WO1999016883A2 - Stabilisation des glycoproteines trimeres d'enveloppe au moyen de liaisons bisulfure introduites dans un ectodomaine de la glycoproteine gp41 - Google Patents

Stabilisation des glycoproteines trimeres d'enveloppe au moyen de liaisons bisulfure introduites dans un ectodomaine de la glycoproteine gp41 Download PDF

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WO1999016883A2
WO1999016883A2 PCT/US1998/020693 US9820693W WO9916883A2 WO 1999016883 A2 WO1999016883 A2 WO 1999016883A2 US 9820693 W US9820693 W US 9820693W WO 9916883 A2 WO9916883 A2 WO 9916883A2
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hiv
gpl20
protein
glycoprotein
derivative
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PCT/US1998/020693
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WO1999016883A3 (fr
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Joseph G. Sodroski
Michael Farzan
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Dana-Farber Cancer Institute
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Priority to CA002305341A priority Critical patent/CA2305341C/fr
Priority to EP98950846A priority patent/EP1019511A2/fr
Priority to AU96785/98A priority patent/AU9678598A/en
Publication of WO1999016883A2 publication Critical patent/WO1999016883A2/fr
Publication of WO1999016883A3 publication Critical patent/WO1999016883A3/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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HIV-1 and type 2 are the etiologic agents of acquired immunodeficiency syndrome (AIDS), which results from the profound depletion of CD4-positive lymphocytes in infected individuals
  • the entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins.
  • the exterior envelope glycoprotein, gpl20, and the transmembrane envelope glycoprotein, gp41, are derived from a gpl ⁇ O precursor (Earl, P.L., et al., J Virol 1984).
  • the gpl ⁇ O glycoprotein results from the addition of N-linked, high mannose sugar chains to the approximately 845-
  • HIV- 1 infects T lymphocytes, monocytes/macrophage, dendritic cells .and, in the centra nervous system, microglia (Gartner et al, 1986; Koenig et al, 1986; Pope et al, 1994; Weissman et al, 1995). All of these cells express the CD4 glycoprotein, which serves as the receptor for HIV-1 and HIV-2
  • Efficient entry of HIV- 1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gpl20, to the CD4-ammo-terminal domain (McDougal et al, 1986; Helseth et al, 1990). After virus binding, the HIV-1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the entry process (Kowalski et al, 1987; Stein et al, 1987; Helseth et al, 1990).
  • Membrane fusion directed by HIV- 1 envelope glycoproteins expressed on the infected cell surface leads to fusion with uninfected CD4-positive cells, resulting in syncytia (Lifson et al, 1986; Sodroski et al, 1986).
  • Host cell factors in addition to CD4 are necessary for effective HIV- 1 envelope glycoprotein-mediated membrane fusion.
  • Some human and animal cells have been shown to be resistant to HIV- 1 infection and syncytium formation even when human CD4 was expressed on the cell surface (Maddon et al, 1986; Ashorn et al, 1990; Chesebro et al, 1990; McKnight et al, 1994).
  • All primary clinical HIV- 1 isolates defined as viruses that have not been passaged on immortalized cell lines, replicate in primary monocytes/macrophages .and in primary T lymphocytes.
  • Two groups of primary HIV- 1 isolates have been defined, based on replication rate in peripheral blood mononu clear cells (PBMC) and the ability to infect and induce the formation of syncytia in immortalized CD4-positive cell lines (Asjo et al, 1986; Cheng-Mayer et al, 1988; Fenyo et al, 1988; Tersmette et al, 1988).
  • PBMC peripheral blood mononu clear cells
  • T cell line-tropic primary viruses (sometimes referred to as "T")
  • T cell line-tropic primary viruses by virtue of their ability to replicate on some immortalized cell lines, serve as precursors to the laboratory- adapted isolates, which have been extensively passaged on such cell lines.
  • V3 region of the gpl20 exterior envelope glycoprotein determine tropism-related phenotypes (Cheng-Mayer et ⁇ l, 1990; O'Brien et ⁇ l, 1990; Hwang et ⁇ l., Westervelt et ⁇ l, 1992; Chesebro et ⁇ l, 1992; Willey et ⁇ l, 1994).
  • V3 region which contains a surface-exposed, disulfide-linked loop (Leonard et ⁇ l, 1990; Moore et ⁇ l, 1994), might act in conjunction with target cell moieties to determine the efficiency of membrane fusion events.
  • HUMSTR HUMSTR
  • LCR-1 LCR-1
  • LESTR LCR-1
  • CXCR4 G protein-coupled seven transmembrane segment receptor
  • HUMSTSR While its natural ligand is currently unknown, HUMSTSR exhibits sequence similarity to the receptor for interleukin-8, an alpha (CXC) chemokine) (Probst et al, 1992).
  • CXC alpha
  • Other G-protein-coupled seven transmembrane segment receptors such as CCR5, CCR3 and CCR2 have been shown to assist cellular entry of other HIV-1 isolates. It is believed that the cellular entry occurs as a result of the interaction of g l20, CD4 and the chemokine receptor.
  • DNA sequences encoding env where we can introduce sequences encoding cysteine residues in a portion encoding the gp 41 transmembrane envelope glycoprotein. These sequences will express proteins that can stably oligomerize in a conformation approaching the native virus. The introduction of these residues creates the molecular contacts between alpha helices that stabilize the trimeric coiled coil, which is responsible for the oligomerization of the HIV-1 envelope glycoprotein. These cysteine residues are introduced in specific locations along these alpha helices. One preferred location is at the residues adjacent to the d and e positions of the coiled coil helix such as positions 576 .and 577 of HIV-1.
  • an adjoining amino acid residue be substituted to provide greater flexibility in the protein backbone; one example is the substitution of a gly at the f position such as 578 of HIV-1.
  • the normally labile HIV-1 g ⁇ l60 envelope glycoprotein was converted into a stable disulfide-linked oligomer that was expressed on the cell surface and had a conformation approaching that of the native glycoprotein as demonstrated by its ability to be recognized by a series of conformationally dependent antibodies.
  • the pattern of hetero- oligomer formation between this construct and an analogous construct lacking portions of the g ⁇ l20 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers.
