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WO1997037005A1 - Methode de prevention de l'infection des cellules cd4+ par le vih-1 - Google Patents

Methode de prevention de l'infection des cellules cd4+ par le vih-1 Download PDF

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Publication number
WO1997037005A1
WO1997037005A1 PCT/US1997/005597 US9705597W WO9737005A1 WO 1997037005 A1 WO1997037005 A1 WO 1997037005A1 US 9705597 W US9705597 W US 9705597W WO 9737005 A1 WO9737005 A1 WO 9737005A1
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WIPO (PCT)
Prior art keywords
agent
chemokine
hiv
cells
fusion
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PCT/US1997/005597
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English (en)
Inventor
Graham P. Allaway
Virginia M. Litwin
Paul J. Maddon
William C. Olson
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Progenics Pharmaceuticals, Inc.
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Application filed by Progenics Pharmaceuticals, Inc. filed Critical Progenics Pharmaceuticals, Inc.
Priority to AU26074/97A priority Critical patent/AU728512B2/en
Priority to JP9535610A priority patent/JP2000507596A/ja
Priority to EP97917856A priority patent/EP0915969A4/fr
Publication of WO1997037005A1 publication Critical patent/WO1997037005A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • 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
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Chemokines are a family of related soluble proteins of molecular weight between 8 and lOKDa, secreted by lymphocytes and other cells, which bind receptors on target cell surfaces resulting in the activation and mobilization of leukocytes, for example in the inflammatory process. Recently, Cocchi et al. demonstrated that the chemokines RANTES, MlP-l ⁇ and MlP-l ⁇ are factors produced by CD8 4 T lymphocytes which inhibit infection by acrophage-tropic primary isolates of HIV-1, but not infection by laboratory-adapted strains of the virus (1) .
  • chemokines are members of the C-C group of chemokines, so named because they have adjacent cysteine residues, unlike the C-X-C group which has a single amino acid separating these residues (2) . While Cocchi et al. found that expression of HIV-1 RNA was suppressed by treatment with the chemokines, they did not identify the site of action of these molecules.
  • a resonance energy transfer (RET) assay of HIV-1 envelope glycoprotein-mediated membrane fusion was used to determine whether fusion mediated by the envelope glycoprotein from the primary acrophage- ropic isolate of HIV-1 JR .
  • FI would be specifically inhibited by chemokines, when compared with fusion mediated by the envelope glycoprotein from the laboratory-adapted T lymphotropic strain HIV-l ⁇ .
  • chemokine receptors are fusion accessory molecules required for HIV-1 infection.
  • unidentified cell surface molecules are required for virus entry in addition to the HIV-1 receptor, CD4.
  • CD4 is required for HIV-1 attachment
  • the accessory molecules are required for the membrane fusion step of entry.
  • These accessory molecules are generally expressed only on human cells, so HIV-1 does not infect non-human CD4 cells (3-6) .
  • non-human CD4" cells by fusing them
  • fusion accessory molecules are found on a subset of human CD4' cells and are required for infection by HIV-1 isolates with particular tropisms .
  • macrophage-tropic primary strains of HIV-l such as HIV-1 JR .
  • FL may have different requirements for accessory molecules compared with laboratory-adapted T lymphotropic strains such as HIV-l ⁇ . This phenomenon may explain differences in tropism between HIV-l strains.
  • the current invention comprises a series of new therapeutics for HIV-l infection. It was demonstrated for the first time that chemokines act at the fusion step of HIV-l entry and specifically inhibit membrane fusion mediated by the envelope glycoprotein of primary macrophage-tropic primary viral isolates, not laboratory-adapted T lymphotrophic strains of the virus. Primary macrophage-tropic isolates of the virus are of particular importance since they are the strains usually involved in virus transmission, and may have particular importance in the pathogenesis of HIV-l infection.
  • RET resonance energy transfer
  • 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 a chemokine receptor in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited.
  • This invention also 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 a chemokine receptor in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited, thereby inhibiting the HIV-l infection.
  • This invention further provides non-chemokine agents capable of binding to the chemokine receptor and inhibiting fusion of HIV-l to CD4 + cells.
  • This invention provides an agent which is capable of binding to fusin and inhibiting infection.
