WO2005116055A1 - Peptides, antibodies and compositions containing same useful for treating hiv virus infection - Google Patents

Peptides, antibodies and compositions containing same useful for treating hiv virus infection Download PDF

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WO2005116055A1
WO2005116055A1 PCT/IL2005/000531 IL2005000531W WO2005116055A1 WO 2005116055 A1 WO2005116055 A1 WO 2005116055A1 IL 2005000531 W IL2005000531 W IL 2005000531W WO 2005116055 A1 WO2005116055 A1 WO 2005116055A1
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peptide
hiv
amino acid
pharmaceutical composition
manufacture
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PCT/IL2005/000531
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French (fr)
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Moshe Kotler
Marina Hutoran
Lea Baraz
Yelena Britan
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
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Publication of WO2005116055A1 publication Critical patent/WO2005116055A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to peptides and antibodies and pharmaceutical composition containing same, which can be utilized for treating and detecting HIV virus infection.
  • the human immunodeficiency virus is the agent responsible for the slow degeneration of the immune system in patients suffering from acquired immune deficiency syndrome (AIDS) [Barre-Sinoussi, F., et al., (1983) Science 220:868-870; Gallo, R., et al., (1984) Science 224:500-503].
  • HIV-1 Barre-Sinoussi, F., et al., (1983), Science 220:868- 870; Gallo, R., et al., (1984), Science 224:500-503
  • HIV-2 HIV-2 [Clavel, F., et al, (1986), Science 233:343-346; Guyader, M , et al, (1987), Nature 326:662-669].
  • HIV-1 Barre-Sinoussi, F., et al., (1983), Science 220:868- 870; Gallo, R., et al., (1984), Science 224:500-503
  • HIV-2 Clavel, F., et al, (1986), Science 233:343-346; Guyader, M , et al, (1987), Nature 326:662-669].
  • Each of these types of viruses displays significant intra-population heterogeneity.
  • HIV replication occurs predominantly in CD4 + T lymphocyte populations, and thus leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death. Since HIV infection is pandemic, HIN-associated diseases represent a major world health problem. Although considerable effort is being put into identifying or designing therapeutics effective in inhibiting HIN replication, current therapeutic approaches fail to eradicate the disease in infected individuals. HIN-1 encodes a number of accessory genes in addition to the canonical gag, pol, and env genes that are expressed by all replication-competent retroviruses.
  • Nif viral infectivity factor, Ratner (1985) Nature 313:277-84
  • the Nif protein of HIN-1 is a basic, 23-kDa protein composed of 192 amino acids. Sequence analysis of viral D ⁇ A from HIN-1 -infected-individuals has revealed that the open reading frame of Nif remains intact. (Sova, P., et al., J. Nirol. 69:2557-2564, 1995; Wieland, U., et al., Virology 203:43-51, 1994; Wieland, U., et al., J. Gen. Nirol. 78:393- 400, 1997).
  • vif-deficient HIN-1 is incapable of infecting certain cells, such as H9 T cells, peripheral blood mononuclear cells, and monocyte- derived macrophages. This has led to classification of these cells as nonpermissive. However, in some cells, such as C8166, Jurkat, SupTl, and HeLa-T4 cells, the vif gene is not essential; these cells have been classified as permissive. (Gabuzda, D. H., et al., J. Virol. 66(ll):6489-95, 1992; von Schwedler, U., et al., J. Virol.
  • permissive cells there may be a cellular compensating factor (e.g., Nif homolog) that can replace Nif function in the virus-producing cells; alternatively, there may be an inhibitor(s) of viral replication in nonpermissive cells that requires Nif to counteract its effect (Trono, D., Cell 82:189-192, 1995).
  • Nif homolog a cellular compensating factor that can replace Nif function in the virus-producing cells
  • an inhibitor(s) of viral replication in nonpermissive cells that requires Nif to counteract its effect (Trono, D., Cell 82:189-192, 1995).
  • Nif plays a role in viral assembly in virus-producing cells or cell-free virions (Blanc, D., et al., Virology 193:186-192, 1993; Gabuzda, D. H., et al., J. Virol. 66:6489-6495, 1992; von Schwedler, U., et al., J. Virol. 67:4945-4955, 1993).