  • the stabilized oligomer can be used to generate a range of antibodies that recognize and interact with a diverse range of HIV strains.
  • the DNA sequence can also be used as a subunit vaccine.
  • Figures 1A and IB show coiled coil regions from env.
  • Figure 1A shows models of coiled coils.
  • the top view of a segment of model dimeric, trimeric and tetrameric coiled coils is shown.
  • the C ⁇ , C ⁇ and C ⁇ atoms for residues for which the interhelical C ⁇ -C ⁇ and C ⁇ -C ⁇ distances are at a minimum are depicted.
  • Typical interhelical distances for the dimer (O'Shea, E., et al., Science 1991) at the d position (shown in dark) of the heptad repeat are 6.1 angstroms for the C ⁇ -C ⁇ and 3.7 angstroms for the C ⁇ -C ⁇ distance.
  • the typical C ⁇ -C ⁇ .and C ⁇ -C ⁇ dist.ance from the d (dark) to e (white) positions in the trimer are 7.2 and 7.1 angstroms, respectively (Harbury, P.B., et al., Nature 1994).
  • the a position (dark) is closest to the g position (white) of an adjacent helix, with a C ⁇ -C ⁇ distance of 6.7 angstroms and a C ⁇ -C ⁇ distance of 4.2 angstroms (Harbury, P.b., et al., Science 1993).
  • Figure IB shows a portion of the gp41 protein containing the amino acid sequence of the coiled coil region of the gp41 (SEQ ID NO: 11), indicating residue number and the position .along the heptad repeat of the coil.
  • Figure 2 shows immunoprecipitation of HIV-1 envelope glycoprotein variants. Plasmids encoding the wild-type HIV-1 envelope glycoproteins and three of the mutant envelope glycoproteins described in Table 1 were tr.ansfected into COS- 1 cells. Cell lysates were immunoprecipitated with the anti-gp41 antibody D61, .and the precipitates were boiled in 2% ⁇ - mercaptoethanol for 3 minutes prior to analysis on an 8% SDS-polyacrylamide gel.
  • Figure 3 shows analysis of wild-type and LQA/CCG envelope glycoproteins. Lysates were immunoprecipitated with the anti-gp41 antibody D61 .and boiled in either 2% or 5% ⁇ -mercaptoethanol for 3 or 10 minutes, as indicated, prior to analysis on an 8% polyacrylamide gel.
  • Figure 4 shows precipitation of LQA/CCG and ⁇ LQA/CCG envelope glycoproteins with antibodies.
  • Lysates containing the LQA/CCG and the ⁇ LQA/CCG envelope glycoproteins were precipitated with HIV-1 -infected patient sera (PSI, PS2), the F105 antibody, the 17b antibody in the presence or absence of soluble CD4, the Cl l antibody, or the G3-519 antibody.
  • the A32 antibody and the anti-gp41 antibodies D ⁇ l, T3 and T4 all recognized both monomeric and higher order forms of LQA/CCG and ⁇ LQA/CCG envelope glycoproteins (data not shown).
  • the 110.4 antibody, directed against the third variable loop of gpl20, recognized the LQA/CCG glycoprotein (data not shown and Figure 5, lane 5).
  • Figure 5 shows formation of hetero-oligomers between LQA/CCG and ⁇ LQA/CCG envelope glycoproteins.
  • Serum from an HIV-1 infected individual was used to precipitate lysates of 293T cells transfected with plasmids encoding LQA/CCG (lane 1) and ⁇ LQA/CCG (lane 4) envelope glycoproteins.
  • plasmids expressing the LQA/CCG and ⁇ LQA/CCG envelope glycoproteins were tr ⁇ ansfected at a 2: 1 ratio, while in lane 3, the LQA/CCG- and ⁇ LQA/CCG- expressing plasmids were transfected in equal amounts.
  • the same cell lysates as those used for the experiment in lane 3 were used for precipitation by the anti-V3 loop .antibody 110.4.
  • Figure 6 shows potenti.al sites for disulfide cross-linking of the HIV- 1 envelope glycoprotein trimer.
  • the structure of the gp41 ectodomain peptides assembled into the helical coiled coil is shown.
  • the sites of intersubunit interactions at the d and e positions of the coiled coil are shaded, as is the site of the LQA/CCG mutant (dark shading) shown to allow cross-linking of gpl60 trimers.
  • Both cys-cys and cys-cys-gly substitutions can be made at the indicated locations along the coiled coil. Substitutions that result in disulfide bridges and trimer stabilization can also be used in combination.
  • gp 160 an improved immunogenic gpl20-gp 41oligomer, sometimes referred to as gp 160 and DNA sequences encoding them.
  • This oligomer is stabilized by the creation of cysteine-SH-cysteine bonds.
  • the resulting oligomer forms spikes similar to that seen in the native wild type virus. Consequently, .antibodies generated by these polypeptides are more likely to recognize .and interact with native virus.
  • the gpl60 glycoprotein is the precursor for gp 120 and gp 41. Following oligomerization of the precursor the gpl60 glycoprotein is transported to the Golgi apparatus where cleavage by a cellular protease generates the g ⁇ l20 and gp41 glycoproteins, which remain associated through non-covalent interactions (Earl, P.L., et al., J Virol 1991, Kowalski, M., et al., Science 1987). In mammalian host cells, addition of complex sugars to selected, preferably surface-exposed, carbohydrate side chains of the envelope glycoproteins occurs in the Golgi apparatus (Leonard, C.K., et al, J Biol Chem 1990).
  • the mature envelope glycoprotein complex is incorporated into virions, where it mediates virus entry into the host cell.
  • the gpl20 exterior envelope glycoprotein binds the CD4 glycoprotein, which serves as a receptor for the virus (Klatzmann, D., et al., Nature 1984, Dalgleish, A.G., et al, Nature 1984). Because gpl20 is external as discussed above it was proposed as a natural target for trying to develop an immune response to prevent viral entry. However, in part due to the numerous variable regions which can mutate rapidly, the wild type gp 120 has not proven to be a successful target.
