, CCR3 and CXCR4 co-receptors, so resist infection by HIV-l in the absence of a transfected co-receptor(2,4, 13, 14) (a few exceptional HIV-l strains will enter U87MG-CD4 cells via an unknown route (15), but we did not use them) .
  • the following mutants were relatively insensitive to the action of one or more of the CC-chemokines: DllA, K22A, R31A (Nt) , H181A, Y184A, K171A/E172A, K191A/N192A (ECL2) , R274A/D276A (ECL3) .
  • DllA did not affect the HIV-l co-receptor function of CCR5, but do affect CC-chemokine-mediated inhibition of this process (Table 6) .
  • gpl20 only partially inhibited (40%) the binding of 2D7-PE to the D2A, DllA and E18A mutants, and was almost ineffective at blocking 2D7-PE binding to the double and triple Nt mutants (25% and 15% inhibition, respectively) (Fig. 7) .
  • those mutants most impaired for HIV-l entry (Fig. 5) were also the ones for which 2D7-PE binding was least sensitive to gpl20 inhibition (Fig. 7) .
  • the most probable explanation of this result is that gpl20 binds to the wild type CCR5 molecule in such a way as to sterically hinder binding of 2D7-PE to ECL2, but binds poorly to the Nt mutants.
  • gpl20 does bind efficiently to the Nt mutants but in an unusual orientation in which it is less able to inhibit 2D7-PE binding to ECL2.
  • the geometry of inter-domain interactions in CCR5 has been altered by the Nt substitutions that impair CCR5 co-receptor function.
  • point substitutions at three negatively-charged residues in the amino-terminal domain that affect the co-receptor function of CCR5, without necessarily interfering with CC-chemokine inhibition of co-receptor function.
  • the same substitutions affect the ability of gpl20 to interact correctly with CCR5, probably by reducing the affinity of the gpl20-CCR5 interaction.
  • the gpl20 binding site on CCR5 is therefore dependent on residues in the Nt, and it is possible that a discrete gpl20-binding domain is actually confined to the Nt.
  • Previous studies using chimeric receptors or deletion mutants indicated the importance of the CCR5 and CXCR4 Nt' s for co-receptor function(8-10,29) .
  • the chimera studies also suggested that the site of interaction between CCR5 and HIV-l is relatively broad and somewhat flexible (8-10) . Although this possibility should not be discounted, alterations in the extracellular loops of receptor chimeras may also indirectly affect the orientation of the CCR5 Nt and hence its ability to interact correctly with gpl20.
  • the CC-chemokine binding site on CCR5 is dependent on residues in both the Nt and the extracellular loops (notably, but not exclusively, ECL2) .
  • ECL2 extracellular loops
  • CCR5 A more detailed understanding of the interactive sites on CCR5 for gpl20 and the CC-chemokines (and on these molecules for CCR5) will be required to define how HIV-l uses CCR5 for entry into its target cells and, perhaps, for the development of inhibitors of this process. It will also be important to determine whether the negatively charged residues that we have identified in the CCR5 Nt interact directly or indirectly with positively charged amino acids in gpl20, in the V3 loop and/or elsewhere.
  • luciferase due and / S-galactosidase activity (OD 420 ) were measured(1, 2) .
  • Standardized luciferase activity is defined as (luc c.p.s / ng/ml p24 / (OD 420 ⁇ control OD 420 ) / (r.f.e. for mutant CCR5 bands ⁇ r.f.e. for wild-type CCR5 bands) (see below) .
  • Luc c.p.s. values ranged from 5xl0 5 to 2xl0 6 for wild-type CCR5.
  • Trkola, A., et al CD4-dependent, antibody-sensitive interactions between HIV-l and its co-receptor CCR5. Nature 384, 184-186 (1996) .
  • HIV-l entry cofactor Functional cDNA cloning of a seven-transmembrane G protein-coupled receptor. Science 272, 872-877 (1996) .
  • This assay has been adapted for drug screening purposes to a 96-well microplate format where binding of the sCD4/gpl20 complexes to CCR5 + /CD4 " cells is measured using a fluorometric plate reader.
  • One method is as follows:
  • Synthetic peptides representing the four extracellular domains of human CCR5 were made by Quality Controlled Biochemicals (Hopkinton, MA) and tested for ability to inhibit membrane fusion mediated by the envelope glycoproteins of the LAI or JR-FL strains of HIV-l using the resonance energy transfer (RET) assay described above. Specific inhibition of fusion mediated by the JR-FL envelope glycoprotein was seen using the ECL2 peptide but not other peptides. ECL2 inhibited fusion between HeLa-env JR-FL cells and PMl cells by 97% at 100 ⁇ g/ml, 65% at 33 ⁇ g/ml and 15% at 11 ⁇ g/ml (mean of two assays) .
  • the bicyclam JM3100 obtained from Dr. J. Moore (Aaron Diamond AIDS Research Center, NY) was tested for ability to inhibit membrane fusion mediated by the envelope glycoproteins of the LAI or JR-FL strains of HIV-l using the resonance energy transfer (RET) assay described above. As illustrated in Fig. 10, this molecule specifically and potently inhibits fusion mediated by gpl20/gp41 from the HIV-1 LAI strain, and not from the HIV-l JR-FL strain. These data suggest that this molecule specifically inhibits HIV fusion by blocking the interaction between HIV-1 ⁇ gpl20 and CXCR4. SEQUENCE LISTING
  • MOLECULE TYPE DNA (genomic)
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  • SEQUENCE DESCRIPTION SEQ ID NO: 11: CTCGAGCATC TGTGTTAGCT GGAGTGAAAA CTTGAAGACT C 41
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 12: GTCTGAGTCT GAGTCCTCGA GCATCTGTGT 30
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  • xi SEQUENCE DESCRIPTION: SEQ ID NO:21: AAGCTTCAGG GAGAAGTGAA ATGACAACC 29
  • MOLECULE TYPE DNA (genomic)
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  • xi SEQUENCE DESCRIPTION: SEQ ID NO:30: GTCTGAGTCT GAGTCCTCGA GTCCGTGTCG CAAGCCCAC 39