  • Vif participates in the nuclear targeting of viral cores by acting as an adaptor, linking the pre-integration complex (PIC) to a cellular transport pathway and allowing viral transport to the nuclear membrane.
  • PIC pre-integration complex
  • CEM 15 the cellular factor, CEM 15, is currently suggested to be responsible for protecting cells against v/ ⁇ negative HIV-1. It was shown that CEM 15 protein is intensively expressed in non-permissive, but not in permissive cells. Moreover, expression of CEM 15 in permissive cells renders them restrictive for replication of vz / ⁇ negative HIV-1. These results strongly suggest that Vif suppresses the anti- viral activity of CEM 15 protein in restrictive cells [Sheehy, Gaddis et al. (2002) Nat. Ned. 9:1404-7]. Sequence analysis of CEM 15 revealed its association with the APOBEC family of proteins, which are involved in RNA editing and function by deaminating cytosine in RNA [Teng, Burant et al. (1993) Science 260:1816-9;
  • APOBEC3G has been shown to act as a DNA mutator in E.coli, with the mutations attributed to dC ⁇ dU [Harris, Petersen-Mahrt et al. (2002) Mol. Cell 10:1247-53]. It has been shown that HIV-1 Vif expressed in cells binds APOBEC3G, leading to its ubiquitination and degradation via the proteosome pathway [Conticello, Harris et al. (2003) Curr. Biol. 13:2009-13; Kao, Khan et al.
  • Vif- deficient particles produced by non-permissive cells differ from wild-type particles by several criteria: Vif-negative particles show non-homogenous packaging of the internal core, instead of the normal dense cone-shaped core structure. Vif-deficient virions are more susceptible to disruption by detergents, high salt concentration and buffers of various pH values [Ohagen and Gabuzda (2000) J. Virol. 74:11055-66; Khan, Akari et al. (2002) J. Virol. 76:9112-23]. These findings strongly suggest an interaction of Vif with PR [Baraz, Hutoran et al. (2002) FEBS Lett. 441:419-26; Blumenzweig, Baraz et al. (2002) 292:832-40].
  • the retroviral protease which is translated as part of a large polyprotein, processes Gag and Gag-Pol precursors, thereby contributing to virus maturation.
  • the HIN-1 PR (GenBank Accession NO. AAL05141, HIV-PR) is a homodimeric aspartic protease composed of two 99 amino acid units.
  • the active form of PR HIV-1 is a homodimer, in which its 4 termini form an anti-parallel ⁇ -sheet interface [Pearl and Taylor (1987) Nature 329:351-4; Meek, Dayton et al. (1989) PNAS 86:1841-5]. Dimerization of the enzyme occurs while PR is still a part of the Gag-Pol precursor, facilitating the autoprocessing of the polyproteins and resulting in virus maturation [Krausslich (1991) Adv. Exp. Med. Biol. 306:417-28. Expression of Vif with truncated Gag-Pol fusion polyproteins in bacterial cells inhibits processing of precursors [Kotler (1997) J. Virol. 71:5774-81].
  • the present inventors have previously found that recombinant Vif prevents degradation of synthetic peptide by PR, and inhibits PR-mediated Pr 55 Gag processing in vitro. This inhibitory activity of Vif was mapped to amino acid coordinates 78-98 [Baraz, Hutoran et al. (2002) J. Gen. Virol. 83:2225-30] pointing at the interaction between Vif and PR to the central region of Vif. While reducing the present invention to practice, the present inventors uncovered that peptides derived from the amino terminus of HIV PR are capable of inhibiting Vif binding to viral PR and thus are capable of inhibiting the production of infectious particles in cells conclusively showing that such peptides can be used as potent therapeutic agents against the HIV infection.
  • a peptide useful as an anti HIV agent comprising at least two consecutive amino acids of the amino acid sequence X-Y-Z, wherein X is an amide derivative of an acidic amino acid, Y is an aliphatic amino acid and Z is a nucleophilic amino acid, the peptide being no more than 25 amino acids and is not as set forth in SEQ ID NOs.: 3, 12, 13 and 24.
  • a pharmaceutical composition comprising as an active ingredient the peptide and a pharmaceutically acceptable carrier.