  • chemokine receptors which are seven- transmembrane G protein-coupled receptors (Feng, Y., et al., Science 1996; Choe, H., et al., Cell 1996; Doranze, et al., Cell 1996; Dragic, et al., Nature 1996; Alkhatib, G., et al., Science 1996).
  • the chemokine receptor interaction is believed to bring the viral envelope glycoprotein complex nearer to the target cell membrane and to trigger additional conformational changes in the envelope glycoproteins (Wu, L., et al., Nature 1996; Trkola, A., et al., Nature 1996). These changes are proposed to result in the interaction of the gp41 glycoprotein with the target cell membrane, culminating in fusion of this membrane with the viral membrane.
  • Such a model is consistent with mutagenic analysis.
  • fusion peptide amino acid changes in the hydrophobic gp41 amino terminus (the "fusion peptide"), in the amino-terminal half of the ectodomain, or in the transmembrane region all result in fusion-defective envelope glycoproteins (Kowalski, M., Science 1987; Freed, E.O., Proc Natl Acad Sci 1990; Cao, J., J Virol 1993). All these factors confirm the importance of the envelope glycoprotein. However, in nature an oligomeric form is seen. Thus, being able to prepare a stable oligomer containing the gp 120 portion is extremely important. Yet, the stable oligomer must approximate the conformation of the oligomer formed natur-ally. This has proven difficult.
  • the HIV- 1 envelope glycoprotein oligomer is naturally labile, disassociating into individual subunits readily.
  • the introduction of cysteine residues in inappropriate positions can result in non-native structures. Since these molecules are folded differently than the native HIV-1 envelope glycoproteins, their utility in raising antibodies that recognize and neutralize the viral envelope spike is limited. We have discovered that there .are only a limited number of positions in the gp 41 portion that can be used to create a stable oligomer that approximates the native conformation.
  • Soluble forms of HIV-1 envelope glycoprotein oligomers should have advantages over monomeric gpl20 preparations as immunogens, since the former are more likely to mimic the native envelope glycoprotein spike on virions (Broder, C.C., et al., Proc Natl Acad Sci USA 1994). Unfortunately, due to the lability of HIV-1 envelope glycoprotein, the preparation of high-quality stable oligomers that maintain high-order states has been difficult.
  • Coiled coils are believed to play a central role in influenza virus entry mediated by the hemagglutinin molecule, where the extension of a trimeric coiled coil in the transmembrane HA 2 subunit is thought to mark the transition to a fusogenic conformation of this protein (Carr, CM., et al., Cell 1993; Bullogh, P.A., Nature 1994). Recently, a crystal structure of an HIV- 1 gp41 ectodomain fragment has been obtained, confirming the existence of a trimeric coiled coil that is bound and stabilized by three monomers of a C-terminal helix (Chan, D.C., Cell 1997).
  • the impaired processing not appear to result from inefficient folding or transport along the secretory pathway.
  • the processing defect could reflect a subtle conformational alteration in the envelope glycoprotein region recognized by the cellular protease, or could suggest that a degree of flexibility at the gp 120/gp41 cleavage site is necessary for efficient processing and is not present in the LQA/CCG mutant.
  • gp 120 polypeptide Traditional approaches at generating antibodies to env have typically focused on the gp 120 polypeptide. However, we found that creating a fusion protein containing a gpl20 portion, preferably a modified gp 120 portion, .and a modified gp 41 portion permits the creation of stable oligomers. As will be discussed in detail below the preferred modified gp 120 portion is a gp 120 protein that has been modified to have variable loops or portions thereof.
  • the HIV-1 envelope glycoprotein oligomer may be stabilized through intersubunit disulfide bonds.
  • One preferred structure has cysteine residues introduced at residues adjacent to the d and e positions of the coiled coil helix in gp 41. See Fig. IB for the amino acid and a nucleotide sequence of this region. These positions correspond to 576 and 577 of HIV- 1. These residues are highly conserved among HIV-1 and HIV-2 strains, indicating that the approach is applicable to both HIV-1 and HIV-2. These positions correspond to 576 and 577 of the HXBc2 isolate of HIV-1. The numbering varies slightly for different HIV-1 isolates, although the sequence in this region of the gp41 coiled coil is largely conserved. Therefore, the equivalently positioned residues .are easily identified in other HIV- 1 and, in fact, in HIV-2 envelope glycoproteins as well.
  • glycines could be introduced adjacent to the introduced cysteines, at positions 557, 564, 571 and 584, respectively.
  • an adjoining amino acid residue with one that provides flexibility in turning.
  • the residue is Gly.
  • substituting gly for ala at position f of the helix in the above example of 576/577 corresponds to position 578.
  • Disulfide crosslinking of the HIV-1 envelope glycoprotein trimer stabilizes otherwise labile neutralization epitopes specific for the oligomer and the form can mask biologically irrelevant epitopes that are exposed on the gp 120 or gpl ⁇ O monomer but buried on the functional oligomer, and lengthen the half-life of the intact vaccine construct in the body.
  • the disulfide crosslinking strategy described herein can be used with other elements of the gp 41 coiled coil based upon our teaching (See Figure 6).
  • Dimers as well as trimers of the mutant may be stabilized by the formation of disulfide bonds.
  • the dimer form of the mutant was less abundant than the trimer and was more sensitive to a disruption by boiling (data not shown).
  • Stable dimers could represent intermediates in the assembly or disassembly of the trimer.
  • the dimer could result from the formation of an alternative disulfide bond between the cysteines in the d positions, excluding the possibility of forming the three d-e disulfide bonds presumably present in the trimer.
  • the oligomer complexes can be used to generate a range of antibodies to gpl20 and gp41.
  • antibodies that affect the interaction with the binding site can be directly screened for example using a direct binding assay.
  • a direct binding assay For example, one can label, e.g. radioactive or fluorescent, a gpl20 protein or derivative and add soluble CD4.
  • soluble CD4s There are various soluble CD4s known in the art including a two-domain (D1D2 sCD4) and a four-domain version.
  • the labeled gpl20, or derivative, e.g., a conformationally intact deletion mutant such as one lacking portions of the variable loops (e.g. V1/V2) and in some instances constant regions and soluble CD4 can be added to medium containing a cell line expressing a chemokine receptor that the antibody will block binding to.