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Abstract

L'invention concerne un polypeptide comprenant un fragment d'un récepteur de la chémokine capable d'inhiber l'infection par le VIH-1. Dans un mode de réalisation, le récepteur de la chémokine est C-C CKR-5. Dans un autre mode de réalisation, le fragment comprend au moins un domaine extracellulaire du récepteur de la chémokine C-C CKR-5. Cette invention porte en outre sur différentes utilisations du récepteur de la chémokine dans l'inhibition de l'infection par le VIH-1.
PCT/US1997/010619 1996-06-14 1997-06-13 Utilisations d'un recepteur de la chemokine dans l'inhibition de l'infection par le vih-1 WO1997047319A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP50189598A JP4183754B2 (ja) 1996-06-14 1997-06-13 Ｈｉｖ―１感染を阻害するためのケモカイン受容体の使用
EP97930120A EP0956044A4 (fr) 1996-06-14 1997-06-13 Utilisations d'un recepteur de la chemokine dans l'inhibition de l'infection par le vih-1
AU34026/97A AU735460B2 (en) 1996-06-14 1997-06-13 Uses of a chemokine receptor for inhibiting HIV-1 infection

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US66509096A 1996-06-14 1996-06-14
US1994196P 1996-06-14 1996-06-14
US60/019,941 1996-06-14
US08/665,090 1996-06-14

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WO1997047319A1 true WO1997047319A1 (fr) 1997-12-18

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PCT/US1997/010233 WO1997047318A1 (fr) 1996-06-14 1997-06-13 Utilisations d'un recepteur de chemokines en vue de l'inhibition de l'infection a vih-1
PCT/US1997/010619 WO1997047319A1 (fr) 1996-06-14 1997-06-13 Utilisations d'un recepteur de la chemokine dans l'inhibition de l'infection par le vih-1

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PCT/US1997/010233 WO1997047318A1 (fr) 1996-06-14 1997-06-13 Utilisations d'un recepteur de chemokines en vue de l'inhibition de l'infection a vih-1

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EP (1) EP0956044A4 (fr)
JP (1) JP4183754B2 (fr)
AU (2) AU3390297A (fr)
CA (1) CA2257991A1 (fr)
WO (2) WO1997047318A1 (fr)