  • the agent is an oligopeptide .
  • the agent is an polypeptide.
  • the agent is an antibody or a portion of an antibody.
  • the agent is a nonypeptidyl agent.
  • This invention provides a composition of matter capable of binding to the chemokine receptor 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.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of the above-described composition of matter effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • This invention provides a composition of matter capable of binding to tne chemokine receptor and inhibiting fusion of HIV-l to CD4 cells comprising a non-chemokme agent linked to a compound capable of increasing the m vivo half-life of the non-chemokme agent.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of a composition of matter comprising a non-chemokme agent linked to a compound capable of increasing the m vivo half-life of the non-chemokme agent effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • This invention provide methods for reducing the likelihood of HIV-l infection a subject comprising administering an above-described pharmaceutical composition to the subject.
  • This invention also provides methods for treating HIV-l infection m a subject comprising administering an above- described pharmaceutical composition to the subject.
  • This invention also provides methods for determining whether a non-chemokme agent is capable of inhibiting the fusion of HIV-l to a CD4 + cell which comprise, (a) contacting (I) a CD4 T cell which is labeled with a first dye and (n) a cell expressing tne HIV-l envelope glycoprotein on its surface which is labeled with a second dye, in the presence of an excess of the agent under conditions permitting the fusion of the CD4" cell to the cell expressing the HIV-l envelope glycoprotein on its surface in the absence of the agent, 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 a reduction of resonance energy transfer, when compared with the resonance energy transfer in the absence of the agent , a decrease in transfer indicating that the agent is capable of inhibiting fusion of HIV-l to CD4 + cells.
  • FIG. 1 Membrane fusion mediated by the HIV-1 ⁇ . ⁇ envelope glycoprotein is inhibited by RANTES, MlP-lor and MIP-l/S.
  • %RET resulting from the fusion of PM1 cells and HeLa-env JR _ FL ( ⁇ ) or ( ⁇ ) was measured in the presence and absence of recombinant human chemokines at a range of concentrations: RANTES
  • Max RET is the %RET value obtained at four hours with HeLa-env cells and CD4-expressing cells in the absence of an inhibitory compound
  • Exp RET is the %RET value obtained for the same cell combination in the presence of an inhibitory compound and Min RET is the background %RET value obtained using HeLa cells in place of HeLa envelope-expressing cells.
  • OKT4A (62 - 0.3 nM) , RANTES (10.3 - 0.3 nM) , MlP-l ⁇ (53.3 - 2.9 nM) , and MIP-lS (25.6 - 0.8 nM) .
  • Inhibitors were added simultaneously with biotinylated HIV-l gpl20 to soluble CD4 coated microtiter plates (Dynatech Laboratories, Inc.,
  • PM1 cells (1 xlO 6 ) were preincubated with RANTES + MIP- l ⁇ + MIP-lS (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.
  • M indicates lkb DNA ladder; 1, 10, 100, 1000 indicate number of reference plasmid (pAD8) copies.
  • the assay can detect 100 copies of reverse transcripts.
  • Figure 4 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 IxlO 7 pfu of vaccinia encoding the T7-polymerase (vFT7.3) 4h post-lipofection (9) . Cells were then cultured overnight in R18-containing media and were tested for their ability to fuse with HeLa-JR-FL cells
  • HeLa cells was between 3% and 4% irrespective of the transfected plasmid.
  • FIG. 5 Membrane fusion mediated by the HIV ⁇ - envelope glycoprotein is inhibited by SDF-1.
  • % RET resulting from the fusion of PM1 cells and HeLa-env JR _ FL or HeLa-env ⁇ cells was measured in the presence of recombinant SDF-l ⁇ (Gryphon Science, San Diego).
  • 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 a chemokine receptor in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited.
  • This invention also 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 a chemokine receptor in an amount and under conditions such that fusion of HIV-l to the CD4 + cells is inhibited, thereby inhibiting the HIV-l infection.
  • a chemokine means RANTES, MIP-1- ⁇ , MIP-1- ⁇ or another chemokine which blocks HIV-l infection.
  • a chemokine receptor means a receptor capable of binding RANTES, MIP-1- ⁇ , MIP-1- / 3 or another chemokine which blocks HIV-l infection.