  • the derivative will blocking binding to CCR5.
  • a derivative from a T cell tropic gpl20 one would use a cell line that expresses CXCR4. Binding can then be directly measured.
  • the antibody of interest can be added before or after the addition of the labeled gpl20 or derivative and the effect of the antibody on binding can be determined by comparing the degree of binding in that situation against a base line standard with that gpl20 or derivative, not in the presence of the antibody.
  • a preferred assay uses the labeled gpl20, or derivative portion, for example a gpl20 protein derived from an M-tropic strain such as JR-FL, iodinated using for instance solid phase lactoperoxidase (in one example having a specific activity of 20 ⁇ Ci/ ⁇ g).
  • the cell line containing the chemokine receptor in this example would be a CCR5 cell line, e.g. LI.2 or membranes thereof. Soluble CD4 would be present.
  • the conformational gp 120 portion should contain a sufficient number of amino acid residues to define the binding site of the gpl20 to the chemokine receptor (e.g. typically from the V3 loop) and a sufficient number of amino acids to maintain the conformation of the peptide in a conformation that approximates that of wild-type gpl20 bound to soluble CD4 with respect to the chemokine receptor binding site.
  • the V3 loop can be removed to remove masking amino acid residues.
  • linker residues that permit potential turns in the polypeptides structure. For example, amino acid residues such as Gly, Pro and Ala. Gly is preferred.
  • the linker residue is as small as necessary to maintain the overall configuration. It should typically be smaller than the number of .amino acids in the variable region being deleted.
  • the linker is 8 amino acid residues or less, more preferably 7 amino acid residues or less. Even more preferably, the linker sequence is 4 amino acid residues or less. In one preferred embodiment the linker sequence is one residue.
  • the linker residue is Gly.
  • the gpl20 portion also contains a CD4 binding site (e.g. from the C3 region residues 368 and 370, and from the C4 region residues 427 and 457).
  • the chemokine binding site is a discontinuous binding site that includes portions of the C2, C3, C4 and V3 regions.
  • deletion of non-essential portions of the gpl20 polypeptide such as deletions of portions of non-essential variable regions (e.g. VI /V2) or portions in the constant regions (e.g. Cl, C5) one can increase exposure of the CD4 binding site.
  • Another embodiment is directed to a gp 120 portion containing a chemokine binding site.
  • the increased exposure enhances the ability to generate an antibody to the CD4 receptor or chemokine receptor, thereby inhibiting viral entry.
  • Remov.al of these regions is done while requiring the derivative to retain an overall conformation approximating that of the wild-type protein with respect to the native gpl20 binding region, e.g. the chemokine binding region when complexed to CD4.
  • Preferred amino acid residues that can be used as linker include Gly and Pro.
  • Other ⁇ amino acids can also be used as part of the linker, e.g. Ala. Examples on how to prepare such peptides are described more fully in Wyatt, R., et al. J. of Virol. 69:5723-5733 (1995); Thali, M., et al, J. of Virol 67:3978-3988 (1993); and U.S. Application Serial No. 07/858, 165 filed March 26, 1992 which are incorporated herein by reference. See for example Wyatt which teaches how to prepare VI /V2 deletions that retain the stem portion of the loop.
  • the gpl20 derivative is designed to be permanently attached at the CD4 binding site to sufficient dom.ains of CD4 to create a conformation of the chemokine binding site approximating that of the native gpl20 CD4 complex.
  • An alternative gpl20 derivative is one wherein the linkers used result in a conformation for the derivative so that the discontinuous binding site with the chemokine receptor approximates the conformation of the discontinuous binding site for the chemokine receptor in the wild-type gpl20/CD4 complex.
  • These derivatives can readily be made by the person of ordinary skill in the art based upon the above described methodologies and screened in the assays shown herein to ensure that proper binding is obtained.
  • the g l20 polypeptide portion is bound to at least a portion of gp41 polypeptide, namely the coiled coil. Some of these derivatives will lack the gp41 transmembrane region and will therefore be made as secreted, soluble oligomers.
  • gp41 portions lacking the transmembrane region but retaining the cytoplasmic region, others truncated beginning with the transmembrane region, and therefore also lacking the cytoplasmic region.
  • those amino acids although being residues that do not bind to the membrane, would be selected to have minimal conformational effect on the polypeptides.
  • These amino acids can readily be selected by the skilled artisan based upon known knowledge in view of the present disclosure. This can be done by standard means using known techniques such as sets directed mulogenesis.
  • the gp41 polypeptide contains the indicated cysteine residues, which result in the formation of the SH bonds between the monomers thereby stabilizing the complex as a trimer having spikes similar to that found in the wild type.
  • These immunogenic oligomers can be used to generate an immune reaction in a host by standard means. For example one can adrninister the trimeric protein in adjuvant.
  • a DNA sequence encoding the gpl20-gp41 complex can be administered by standard techniques. The approach of administering the protein is presently preferred.
  • the protein is preferably administered with an adjuvant.
  • Adjuvants are well known in the art and include aluminum hydroxide, Ribi adjuvant, etc.
  • the administered protein is typically an isolated and purified protein.
  • the protein is preferably purified to at least 95% purity, more preferably at least 98% pure, and still more preferably at least 99% pure. Methods of purification while retaining the conformation of the protein are known in the art.
  • the purified protein is preferably present in a pharmaceutical composition with a pharmaceutically acceptable carrier or diluent present.
  • DNA sequences encoding these proteins can readily be made.
  • native gp 160 of any of a range of HIV-1 str.ains which are well known in the art and can be modified by known techniques such to deleted the undesired regions such as variable loops and to insert desired coding sequences such as cysteines and linker segments.
  • desired coding sequences such as cysteines and linker segments.
  • codons for the various amino acid residues are known and one can readily prepare alternative coding sequences by standard techniques.
  • DNA sequences can be used in a range of animals to express the monomer, which then forms into the trimer and generates an immune reaction.
  • DNA sequences can be administer to a host animal by numerous methods including vectors such as viral vectors, naked DNA, adjuvant assisted DNA catheters, gene gun, liposomes, etc.