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US6448021B1 (en) 1998-07-23 2002-09-10 Millenium Pharmaceuticals, Inc. Method of inhibiting cell function associated with CCR2 by anti-CCR2 amino-terminal domain antibodies
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US6696550B2 (en) 1998-07-23 2004-02-24 Millennium Pharmaceuticals, Inc. Humanized anti-CCR2 antibodies and methods of use therefor
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US7060273B2 (en) 2001-04-06 2006-06-13 Progenics Pharmaceuticals, Inc. Methods for inhibiting HIV-1 infection
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US7138119B2 (en) 2000-09-15 2006-11-21 Progenics Pharmaceuticals, Inc. Compositions and methods for inhibition of HIV-1 infection
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
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US7160546B2 (en) 1995-06-06 2007-01-09 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
US6759519B2 (en) 1995-06-06 2004-07-06 Human Genome Sciences, Inc. Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor)
US7862994B2 (en) 1995-06-07 2011-01-04 Progenics Pharmaceuticals Inc. Methods for inhibiting HIV-1 envelope glycoprotein-medicated membrane fusion
US6797811B1 (en) 1995-12-20 2004-09-28 Icos Corporation Antibodies to chemokine receptor 88C
US7662548B2 (en) 1995-12-20 2010-02-16 Euroscreen S.A. ICOS Corporation Method of screening for modulators of HIV infection
US6265184B1 (en) 1995-12-20 2001-07-24 Icos Corporation Polynucleotides encoding chemokine receptor 88C
US6268477B1 (en) 1995-12-20 2001-07-31 Icos Corporation Chemokine receptor 88-C
US7118859B2 (en) 1996-01-17 2006-10-10 Progenics Pharmaceuticals, Inc. Methods for inhibiting HIV-1 infection
US6528625B1 (en) 1996-10-28 2003-03-04 Millennium Pharmaceuticals, Inc. Anti-CCR5 antibodies and kits comprising same
WO1998054317A1 (fr) * 1997-05-30 1998-12-03 Fondation Mondiale Recherche Et Prevention Sida Variants de co-recepteur du virus de l'immunodeficience humaine associes a une resistance a une infection par le virus
US6399078B1 (en) 1998-06-01 2002-06-04 University Of Maryland Biotechnology Institute Chemokine—glycosaminoglycan complexes and their use in treating or preventing receptor mediated diseases
US6451522B2 (en) 1998-07-23 2002-09-17 Millennium Pharmacueticals, Inc. Anti-CCR2 antibodies and methods of use therefor
US7566539B2 (en) 1998-07-23 2009-07-28 Millennium Pharmaceuticals, Inc. Anti-CCR2 antibodies and methods of use therefor
US6491915B2 (en) 1998-07-23 2002-12-10 Millennium Pharmaceuticals, Inc. Anti-CCR2 antibodies and methods of use therefor
US6458353B1 (en) 1998-07-23 2002-10-01 Millennium Pharmaceuticals, Inc. Anti-CCR2 antibodies and methods of use therefor
US6448021B1 (en) 1998-07-23 2002-09-10 Millenium Pharmaceuticals, Inc. Method of inhibiting cell function associated with CCR2 by anti-CCR2 amino-terminal domain antibodies
US7473421B2 (en) 1998-07-23 2009-01-06 Millennium Pharmaceuticals, Inc. Humanized anti-CCR2 antibodies and methods of use therefor
US8227211B2 (en) 1998-07-23 2012-07-24 Millennium Pharmaceuticals, Inc. Humanized anti-CCR2 antibodies and methods of use therefor
US6696550B2 (en) 1998-07-23 2004-02-24 Millennium Pharmaceuticals, Inc. Humanized anti-CCR2 antibodies and methods of use therefor
US7442775B2 (en) 1998-07-23 2008-10-28 Millennium Pharmaceuticals, Inc. Humanized anti-CCR2 antibodies and methods of use therefor
US7566450B2 (en) 1998-07-23 2009-07-28 Millennium Pharmaceuticals, Inc. Humanized anti-CCR2 antibodies and methods of use therefor
WO2000047609A1 (fr) * 1999-02-10 2000-08-17 Nissui Pharmaceutical Co., Ltd. Peptides cycliques et vaccins contre le sida
US7378490B1 (en) 1999-02-10 2008-05-27 Nissui Pharmaceutical Co., Ltd. Cyclic peptides and aids vaccines
WO2001030809A1 (fr) * 1999-10-28 2001-05-03 Nissui Pharmaceutical Co., Ltd. Peptides cycliques et vaccins contre le sida
US6943233B1 (en) 1999-10-28 2005-09-13 Nissui Pharmaceutical Co., Ltd. Cyclic peptides and aids vaccines
WO2001045738A2 (fr) * 1999-12-22 2001-06-28 King's College London Utilisation de proteines de choc thermique
WO2001045738A3 (fr) * 1999-12-22 2001-12-27 King S College London Utilisation de proteines de choc thermique
US7070785B2 (en) 1999-12-22 2006-07-04 Thomas Lehner Heat shock proteins for use in enhancing cellular factor production
US7138119B2 (en) 2000-09-15 2006-11-21 Progenics Pharmaceuticals, Inc. Compositions and methods for inhibition of HIV-1 infection
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7862818B2 (en) 2001-02-09 2011-01-04 Human Genome Sciences, Inc. Method of inhibiting human G-protein chemokine receptor (CCR5) HDGNR10
US7060273B2 (en) 2001-04-06 2006-06-13 Progenics Pharmaceuticals, Inc. Methods for inhibiting HIV-1 infection
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7122185B2 (en) 2002-02-22 2006-10-17 Progenics Pharmaceuticals, Inc. Anti-CCR5 antibody
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10

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JP4183754B2 (ja) 2008-11-19
EP0956044A4 (fr) 2005-01-05
AU735460B2 (en) 2001-07-12
JP2001503608A (ja) 2001-03-21
EP0956044A1 (fr) 1999-11-17
CA2257991A1 (fr) 1997-12-18
AU3402697A (en) 1998-01-07
WO1997047318A1 (fr) 1997-12-18
AU3390297A (en) 1998-01-07

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