  • an article-of-manufacture comprising packaging material and a pharmaceutical composition identified for treating or preventing HIV infection, being contained within the packaging material, the pharmaceutical composition including, as an active ingredient, the peptide and a pharmaceutically acceptable carrier.
  • the peptide as a pharmaceutical.
  • use of the peptide for the manufacture of a medicament identified for treating HIV infection there is provided a method of inhibiting HIV propagation in cells, the method comprising exposing the cells to the peptide, thereby inhibiting the HIV propagation in the cells.
  • a method of treating HIV infection in a subject comprising administering to a subject in need thereof, a therapeutically effective amount of the peptide, thereby treating the HIV infection in the subject.
  • the administering is effected by: (i) administering the peptide to the subject; and/or (ii) administering an expression construct encoding the peptide to the subject.
  • the amide derivative of the acidic amino acid is selected from the group consisting of asparagine and glutamine or functional mimetics thereof.
  • the aliphatic amino acid is selected from the group consisting of leucine, isoleucine and valine or functional mimetics thereof.
  • the nucleophilic amino acid is a hydroxylic amino acid or a functional mimetic thereof.
  • the hydroxylic amino acid is selected from the group consisting of threonine, serine and tyrosine.
  • the nucleophilic amino acid is selected from the group consisting of threonine, serine, proline and tyrosine or functional mimetics thereof.
  • At least one amino acid of the no more than 25 amino acids of the peptide is a D stereoisomer. According to still further features in the described preferred embodiments at least one amino acid of the no more than 25 amino acids of the peptide is an L stereoisomer. According to still further features in the described preferred embodiments the peptide is a linear or cyclic peptide. According to still further features in the described preferred embodiments the no more than 25 amino acids are in retro orientation. According to still further features in the described preferred embodiments the peptide is selected from the group consisting of SEQ ID NOs. 4, 10-11, 14-17, 19-23,
  • the peptide is provided at medical grade purity.
  • the peptide further comprises a tripeptide sequence as set forth in SEQ ID NO: 50 or functional mimetics thereof attached to the Z.
  • the peptide further comprises a proline, glycine or tryptophan residue attached to the X.
  • the peptide is 3 amino acids in length.
  • the peptide is 2 amino acids in length.
  • At least one amino acid of the no more than 25 amino acids of the peptide is a synthetic amino acid. According to still further features in the described preferred embodiments at least one amino acid of the no more than 25 amino acids of the peptide is a natural amino acid. According to still further features in the described preferred embodiments the peptide further comprises a stabilizing moiety at an N- terminus and/or C-terminus thereof.
  • the stabilizing moiety is selected from the group consisting of N- ⁇ -trans L-4 hydroxyproline (Hyp), cis-4-amino-L-proline (Pam), PhOxalidine (Oxa), 4- thiazolidine carboxylic acid (Thiazole) and a stabilizing group.
  • the stabilizing group is selected from the group consisting of amido group, acetyl group, benzyl group, phenyl group, tosyl group, alkoxycarbonyl group, alkyl carbonyl group and benzyloxycarbonyl group.
  • the peptide further comprises a hydrophobic moiety at an N- terminus and/or C-terminus thereof. According to still further features in the described preferred embodiments the peptide further comprises at least one negatively charged ammo acid at an N- terminus and/or C-terminus thereof. According to a further aspect of the present invention there is provided an antibody or antibody fragment comprising an antigen binding site capable of specifically binding with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-4 and 10-23.
  • a method of treating HIV infection in a subject comprising administering to a subject in need thereof, a therapeutically effective amount of the antibody or antibody fragment, thereby treating the HIV infection in the subject.
  • a method of identifying putative anti HIV agents comprising, identifying agents being capable of preventing formation of an HIV protease- Vif complex or capable of dissociating the complex, thereby identifying the putative anti HIV agents.
  • a method of identifying putative anti HIV agents comprising, identifying agents being capable of preventing formation of an HIV protease- APOB EC complex or capable of dissociating the complex, thereby identifying the putative anti HIV agents.