  • the DNA sequence is administered to a human host as either a prophylactic or therapeutic treatment to stimulate an immune response, most preferably as a prophylactic.
  • Vectors include chemical conjugates such as described in WO 93/04701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, etc.
  • the vectors can be chromosomal, non-chromosomal or synthetic.
  • Retroviral vectors include moloney murine leukemia viruses and HIV-based viruses.
  • One preferred HIV-based viral vector comprises at least two vectors wherein the gag .and pol genes are from an HIV genome and the env gene is from another virus.
  • These vectors include herpes virus vectors such as a herpes simplex I virus (HSV) vector [Geller, A.I. et al. J. Neurochem 64: 487 (1995); Lim, F. et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A.I.
  • HSV herpes simplex I virus
  • the DNA sequence would be operably linked to a promoter that would permit expression in the host cell.
  • promoters are well known in the .art and can readily be selected.
  • Stabilized forms of these complexes can readily be made, for example, by conjugates such as a poly(alkylene oxide) conjugate.
  • the conjugate is preferably formed by covalently bonding the hydroxyl terminals of the poly(alkylene oxide) and a free amino group in the gpl20 portion that will not affect the conformation of the discontinuous binding site.
  • Other art recognized methods of conjugating these materials include amide or ester linkages. Covalent linkage as well as non-covalent conjugation such as lipophilic or hydrophilic interactions can be used.
  • the conjugate can be comprised of non-antigenic polymeric substances such as dextran, polyvinyl pyrrolidones, polysaccharides, st.arches, polyvinyl alcohols, polyacryl amides or other similar substantially non-immunogenic polymers.
  • Polyethylene glycol(PEG) is preferred.
  • Other poly(alkylenes oxides) include monomethoxy-polyethylene glycol polypropylene glycol, block copolymers of polyethylene glycol, and polypropylene glycol and the like.
  • the polymers can also be distally capped with Cl-4 alkyls instead of monomethoxy groups.
  • the poly(alkylene oxides) used must be soluble in liquid at room temperature. Thus, they preferably have a molecular weight from about 200 to about 20,000 daltons, more preferably about 2,000 to about 10,000 and still more preferably about 5,000.
  • these stabilized compounds can be administered to individuals by a variety of means.
  • these .antibodies can be included in vaginal foams or gels that are used as preventives to avoid infection .and applied before people have sexual contact.
  • the peptides or antibodies when used for administration are prepared under aseptic conditions with a pharmaceutically acceptable carrier or diluent.
  • Doses of the pharmaceutical compositions will vary depending upon the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 ⁇ g/kg a day, more preferably 1 to 10,000 ⁇ g/kg.
  • Routes of administration include oral, parenteral, rectal, intravaginal, topical, nasal, ophthalmic, direct injection, etc.
  • V3 region of the g ⁇ l20 exterior envelope glycoprotein determine tropism-related phenotypes (Cheng-Mayer et al, 1990; O'Brien et al, 1990;
  • the inhibition of HIV infection means that as compared to a control situation infection is reduced, inhibited or prevented. Infection is preferably at least 20% less, more preferably at least 40% less, even more preferably at least 50% less, still more preferably at least 75% less, even more preferably at least 80% less, and yet more preferably at least 90% less than the control.
  • antibodies can be included in ointments, foams, creams that can be used during sex. For example, they can be administered preferably prior to or just after sexual contact such as intercourse.
  • One preferred composition would be a vaginal foam containing one of the antibodies.
  • Another use would be in systemic administration to block HIV- 1 replication in the blood and tissues.
  • the antibodies could also be administered in combination with other HIV treatments.
  • An exemplary pharmaceutical composition is a therapeutically effective amount of a the oligomer, antibody etc. that for examples affects the ability of the receptor to facilitate HIV infection or for the DNA sequence or the oligomer that can induce an immune reaction, thereby acting as a prophylactic immunogen, optionally included in a pharmaceutically-acceptable and compatible carrier.
  • pharmaceutically-acceptable and compatible carrier includes (i) one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal, and/or (ii) a system, such as a retroviral vector, capable of delivering the molecule to a target cell.
  • the term “carrier” thus denotes an organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention .are combined to facilitate application.
  • the term “therapeutic-ally-effective amount” is that amount of the present pharmaceutical compositions which produces a desired result or exerts a desired influence on the particul.ar condition being treated. For example, the amount necessary to raise an immune reaction to provide prophylactic protection.
  • at least one "boost” will be administered at a periodic internal after the initial administration.
  • concentrations may be used in preparing compositions incorporating the same ingredient to provide for variations in the age of the patient to be treated, the severity of the condition, the duration of the treatment and the mode of administration.
  • compatible means that the components of the pharmaceutical compositions are capable of being commingled with a small molecule, nucleic acid and /or polypeptides of the present invention, and with each other, in a manner such that does not substantially impair the desired pharmaceutical efficacy.
  • Dose of the pharmaceutical compositions of the invention will vary depending on the subject and upon particular route of administration used. Dosages can range from 0.1 to 100,000 ⁇ g/kg per day, more preferably 1 to 10,000 ⁇ g/kg. By way of an example only, an overall dose range of from about, for example, 1 microgram to about 300 micrograms might be used for human use. This dose can be delivered at periodic intervals based upon the composition. For example on at least two separate occasions, preferably spaced apart by about 4 weeks. Other compounds might be administered daily. Pharmaceutical compositions of the present invention can also be administered to a subject according to a variety of other, well-characterized protocols.
  • certain currently accepted immunization regimens can include the following: (i) administration times are a first dose at elected date; a second dose at 1 month after first dose; and a third dose at 5 months after second dose. See Product Information, Physician's Desk Reference, Merck Sharp & Dohme (1990), at 1442-43. (e.g., Hepatitis B Vaccine-type protocol); (ii) Recommended administration for children is first dose at elected date (at age 6 weeks old or older); a second dose at 4-8 weeks after first dose; a third dose at 4-8 weeks after second dose; a fourth dose at 6-12 months after third dose; a fifth dose at age 4-6 years old; and addition ⁇ boosters every 10 years after last dose.
  • Desired time intervals for delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation.