  • the receptor "fusin” is also named CXCR4 and the chemokine receptor C-C CKR5 is also named CCR5.
  • the HIV-l used in this application unless specified will mean clinical or primary or field isolates or HIV-l viruses which maintain their clinical characteristics.
  • the HIV-l clinical isolates may be passaged in primary peripheral blood mononuclear cells.
  • the HIV-l clinical isolates may be macrophage-trophic.
  • non-chemokine agents of this invention are capable of binding to chemokine receptors and inhibiting fusion of HIV-
  • 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 agents include multimeric forms of the chemokine fragments and chemokine derivatives and analogues or fusion molecules which contain chemokine fragments, derivatives and analogues linked to other molecules.
  • 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 an antibody or a portion thereof. Antibodies against the chemokine receptor may easily be generated by routine experiments. It is also within the level of ordinary skill to synthesize fragments of the antibody capable of binding to the chemokine receptor. In a further embodiment, the non-chemokine agent is a nonpeptidyl agent.
  • Non-chemokine agents which are purely peptidyl in composition can be either chemically synthesized by solid- phase methods (Merrifield, 1966) or produced using recombinant technology in either prokaryotic or eukaryotic systems.
  • the synthetic and recombinant methods are well known in the art .
  • Non-chemokine agents which contain biotin or other nonpeptidyl groups can be prepared by chemical modification of synthetic or recombinant chemokines or non-chemokine agents.
  • One chemical modification method involves periodate oxidation of the 2-amino alcohol present on chemokines or non-chemokine agents possessing serine or threonine as their N-terminal amino acid (Geophegan and Stroh, 1992) .
  • the resulting aldehyde group can be used to link peptidyl or non-peptidyl groups to the oxidized chemokine or non- chemokine agent by reductive amination, hydrazine, or other chemistries well known to those skilled in the art.
  • a N-terminus of a protein should mean the terminus of the protein after it has been processed.
  • the N-terminus of a secretory protein should be the terminus after the cleavage of a signal peptide.
  • This invention provides a method of identifying these non- chemokine agents.
  • One way of identifying such agents, including non-peptidyl agents, that bind to a chemokine receptor and inhibit fusion of HIV-l to CD4 + cells is to use the following assay: 1) Incubate soluble CD4 with biotinylated gpl20 from HIV-1 JR _ FL or HIV-l ⁇ ; 2) Incubate this complex with CCR5 or CXCR4-expressing cells (for HIV- !
  • This invention further provides the non-chemokine agents identified by the above methods.
  • This invention provides a non-chemokine agent capable of binding to the chemokine receptor and inhibiting fusion of HIV-l to CD4 * cells.
  • the non-chemokine is a polypeptide.
  • this polypeptide is a fragment of the chemokine RANTES (Gong et al . , 1996) .
  • the polypeptide may also comprise the RANTES sequence with deletion of the N-terminal amino acids of said sequence. The deletion may be the first eight N-terminal amino acids of the RANTES sequence (SEQ ID NO: 5) .
  • the polypeptide may comprise the MIP-l ⁇ sequence with deletion of the N-terminal amino acids of said sequence.
  • the deletion may be the first seven, eight, nine or ten N-terminal amino acids of the MIP-l ⁇ sequence.
  • the polypeptide comprises the MIP-l ⁇ sequence with the N- terminal sequence modified by addition of an amino acid or oligopeptide .
  • the polypeptide comprises the MIP-l ⁇ sequence with the N-terminal sequence modified by removing the N-terminal alanine and replaced it by serine or threonine and additional amino acid or oligopeptide or nonpeptidyl moiety.
  • the additional amino acid is methionine.
  • fusin a cofactor for HIV-l fusion and entry was identified and designated "fusin" (Feng et al . , 1996) .
  • This invention provides an agent which is capable of binding to fusin and inhibiting infection.
  • the agent is an oligopeptide.
  • the agent is an polypeptide.
  • the polypeptide comprises SDF-1 with deletion of the N-terminal amino acids of said sequence. The deletion may be the first six, seven, eight, or nine N-terminal amino acids of the SDF-1 sequence.
  • This invention also provides the above non-chemokine agent, wherein the polypeptide comprises SDF-1 sequence with the N- terminal sequence modified to produce antagonistic effect to SDF-1.