  • the agents are selected from the group consisting of chemicals, antibodies, aptamers, peptides and combinations thereof.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing peptides, antibodies and pharmaceutical compositions containing same which can be utilized to diagnose and treat HIV infection. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • FIG. 1 is a schematic illustration showing mature wild-type HIV PR and
  • FIGs. 2A-B are graphs depicting dose-dependent binding of wild-type and chimeric proteases to solid-phase bound Vif ( Figure 2A) or binding of Vif to solid- phase bound proteases ( Figure 2B).
  • FIG. 2A Microwells of an ELIS A plate were coated with 0.2 ⁇ M of purified Vif, blocked with low fat milk for 2 h and incubated with 0, 25, 50, 100, and 200 nM of PR HIV-1 (closed circles), PR HIV-1 ASLV (open circles), PR ASLV (closed squares) and PR ASLVTM 7"1 (open squares). Bound PRs were detected by using rabbit anti-PR and alkaline phosphatase conjugated goat anti- rabbit IgG and quantified in an ELISA reader at 405 nm.
  • FIG. 2B Microwells of ELISA plate were coated with 90 nM of PR HIV-1 (closed circles), PR HIV-1 ASLV (open circles), PR ASLV (closed squares), PR ASLV HIY"1 (open squares) and BSA (triangles), incubated with low fat milk for 2 h and incubated with 0, 20, 40, 80, and 160 nM of purified Vif protein. Bound Vif was detected by anti- Vif and alkaline phosphatase conjugated goat anti-rabbit IgG and quantified in an ELISA reader at 405 nm.
  • FIG. 3 is a bar graph showing the effect of soluble PR on Vif binding to solid- phase bound PR chimeras, as determined by an ELISA assay.
  • FIG. 4 is a bar graph showing the effect of PR1-9 (SEQ ID NO:4) on binding of Vif to PR HIV-1 and PR ASLVTM 7"1 , as determined by ELISA.
  • FIGs. 5A-C are graphs depicting the effect of PR1-9 (SEQ ID NO:4) on HIV- 1 replication in restrictive and permissive cells.
  • FIGs. 6A-C are graphs showing the ability of PR1-9 (SEQ ID NO:4) to inhibit
  • FIGs. 1-10 show 293 T cells co-transfected with wild-type HIV-1 and APOBEC3G.
  • Figure 6B shows 293T cells transfected with wild-type HIV-1.
  • Figure 6C shows 293T cells transfected with HIV-1 ⁇ vif.
  • FIGS. 7A-B are bar graphs depicting binding of PR1-9 and deletions thereof (SEQ ID NOs: 3, 10, 11, 12, 13, 15-19 and 24) to Vif protein. Microwells were coated with Vif protein ( Figure 7A) or PR protein( Figure 7B) and blocked with low fat milk. Figure 7A - Peptides were added to immobilized Vif and incubated for 2 hours at room temperature. Wells were washed and incubated with PR. Figure 7B - Vif was preincubated (for 18 hours at 4 °C) with PR-derived peptide. The solution was then added to immobilized PR for 2 and left to incubate for 2 hours at room temperature.
  • FIG. 8 is a bar graph depicting inhibition of HIV-1 production in H9 cells /infected with wild-type (wt) HIV by PR derived peptides. Samples of media from days 4, 7 and 10 were collected and analyzed for production of infectious virus by MAGI assay [Hutoran Virology. 2004 330(l):261-70]. FIGs.
  • FIG. 9A-B are bar graphs depicting binding of glycine substituted PR-derived peptides (SEQ ID NOs: 52-56) to Vif protein, as described in Figures 7A-B.
  • FIG. 10 is a bar graph depicting inhibition of HIV-1 production in H9 cells infected with HIV-1 by PR derived peptides. Assay was effected as described in
  • FIGs. 11A-B are bar graphs depicting binding of various PR-derived peptide configurations including amino acid sequence repeats and retro analogues thereof
  • FIGs. 12A-B are bar graphs depicting binding of various PR-derived peptide configurations conferring bioavailability (SEQ ID NOs: 32-37) to Vif protein, as described in Figures 7A-B.
  • FIGs. 13A-B are bar graphs depicting binding of various PR-derived peptide configurations (SEQ ID NOs: 38-43) to Vif protein, as described in Figures 7A-B.
  • FIGs. 14A-B are bar graphs depicting binding of various PR-derived peptide configurations including conservative amino acid substitutions (SEQ ID NOs: 44-49) to Vif protein, as described in Figures 7A-B.