  • the antibodies, DNA sequences or oligomers of the invention may also be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention.
  • Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene- 2 -sulfonic, and benzenesulphonic.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • the present invention also provides pharmaceutical compositions, for medical use, which comprise nucleic acid and/or polypeptides of the invention together with one or more pharmaceutically acceptable carriers thereof and optionally any other therapeutic ingredients.
  • compositions include those suitable for oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral administration, all of which may be used as routes of administration using the materials of the present invention.
  • Other suitable routes of administration include intrathecal administration directly into spinal fluid (CSF), direct injection onto an arterial surface and intraparenchymal injection directly into targeted areas of an organ.
  • Compositions suitable for parenteral administration are preferred.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing the active ingredients of the invention into association with a carrier which constitutes one or more accessory ingredients.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the nucleic acid and /or polypeptide of the invention in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the molecule of the invention which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenteralfy-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution .and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectibles.
  • Antibodies are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may
  • antibodies is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by eukaryotic nucleotide sequences of the present invention.
  • selectively reactive refers to those antibodies that react with one or more antigenic determinants on e.g. gpl20 and do not react with other polypeptides.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibodies can be used for diagnostic applications or for research purposes, as well as to block bindiner interactions.
  • cDNA clone encoding a gpl20-gp41 complex of the present invention may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York: 1989) such that 5-20% of the total protein that can be recovered from the host is the desired protein. Recovered proteins can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel. The desired protein sample can then be eluted from the gel slice and prepared for immunization. Preferably, one would design a stable cell could expressing high levels of the proteins which be selected and used to generate antibodies
  • mice can be immunized twice intraperitoneally with approximately 50 micrograms of protein immunogen per mouse. Sera from such immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by ELISA ⁇ with the expressed polypeptide.
  • active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin-peroxidase .and a chromogenic peroxidase substrate. Preparations of such reagents are commercially available; for example, from Zymad Corp., San Francisco, California.
  • mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production. Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.
  • amino acid sequence of polypeptides encoded by a eukaryotic nucleotide sequence of the present invention may be analyzed in order to identify desired portions of amino acid sequence which may be associated with receptor binding.
  • polypeptide sequences may be subjected to computer analysis to identify such sites.
  • any technique that provides for the production of antibody molecules by continuous cell lines may be used.
  • SCA single-chain antibody
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • the present invention provides for antibody molecules as well as fragments of such antibody molecules.
  • Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities.
  • This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.
  • the preferred binding is, however, covalent binding.
  • Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules.
  • representative coupling agents can include organic compounds such as thioesters, carbodii ides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines.
  • linkers include: (i) EDC (l-et ⁇ yl-3-(3-dimeti ylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- ⁇ yridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); ( ⁇ i) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem.
  • linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties.
  • sulfo-NHS esters of alkyl carboxylates are more stable than sulfo- NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.
  • Disulfide linkages are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
  • Sulfo- NHS in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings when used in conjunction with sulfo- NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • Antibodies of the present invention can be detected by appropriate assays, such as the direct binding assay discussed earlier and by other convention ⁇ types of immunoassays.
  • a sandwich assay can be performed in which the receptor or fragment thereof is affixed to a solid phase. Incubation is maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase is separated from the sample.
  • the solid phase is washed to remove unbound materials and interfering substances such as non-specific proteins which may also be present in the sample.
  • the solid phase containing the antibody of interest bound to the immobilized polypeptide of the present invention is subsequently incubated with labeled antibody or antibody bound to a coupling agent such as bio tin or avidin.
  • Labels for antibodies are well-known in the art and include radionuclides, enzymes (e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biot ⁇ n, and the like.
  • the labeled antibodies are incubated with the solid and the label bound to the solid phase is measured, the amount of the label detected serving as a measure of the amount of anti-urea transporter antibody present in the sample.
  • the ⁇ V1/V2/V3 (tail-) 576/7/8 LQA/CCG construct was made by introducing the 576/7/8 LQA/CCG mutation into a previously described HIV-1 envelope glycoprotein construct (Wyatt, R., et al., J Virol 1995), in which residues 128- 194 and 298-303 were replaced by glycine-alanine-glycine connectors, and a stop codon was introduced to produce an envelope glycoprotein truncated after residue 712 (Mammano, F., J Virol 1995).
  • Transfections, Metabolic Labeling and Analysis of Envelope Glycoproteins Cells were transfected by the calcium phosphate method, using 25 ⁇ g of the pSVIIIenv plasmid expressing wild-type or mutant envelope glycoproteins, as described (Cao, J., J Virol 1993). Transfected cells were labeled with 35 S- cysteine and used for analysis of envelope glycoproteins. For studying expression and the presence of higher-order forms of the envelope glycoproteins, labeled cells were lysed in NP40 buffer (0.5% NP40, 0.5 M NaCl, 10 mM Tris, pH 7.5) and used for immunoprecipitation by serum from an HIV-1 infected individual.
  • NP40 buffer 0.5% NP40, 0.5 M NaCl, 10 mM Tris, pH 7.5
  • Precipitates were boiled in sample buffer containing from 0 to 5% ⁇ -mercaptoethanol for 3 to 10 minutes prior to analysis on 7 or 10% SDS-polyacrylamide gels. In some experiments, 10 mM iodoacetamide was included in lysis and sample buffers and in these cases, no ⁇ -mercaptoethanol was added to the sample buffer prior to analysis on SDS-polyacrylamide gels. For analysis of the conformation of the mutant envelope glycoproteins, radiolabeled cell lysates in NP40 buffer were precipitated with the antibodies described above. Precipitates were analyzed on an 8 percent SDS- polyacrylamide gel after boiling in sample buffer containing 0.4% ⁇ - mercaptoethanol .
  • Cell surface expression was also assessed by FACS analysis of 293T cells that were either mock-transfected or transfected with pSVIIIenv plasmid encoding wild-type or mutant envelope glycoproteins.