  • the polypeptide comprises SDF-1 sequence with the N- terminal sequence modified to produce antagonistic effect to SDF-1.
  • One modification is to replace the N-terminal glycine of SDF-1 by serine and derivatized with biotin.
  • Another modification is to replace the N-terminal glycine of SDF-1 by serine and derivatized with methionine .
  • a further modification is to add the N-terminus of SDF-1 with a methionine before the terminal glycine .
  • the agent is an antibody or a portion of an antibody.
  • the agent is a nonpeptidyl agent .
  • the agents capable of binding to fusin may be identified by screening different compounds for their capability to bind to fusin in vitro.
  • yeast cells having a pheromone system are engineered to express a heterologous surrogate of a yeast pheromone system protein.
  • the surrogate incorporates fusin and under some conditions performs in the pheromone system of the yeast cell a function naturally performed by the corresponding yeast pheromone system protein.
  • Such yeast cells are also engineered to express a library of peptides whereby a yeast cell containing a peptide which binds fusin exhibits modulation of the interaction of surrogate yeast pheromone system protein with the yeast pheromone system and this modulation is a selectable or screenable event. Similar approaches may be used to identify agents capable of binding to both fusin and the chemokine receptor C-C CKR-5.
  • This invention also provides pharmaceutical compositions comprising an amount of such non-chemokine agents or agents capable of binding to fusin effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • Such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • This invention provides a composition of matter capable of binding to the chemokine receptor 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 T 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 is an antibody or a portion of an antibody.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of an above-described composition of matter effective to inhibit fusion of HIV-l to CD4 + cells and a pharmaceutically acceptable carrier.
  • This invention provides a composition of matter capable of binding to the chemokine receptor 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 also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of a composition of matter 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 provide methods for reducing likelihood of HIV-l infection in a subject comprising administering the above-described pharmaceutical compositions to the subject.
  • This invention also provides methods for treating HIV-l infection in a subject comprising administering the above- described pharmaceutical compositions to the subject.
  • This invention also provides methods for determining whether a non-chemokine agent is capable of inhibiting the fusion of HIV-l to a CD4 * cell which comprise: (a) contacting (i) a CD4 * cell which is labeled with a first dye and (ii) a cell expressing the HIV-l envelope glycoprotein on its surface which is labeled with a second dye, in the presence of an excess of the agent under conditions permitting the fusion of the CD4 + cell to the cell expressing the HIV-l envelope glycoprotein on its surface in the absence of the agent, 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 a reduction of resonance energy transfer, when compared with the resonance energy transfer in the absence of the agent, a decrease in transfer indicating that the agent is capable of inhibiting fusion of HIV-l to CD4- cells.
  • HIV-l only fuses with appropriate CD4 + cells.
  • laboratory-adapted T lymphotropic HIV-l strains fuse with most CD4 + human cells.
  • Clinical HIV-l isolates do not fuse with most transformed CD4 * human cell lines but do fuse with human primary CD4 + cells such as CD4 + T lymphocytes and macrophages. Routine experiments may be easily performed to determine whether the CD4 * cell is appropriate for the above fusion assay.
  • the non-chemokine agent is an oligopeptide.
  • the non-chemokine agent is a polypeptide.
  • the agent is an antibody or a portion thereof.
  • the non-chemokine agent is a nonpeptidyl agent.
  • the CD4 + cell is a PM1 cell.
  • the cell expressing the HIV-l envelope glycoprotein is a HeLa cell expressing HIV-1 JR _ FL gpl20/gp41.
  • Chemokines inhibit fusion mediated bv the envelope glycoprotein from a macrophage-tropic primary isolate of HIV-l but not from a laboratory-adapted T- lymphotrophic strain of the virus
  • the chemokines RANTES, MlP-l ⁇ and MIP-l / S were obtained from R & D systems (Minneapolis, MN) . They were tested in the RET assay for ability to inhibit fusion between HeLa-env JR _ FL cells (expressing gpl20/gp41 from the macrophage tropic isolate HIV-1 JR _ FL ) and PM1 cells, or for inhibition of fusion between HeLa-env ul cells (expressing gpl20/gp41 from the laboratory-adapted strain HIV-l ⁇ ) and various CD4' T lymphocyte cell lines. As shown in Figure 1, all three chemokines inhibited fusion mediated by the macrophage tropic virus envelope glycoprotein, but not that mediated by the laboratory-adapted strain envelope glycoprotein.