  • FIGs. 15A-B are bar graphs depicting inhibition of HIV-1 production in H9 cells infected with HIV-1 wild type by the PR derived peptides of Figures 14A-B.
  • FIGs. 16A-B are bar graphs depicting inhibition of HIV-1 production in 293 T cells trancfected with Apo3G by PR-derived peptides of Figures 14A-B provided at
  • the present invention is of peptides, antibodies, and pharmaceutical compositions, which can be used for treating and detecting HIV virus infection in mammals, such as humans.
  • the principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • Vif a basic, 23 -kDa protein composed of 192 amino acids which is expressed by most Lentiviruses (for further detail see, for example, U.S. Pat. No. 6,653,443).
  • PR HIV protease
  • Vif 35-50 and Vif 78-98 interacts with the protease [Baraz, Hutoran et al. (2002) J. Gen. Virol. 83:2225-30;
  • HIV PR inhibit Vif/PR binding; (iii) such synthetic peptides inhibit the propagation of
  • PR-derived peptides of the present invention inhibit Vif in anti HIV therapy.
  • HIV-PR derived peptides were previously described, such as be Zutshi and Chmielewski [(2000) Bioorganic & Midicinal Chemistry Letters 10:1901-1903].
  • prior art peptides consist of C-terminus derived PR peptides or such peptides linked to N-terminus derived PR peptides which are aimed at inhibiting HIV-PR rather than Vif, as they are directed at the dimerization interface of HIV-PR (i.e., by targeting a Cys residue present at the dimerization interface).
  • an anti HIV agent refers to an agent which is capable of preventing HIV infection and/or at least alleviating symptoms associated with HIV infection.
  • HIV refers to type I and type II human immunodeficiency virus (HIV). It will be appreciated that anti HIV agents of the present invention may also be effective in combating HIV-related viruses such as simian immunodeficiency virus (SrV) and feline immunodeficiency virus (FIV).
  • the peptide according to this aspect of the present invention comprises at least two consecutive amino acids of the amino acid sequence X-Y-Z, wherein X is an amide derivative of an acidic amino acid, Y is an aliphatic amino acid and Z is a nucleophilic amino acid, is not as set forth in SEQ ID NOs.: 3, 12, 13 and 24.
  • the peptide of this aspect of the present invention is preferably no more than 50 amino acids in length, preferably no more than 40 amino acids in length, preferably no more than 30 amino acids in length, preferably no more than 25 amino acids in length, preferably no more than 20 amino acids in length, preferably no more than 15 amino acids in length, preferably no more than 12 amino acids in length, preferably no more than 10 amino acids in length, preferably no more than 9 amino acids in length, preferably no more than 8 amino acids in length, preferably no more than 7 amino acids in length, preferably no more than 6 amino acids in length, preferably no more than 5 amino acids in length, preferably no more than 4 amino acids in length, preferably no more than 3 amino acids in length, preferably no more than 2 amino acids in length.
  • an amide derivative of an acidic amino acid refers to the uncharged derivatives of glutamate and aspartate, that is glutamine and asparagine, which contain a terminal amide group in place of a carboxylate.
  • the term refers also to functional mimetics (i.e., synthetic) of these amino acids, such as provided in Table
  • an aliphatic amino acid refers to amino acids having straight or branched side chain structures.
  • examples of aliphatic amino acids which can be used in accordance with this aspect of the present invention include, but are not limited to, leucine, isoleucine and valine or functional mimetics thereof, such as provided in Table 2 below.
  • a nucleophilic amino acid refers to amino acids having side chains capable of donating electrons to a species known as an electrophile in order to form a chemical bond.
  • nucleophilic amino acids which can be used in accordance with this aspect of the present invention include, but are not limited to, hydroxylic amino acids (e.g., threonine, serine and tyrosine) and proline or functional mimetics thereof, such as provided in Table 2 below.
  • the peptide of this aspect of the present invention may be modified or further comprise amino acids which enhance its interaction with Vif.
  • the peptide may comprise a peptide sequence, such as set forth in SEQ ID NO: 50 or functional mimetics thereof attached to the above described nucleophilic amino acid. Inclusion of such a peptide sequence was shown by the present inventors to promote interaction with Vif and inhibit HIV propagation (see Example 4 of the Examples section which follows).