  • Cells were incubated for one hour at 4° C, with 0.5 ⁇ g of F105, 110.4, Cl l or 212A antibodies, washed in PBS, and subsequently incubated with 1 ⁇ l/ml phycoeiythrin-conjugated goat anti-hum.an IgG (sigma, St. Louis, MO). Cells were washed and fixed in 2% formaldehyde in PBS and analyzed on a Becton-Dickenson FACS analyzer.
  • Modeling and visualization of model coiled-coils were done with Slimm, using Silcon Graphics.
  • the illustrations in Figure 1 were constructed with Molscript (Kraulis, P., JAppl Crstallogr 1991).
  • a disulfide bond has been previously introduced in a model dimeric coiled coil by substitution of cysteines at the d position of the helical repeat structure (Zhou, N.E., Biochemistry 1993.
  • distance requirements for disulfide bond formation could be met by introduction of cysteines at the g and a positions.
  • no simple substitution of cysteines met the ideal distance requirements for the formation of a disulfide bond.
  • Table 1 shows the mutant HIV-1 envelope glycoproteins and the observed phenotypes. Most of the envelope glycoproteins were defective in processing of the gpl ⁇ O precursor tomature gpl20 and gp41 glycoproteins ( Figure 1 and data no shown).
  • LQA/CCG 576/7/8 LQA/CCG
  • the LQA/CCG mutant was expressed on the surface of transfected cells at levels comparable to those of the wild-type envelope glycoproteins, as assessed by FACS analysis and by a surface immunoprecipitation assay (data not shown). Moreover, the higher order forms of the LQA/CCG mutant were precipitated by a number of monoclonal antibodies that recognize discontinuous epitopes on the HIV-1 gpl20 envelope glycoprotein (Moore, J.P., et al., J Virol 1996). These include the F105 antibody, which recognizes the CD4 binding site, the 17b antibody, which recognizes a CD4-induced epitope, and antibodies directed against the third variable loop of gpl20 ( Figure 4 and Figure 4 legend). It is noteworthy that the 17b epitope represents the discontinuous epitope most sensitive to disruption by detergent (Thaili, M., J Virol 1993). These results suggest that the LQA/CCG mutant does not exhibit global defects in folding or transport.
  • ⁇ V1/V2/V3 (tail-) 576/7/8 LQA/CCG (hereafter referred to as ⁇ LQA/CCG)
  • ⁇ LQA/CCG is identical to the LQA/CCG mutant except that it lacks the V1/V2 and V3 gpl20 loops and a large portion of the gp41 cytoplasmic tail.
  • the ⁇ LQA/CCG glycoprotein was efficiently expressed on the cell surface as judged by FACS analysis, and was recognized by a number of monoclonal antibodies with conformation- dependent epitopes (Figure 4 and data not shown).
  • the ⁇ LQA/CCG envelope glycoprotein precursor migrated with an apparent molecular mass of 110 kD, presumably a monomer, and two apparently higher-order forms resistant to boiling and gentle reduction.
  • the smaller of these higher-order forms migrated slightly slower than the 200 kD marker protein, suggesting that it represents a dimer of the ⁇ LQACCG protein (Figure 4).
  • the larger of the two high-order forms of the ⁇ LQA/CCG protein comigrated with the smaller of the two higher-order forms of the LQA/CCG protein ( Figures 4 and 5). This is consistent with the expected molecular mass of approximately 330 kD for a ⁇ LQA/CCG trimer and an expected molecular mass of 320 kD for a LQA/CCG dimer.
  • the LQA/CCG and ⁇ LQA/CCG proteins were expressed in the same cells by cotransfection of their respective expresser plasmids. We anticipated that these two proteins would form hetero-oligomers and that the pattern of bands formed would allow a determination of the number of subunits in the assembled oligomers. For example, if the oligomer were a trimer, one would expect to observe two different species of heterotrimers of 380 and 430 kD, in addition to the 480 and 330 kD homotrimers. In addition to the monomers and 220 and 320 kD homodimers, a heterodimer of 270 kD would be expected. Markedly different patterns of hetero-oligomers would be observed if the assembled oligomer were a tetramer.
  • the density of the heterotrimeric forms reflects that expected from the relative expression of each of the mutants present in the transfected cell.
  • the identity of the components in each band was further confirmed by precipitating the lysate shown in lane 3 with an antibody, 110.3, against the gp 120 V3 loop ( Figure 5, lane 5).
  • this antibody recognized only oligomeric forms proposed to contain the LQA/CCG protein.
  • the decreasing order of efficiency with which the 1 10.3 antibody precipitated the 480, 430, 380 and 330 kD proteins is consistent with the proposed content of 3,2,1 and 0 LQA/CCG monomers, respectively, in the trimer.
  • the LQA/CCG and ⁇ LQA/CCG proteins form disulfide bonds to stabilize a trimer.
  • HIV-Envelope Glycoprotein Mutants and Phenotypes The HIV- 1 envelope glycoprotein mutants, the location of the cysteines in the heptad repeat and the presence of higher order forms after boiling for 3 minutes in the presence of 0.2% ⁇ -mercaptoethanol are shown.

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Abstract

La présente invention se rapporte à des glycoprotéines trimères d'enveloppe stabilisés. Ces trimères sont stabilisés par l'introduction de liaisons bisulfure dans certains sites de l'ectodomaine de gp41. On peut utiliser les molécules d'ADN codant ces trimères pour déclencher une réponse immunogénique.