  • the non-chemokines include chemokine fragments and chemokine derivatives that are tested in the RET assay to determine which are active in inhibiting HIV-l membrane fusion. Particular attention is focused on fragments or derivatives that inhibit HIV-l fusion but do not activate leukocyte responses.
  • These non-chemokines include:
  • N-terminal derivatives of the chemokines Addition of residues to the N-terminus of chemokines inhibits the function of these proteins without significantly reducing their ability to bind chemokine receptors.
  • Met-RANTES RANTES with an N-terminal methionine
  • the mechanism of antagonism appears to be competition for receptor binding (9) .
  • Similar results were found using other derivatives of the N terminus of RANTES (9) and also by N-terminal modification of other chemokines, such as IL-8 (a member of the C-X-C chemokines) (10) .
  • the current invention includes Met-RANTES and other chemokines derivatised by the addition of methionine, or other residues, to the N-terminus so that they inhibit fusion mediated by the envelope glycoprotein of HIV-1 JR _ FL , and inhibit infection by many isolates of HIV-l, yet do not activate the inflammatory response.
  • Chemokines with N-terminal amino acids deleted have been generated by deleting amino acids in the N-terminal region. For example, deletion of up to 8 amino acids at the N-terminus of the chemokine MCP-1 (a member of the C-C chemokine group) , ablated the bioactivity of the protein while allowing it to retain chemokine receptor binding and the ability to inhibit activity of native MCP-1 (11,12) .
  • the current invention includes N-terminal deletants of RANTES, MlP-l ⁇ and MIP-1/3, lacking the biological activity of the native proteins, which inhibit HIV-l fusion and HIV-l infection.
  • Other peptides A series of overlapping peptides (e.g. of 20-67 residues) from all regions of RANTES, MIP-l ⁇ and MIP-1/3 are screened by the same approaches to identify peptides which inhibit HIV-l fusion most potently without activating leukocytes. Activation of leukocyte responses is measured following routine procedures (9, 10, 11, 12) . 3) Cloning the chemokine receptors
  • Chemokine receptors required for HIV-l fusion are cloned by the following strategy.
  • First a cDNA library is made in a mammalian expression vector (e.g. pcDNA3.1 from Invitrogen Corp. San Diego, CA) using mRNA prepared from the PM1 cell line or CD4 * T-lymphocytes or macrophages.
  • a mammalian expression vector e.g. pcDNA3.1 from Invitrogen Corp. San Diego, CA
  • Degenerate oligonucleotide probes are used to identify members of the cDNA library encoding members of the chemokine receptor family, for example following previously published methods
  • the vectors containing chemokine receptor cDNAs are then individually expressed in one of several mammalian cell lines which express human CD4 but do not fuse with
  • FL cells e.g. HeLa-CD4, CHO-CD4 or COS-CD4
  • HeLa-env ⁇ ! cells e.g. CHO-CD4 or COS-CD4
  • clones which gain the ability to fuse with HeLa-env JR . FL or HeLa-env, ⁇ are identified and the coding sequences recovered, for example by PCR amplification, following procedures well known to those skilled in the art. DNA sequencing is then performed to determine whether the cDNA recovered encodes a known chemokine receptor.
  • monoclonal and polyclonal antibodies are prepared and tested for ability to inhibit infection by a panel of HIV-l isolates. References of the First Series of Experiments
  • HIV-l isolates in CD4 + T-cells is inhibited by the C-C ⁇ -chemokines MlP-l ⁇ , MIP-l ⁇ and RANTES (1,2) , but T-cell line-adapted (TCLA) or syncytium-inducing (SI) primary strains are insensitive (2,3) .
  • the ⁇ -chemokines are small
  • Fusin 7-membrane-spanning, G-protein-linked superfamily, one of which (the LESTR orphan receptor) has been identified as the second receptor for TCLA HIV-l strains, and is now designated fusin (9) . Fusin is not known to be a ⁇ -chemokine receptor (7-9) .
  • ⁇ -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) .
  • MIP-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 BaL (Table la) .
  • 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/ l; Hofmann LaRoche , Nutley, NJ) , as described previously (3).
  • Target cells (1-2x10 s ) were infected with supernatants (10-50ng of HIV-l p2 4 ) from 293-cells co- transfected with an 0 NL4/3Aenv-luciferase vector and a HIV-l env- express ing vector (10,11) .
  • ⁇ -Chemokines (R & D Systems, Minneapolis) were added to the target cells simultaneously with virus, at the final concentrations (ng/ml) indicated in parentheses in the first column.
  • the ⁇ - chemokine concentration range was selected based on prior s tudies (2 , 3 ) .
  • Luciferase activi ty in cell lysa tes was measured as described previously (10 , 11 ) .
  • the values indica ted represent luciferase activi ty (cpm) /ng p24/mg protein, expressed rela tive to tha t in virus- control cul tures lacking ⁇ - chemokines (100%) , and are the means of duplica te or sextuplica te determina tions . nd, not done .
  • the env-complementation assay was used to assess HIV-l entry intc 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 MIP-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.3a) .
  • 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.3a) .
  • 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.3b) .
  • ⁇ -chemokines requires their presence during at least one of the early stages of HIV-l replication: virus attachment, fusion and early reverse transcription.
  • ⁇ -chemokines inhibited binding of JR-FL or BRU (LAI) gpl20 to soluble CD4, or of tetrameric CD4-IgG2 binding to HeLa-JR-FL cells expressing oligomeric envelope glycoprotein ⁇ (17) .
  • LAI JR-FL or BRU
  • tetrameric CD4-IgG2 binding to HeLa-JR-FL cells expressing oligomeric envelope glycoprotein ⁇
  • ⁇ -chemokines inhibit a step after CD4 binding, when conformational changes in the envelope glycoproteins lead to fusion of the viral and cellular membranes (18) .
  • RET resonance energy transfer
  • MlP-l ⁇ strongly inhibited membrane fusion of HeLa-JR-FL cells with PMl cells, whereas fusion between PMl cells and
  • HeLa-BRU cells was insensitive to these ⁇ -chemokines (Fig.
  • CD4* target cells mi togen-activated CD4 * lymphocytes or PMl 0 cells
  • wi th octadecyl rhodamine Mol ecular Probes, Eugene, OR
  • HeLa -JR-FL cells HeLa -BRU cells (or control 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 5 cells were mixed in 96-well pla tes and ⁇ - chemokines (or CD4 MAb OKT4a) were added at the final concen tra tions (ng/ml ) indica ted 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 that excitation of fluorescein at 450nm results in resonance energy transfer (RET) and emission by rhodamine at 590nm. Percentage fusion is defined as equal to 100 x [(Exp RET - Min RET) / (Max RET - Min
  • RET %RET obtained when HeLa- Env and CD4* cells are mixed
  • Exp RET %RET obtained when HeLa-Env and CD4 * cells are mixed in the presence of fusion- inhibitory compounds
  • Min RET %RET obtained when HeLa cells (lacking HIV-l envelope glycoproteins) and CD4' cells are mixed.
  • the %RET value is defined by a calculation described elsewhere (17 ⁇ , and each is the mean of triplicate determinations. These values were, for HeLa-JR-FL and HeLa-BRU cells respectively: PMl cells 11.5%, 10.5%; LW5 CD4 * cells, 6.0%, 10.5%; R/M /M ⁇ , RANTES + MI P- lot + MIP-l ⁇ .
  • C-C CKR-1 and, especially, C-C CKR-5 were identified as the most likely candidates, based on tissue expression patterns and their abilities to bind MlP-l ⁇ , MlP-l ⁇ and RANTES (4,7,8,15,22) .
  • C-C CKR-1, C-C CKR-5 and LESTR are each expressed at the mRNA level in PMl cells and primary macrophages (data not shown) . These and other ⁇ -chemokine receptors were therefore PCR-amplified, cloned and expressed.
  • 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.