PCT/US1998/020693 1997-10-01 1998-10-01 Stabilisation des glycoproteines trimeres d'enveloppe au moyen de liaisons bisulfure introduites dans un ectodomaine de la glycoproteine gp41 WO1999016883A2 (fr)

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CA002305341A CA2305341C (fr) 1997-10-01 1998-10-01 Stabilisation des glycoproteines trimeres d'enveloppe au moyen de liaisons bisulfure introduites dans un ectodomaine de la glycoproteine gp41
EP98950846A EP1019511A2 (fr) 1997-10-01 1998-10-01 Stabilisation des glycoproteines trimeres d'enveloppe au moyen de liaisons bisulfure introduites dans un ectodomaine de la glycoproteine gp41
AU96785/98A AU9678598A (en) 1997-10-01 1998-10-01 Stabilization of envelope glycoprotein trimers by disulfide bonds introduced into a gp41 glycoprotein ectodomain

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WO2000008167A1 (fr) * 1998-07-31 2000-02-17 Aventis Pasteur Trimere du produit d'expression du gene env de hiv
EP1214333A2 (fr) * 1999-09-17 2002-06-19 Dana-Farber Cancer Institute, Inc. Trimeres stabilises solubles de glycoproteines
EP1345623A2 (fr) * 2000-12-27 2003-09-24 Dana-Farber Cancer Institute Inc. Proteoliposomes immunogenes et applications de ceux-ci
US7179468B1 (en) 2000-06-08 2007-02-20 Cornell Research Foundation, Inc. Antigen for developing neutralizing antibodies to human immunodeficiency virus
EP1755667A2 (fr) * 2004-06-01 2007-02-28 Merck & Co., Inc. Peptide stable mimétique d'intermédiaire de fusion du gp4 du vih
WO2008122687A1 (fr) * 2007-04-04 2008-10-16 Consejo Superior De Investigaciones Científicas Polypeptide de la protéine gp120 permettant d'inhiber le cycle vital du virus du sida
EP2161278A1 (fr) * 2008-09-08 2010-03-10 Complix N.V. Échafaudage à bobine à une seule chaîne
US9217011B2 (en) 2007-09-07 2015-12-22 Complix Nv Non-natural proteinaceous scaffold made of three non-covalently associated peptides
CN110184298A (zh) * 2019-05-15 2019-08-30 武汉璟泓万方堂医药科技股份有限公司 Hiv突变型表面糖蛋白及其纳米化抗原与制备方法
US10851133B2 (en) 2015-06-08 2020-12-01 New York University Stabilized minimal coiled-coil mimetics
US11891422B2 (en) 2018-11-16 2024-02-06 New York University NEMO coiled coil mimics and methods of using same

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EP1411770A4 (fr) 2001-07-05 2006-05-10 Chiron Corp Polynucleotides codant des polypeptides de type c du vih antigeniques, polypeptides et leurs utilisations

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Cited By (25)

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US6737067B1 (en) 1998-07-31 2004-05-18 Aventis Pasteur S.A. Trimer of HIV env gene expression product
WO2000008167A1 (fr) * 1998-07-31 2000-02-17 Aventis Pasteur Trimere du produit d'expression du gene env de hiv
US7429653B2 (en) 1999-09-17 2008-09-30 Dana-Farber Cancer Institute, Inc. Stabilized soluble glycoprotein trimers
EP1214333A4 (fr) * 1999-09-17 2005-01-19 Dana Farber Cancer Inst Inc Trimeres stabilises solubles de glycoproteines
EP1214333A2 (fr) * 1999-09-17 2002-06-19 Dana-Farber Cancer Institute, Inc. Trimeres stabilises solubles de glycoproteines
US6911205B2 (en) 1999-09-17 2005-06-28 Dana-Farber Cancer Institute, Inc. Stabilized soluble glycoprotein trimers
US7179468B1 (en) 2000-06-08 2007-02-20 Cornell Research Foundation, Inc. Antigen for developing neutralizing antibodies to human immunodeficiency virus
EP1345623A4 (fr) * 2000-12-27 2005-05-11 Dana Farber Cancer Inst Inc Proteoliposomes immunogenes et applications de ceux-ci
EP1345623A2 (fr) * 2000-12-27 2003-09-24 Dana-Farber Cancer Institute Inc. Proteoliposomes immunogenes et applications de ceux-ci
US7276579B2 (en) 2000-12-27 2007-10-02 Dana-Farber Cancer Institute, Inc. Immunogenic proteoliposomes, and uses thereof
US7741024B2 (en) 2000-12-27 2010-06-22 Dana-Farber Cancer Institute, Inc. Immunogenic proteoliposomes, and uses thereof
AU2005250430B2 (en) * 2004-06-01 2011-10-13 Msd Italia S.R.L. Stable peptide mimetic of HIV gp41 fusion intermediate
EP1755667A4 (fr) * 2004-06-01 2008-09-03 Merck & Co Inc Peptide stable mimetique d'intermediaire de fusion du gp4 du vih
JP2008501028A (ja) * 2004-06-01 2008-01-17 メルク　エンド　カムパニー　インコーポレーテッド ＨＩＶｇｐ４１融合中間物質の安定したペプチド模倣薬
US7811577B2 (en) 2004-06-01 2010-10-12 Merck Sharp & Dohme Corp. Covalently stabilized chimeric coiled-coil HIV gp41 N-peptides with improved antiviral activity
EP1755667A2 (fr) * 2004-06-01 2007-02-28 Merck & Co., Inc. Peptide stable mimétique d'intermédiaire de fusion du gp4 du vih
EP2354153A3 (fr) * 2004-06-01 2012-05-30 Merck Sharp & Dohme Corp. Mimétique de peptide stable d'intermédiaire fusion gp41 du VIH
WO2008122687A1 (fr) * 2007-04-04 2008-10-16 Consejo Superior De Investigaciones Científicas Polypeptide de la protéine gp120 permettant d'inhiber le cycle vital du virus du sida
ES2324755A1 (es) * 2007-04-04 2009-08-13 Consejo Superior De Investigaciones Cientificas (Csic) Polipeptido de la proteina gp120 capaz de inhibir el ciclo vital del virus del sida.
US9217011B2 (en) 2007-09-07 2015-12-22 Complix Nv Non-natural proteinaceous scaffold made of three non-covalently associated peptides
EP2161278A1 (fr) * 2008-09-08 2010-03-10 Complix N.V. Échafaudage à bobine à une seule chaîne
US10851133B2 (en) 2015-06-08 2020-12-01 New York University Stabilized minimal coiled-coil mimetics
US11440938B2 (en) 2015-06-08 2022-09-13 New York University Stabilized minimal coiled-coil mimetics
US11891422B2 (en) 2018-11-16 2024-02-06 New York University NEMO coiled coil mimics and methods of using same
CN110184298A (zh) * 2019-05-15 2019-08-30 武汉璟泓万方堂医药科技股份有限公司 Hiv突变型表面糖蛋白及其纳米化抗原与制备方法

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