  • L/5-1 AAG CTT GGA GAA CCA GCG GTT ACC ATG GAG GGG
  • L/3-1 CTC GAG CAT CTG TGT TAG CTG GAG TGA AAA CTT GAA GAC
  • L/3-2 GTC TGA GTC TGA GTC CTC GAG CAT CTG TGT (SEQ ID NO: 9) ;
  • CKR-1.-C1/5-1 AAG CTT CAG AGA GAA GCC GGG ATG GAA ACT CC
  • CKR-2a:C2/5-l AAG CTT CAG TAC ATC CAC AAC ATG CTG TCC AC (SEQ ID NO: 14) ;
  • C2/5-2 GTC TGA GTC TGA GTC AAG CTT CAG TAC ATC (SEQ ID NO: 14)
  • 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: 16);
  • CKR-4: C4/5-1 AAG CTT CTG TAG AGT TAA AAA ATG AAC CCC ACG
  • CKR-5 GTC TGA GTC TGA GTC AAG CTT AAC AAG ATG GAT
  • CAC (SEQ ID NO: 37) .
  • 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 p24/10 e cells) in the presence or absence of ⁇ -chemokines (400ng/ml each of RANTES, MlP-lot 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 COS-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.4) .
  • 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.
  • the chemokine SDF-1 (stromal cell-derived factor 1) is the natural ligand for Fusin/CXCR4 and blocks infection by laboratory-adapted strains of HIV-l (Ref. 1 and 2) .
  • SDF-1 exists as at least two forms, SDF-l ⁇ and SDF-l ⁇ based on variable splicing of the SDF-1 gene (Ref. 1 and 3)
  • this chemokine specifically inhibits membrane fusion mediated by gpl20/gp41 form the laboratory-adapted strain HIV ⁇ but not by gpl20/gp41 from the macrophage- tropic isolate HIV-1 JR . FL as shown in Figure 5.
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:11: GTCTGAGTCT GAGTCAAGCT TCAGAGAGAA 30
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide -
  • SEQUENCE DESCRIPTION SEQ ID NO:14: AAGCTTCAGT ACATCCACAA CATGCTGTCC AC 32
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide (x ) SEQUENCE DESCRIPTION: SEQ ID NO:16. CTCGAGCCTC GTTTTATAAA CCAGCCGAGA C 31
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:17- GTCTGAGTCT GAGTCCTCGA GCCTCGTTTT 30
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:23: GTCTGAGTCT GAGTCAAGCT TCTGTAGAGT 30
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotides
  • oligonucleotides SEQUENCE DESCRIPTION: SEQ ID NO:27: GTCTGAGTCT GAGTCCTCGA GTCCGTGTCA CAAGCCCAC 39

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Abstract

L'invention concerne une méthode permettant d'inhiber la fusion du VIH-1 et des cellules CD4+ et consistant à mettre les cellules CD4+ en contact avec un agent non-chémokine capable de se lier à un récepteur de chémokine dans des conditions et en quantités suffisantes pour inhiber la fusion du VIH-1 et des cellules CD4+. L'invention concerne également une méthode permettant d'inhiber l'infection des cellules CD4+ par le VIH-1 et consistant à mettre les cellules CD4+ en contact avec un agent non-chémokine capable de se lier à un récepteur de chémokine dans des conditions et en quantités suffisantes pour inhiber la fusion du VIH-1 et des cellules CD4+, ce qui permet d'inhiber l'infection par le VIH-1. De même, l'invention décrit des agents non-chémokines capables de se lier au récepteur de chémokine et d'inhiber la fusion du VIH-1 et des cellules CD4+. L'invention concerne finalement des compositions pharmaceutiques comprenant une certaine quantité d'agent non-chémokine capable de se lier au récepteur de chémokine et d'inhiber la fusion du VIH-1 et des cellules CD4+, ainsi qu'un excipient acceptable en pharmacologie.
PCT/US1997/005597 1996-04-02 1997-04-02 Methode de prevention de l'infection des cellules cd4+ par le vih-1 WO1997037005A1 (fr)

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WO1997045543A3 (fr) * 1996-05-28 1998-05-07 Us Health Recepteur 5 de cc chemokine, anticorps diriges contre ce dernier et animaux transgeniques
WO1999027122A1 (fr) * 1997-11-21 1999-06-03 Transgene S.A. Vecteurs inhibant ou retardant la liaison d'un virus d'immunodeficience aux cellules
EP1009435A1 (fr) * 1997-06-13 2000-06-21 Progenics Pharmaceuticals, Inc. Prevention de l'infection des cellules cd4?+ par le hiv-1
EP1025229A1 (fr) * 1997-10-22 2000-08-09 Genetics Institute, Inc. Chemokines avec modifications n-terminales
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
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6582920B2 (en) 2000-09-01 2003-06-24 Gen-Probe Incorporated Amplification of HIV-1 RT sequences for detection of sequences associated with drug-resistance mutations
US6623920B1 (en) * 1999-07-09 2003-09-23 Gen-Probe Incorporated Detection of HIV-1 by nucleic acid amplification
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US7060273B2 (en) 2001-04-06 2006-06-13 Progenics Pharmaceuticals, Inc. Methods for inhibiting HIV-1 infection
EP0910659B1 (fr) * 1996-06-03 2006-08-09 United Biomedical, Inc. Anticorps diriges contre un complexe de cd4 et d'un domaine de recepteurs des chemokines, et leur utilisation pour lutter contre les infections a vih
US7118859B2 (en) 1996-01-17 2006-10-10 Progenics Pharmaceuticals, Inc. Methods for inhibiting HIV-1 infection
US7122185B2 (en) 2002-02-22 2006-10-17 Progenics Pharmaceuticals, Inc. Anti-CCR5 antibody
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
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7935797B2 (en) 1996-04-01 2011-05-03 Progenics Pharmaceuticals Inc. CCR5 chemokine receptor-specific monoclonal antibodies capable of inhibiting HIV-1 cell fusion
US8821877B2 (en) 2005-07-22 2014-09-02 Cytodyn Inc. Methods for inhibiting HIV-1 replication involving the administration of an anti-CCR5 receptor monoclonal antibody and small molecule CCR5 receptor antagonist
US20160250300A1 (en) * 2013-08-29 2016-09-01 Temple University Of The Commonwealth System Of Higher Education Methods and compositions for rna-guided treatment of hiv infection

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US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
US7160546B2 (en) 1995-06-06 2007-01-09 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6759519B2 (en) 1995-06-06 2004-07-06 Human Genome Sciences, Inc. Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor)
US7345153B2 (en) 1996-01-17 2008-03-18 Progenics Pharmaceuticals, Inc. Compounds capable of inhibiting HIV-1 infection
US7118859B2 (en) 1996-01-17 2006-10-10 Progenics Pharmaceuticals, Inc. Methods for inhibiting HIV-1 infection
US7935797B2 (en) 1996-04-01 2011-05-03 Progenics Pharmaceuticals Inc. CCR5 chemokine receptor-specific monoclonal antibodies capable of inhibiting HIV-1 cell fusion
US8198042B2 (en) 1996-05-28 2012-06-12 The United States Of America, Represented By The Secretary, Department Of Health And Human Services CC chemokine receptor 5 DNA, new animal models and therapeutic agents for HIV infection
WO1997045543A3 (fr) * 1996-05-28 1998-05-07 Us Health Recepteur 5 de cc chemokine, anticorps diriges contre ce dernier et animaux transgeniques
US7374872B2 (en) 1996-05-28 2008-05-20 The United States Of America As Represented By The Department Of Health And Human Services CC chemokine receptor 5 DNA, new animal models and therapeutic agents for HIV infection
US7151087B2 (en) 1996-05-28 2006-12-19 The United States Of America As Represented By The Department Of Health And Human Resources CC chemokine receptor 5 DNA, new animal models and therapeutic agents for HIV infection
EP0910659B1 (fr) * 1996-06-03 2006-08-09 United Biomedical, Inc. Anticorps diriges contre un complexe de cd4 et d'un domaine de recepteurs des chemokines, et leur utilisation pour lutter contre les infections a vih
EP1009435A4 (fr) * 1997-06-13 2003-04-09 Progenics Pharm Inc Prevention de l'infection des cellules cd4?+ par le hiv-1
EP1009435A1 (fr) * 1997-06-13 2000-06-21 Progenics Pharmaceuticals, Inc. Prevention de l'infection des cellules cd4?+ par le hiv-1
EP1025229A4 (fr) * 1997-10-22 2003-06-18 Inst Genetics Llc Chemokines avec modifications n-terminales
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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
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