  • the peptide may further comprise at least one negatively charged amino acid residue which promotes association with a positively charged amino acid sequence of Vif (i.e., RKKR).
  • the peptide may comprise an amino acid residue N-terminally of the above-described amide derivative of an acidic amino acid. Examples of such amino acids include, but are not limited to, glycine, tryptophan or proline. It will be appreciated that each amino acid sequence of the peptide (or a portion thereof) may be presented at least once (e.g., twice, see SEQ ID NOs.: 25-30) in the peptide of this aspect of the present invention.
  • peptide linkers e.g., Ala-Ala
  • spacers may be included in the peptide sequence.
  • a spacer is inserted in a papeitde sequence to generate a space between two moieties (e.g., peptide moieties).
  • Typical linkers which may be used in accordance with this aspect of the present invention include, but are not limited to, alpha, omega- bifunctional moiety, e.g., hydrocarbon such as alkyl, cycloalkyl, aryl, alkenyl, alkynyl, substituted by two functional groups that can form a bond with the N-terminus and/or the C-terminus of the peptide.
  • Typical spacers which may be used in accordance with this aspect of the present invention include, but are not limited to, alkyl, cycloalkyl, aryl and O-alkyl.
  • the peptide includes the amino acid sequence of SEQ ID NO: 4, 10-11, 14-17, 19-23, 25-49, 52-57, 59-64, 67, 68, 71-75 or 76.
  • the term "mimetics" when made in reference to peptides refers to molecular structures, which serve as substitutes for the peptides of the present invention in interaction with HIV- Vif (Morgan et al. (1989) Ann. Reports Med. Chem.
  • Peptide mimetics include synthetic structures (known and yet unknown), which may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of a peptide ligand. Types of amino acids which can be utilized to generate mimetics are further described hereinbelow.
  • the term, "peptide mimetics” also includes peptoids and oligopeptoids, which are peptides or oligomers of N-substituted amino acids [Simon et al. (1972) Proc. Natl. Acad. Sci. USA 89:9367-9371].
  • peptide mimetics are peptide libraries, which are collections of peptides designed to be of a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto. Methods for the production of peptide mimetics are described hereinbelow.
  • peptide libraries are collections of peptides designed to be of a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto. Methods for the production of peptide mimetics are described hereinbelow.
  • the term “functional” refers to the ability to interfere with HIV infection.
  • functional mimetic refers to the above-described mimetics which function to interfere with HIV infection.
  • peptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and as mentioned hereinabove, peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids. Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non- conventional or modified amino acids (Table 2) which can be used with the present invention.
  • the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • peptides of this aspect of the present invention may include a hydrophobic moiety which promotes cell penetration.
  • hydrophobic moiety refers to any substance which is nonpolar and generally immiscible with water.
  • a hydrophobic moiety according to the present invention is preferably a hydrophobic residue (portion) of a hydrophobic substance.
  • hydrophobic moiety of the present invention can be attached to the amino acid sequence via covalent interactions.
  • hydrophobic substances from which the hydrophobic moiety of the present invention can be derived include, but are not limited to, substituted and unsubstituted, saturated and unsaturated hydrocarbons, where the hydrocarbon can be an aliphatic, an alicyclic or an aromatic compound.
  • the hydrocarbon bears a functional group which enables attachment thereof to an amino acid residue.
  • the hydrophobic moiety of the present invention may be a fatty acid, such as myristic acid, lauric acid, palmitic acid, stearic acid (C18), oleic acid, linolenic acid and arachidonic acid
  • the hydrophobic moiety can be an amino acid residue that is modified to include a fatty acid residue, or any other residue of a hydrophobic substance as described hereinabove, such that this modified amino acid residue is attached to the amino acid sequence via a peptide bond or a substituted peptide bond, as is described hereinabove.
  • the hydrophobic moiety can be a short peptide in which one or more amino acid residues are modified to include a fatty acid residue or any other residue of a hydrophobic substance as described hereinabove.
  • the peptide of the present invention can also include a hydrophobic peptide sequence attached to the amino acid sequence described above. This hydrophobic peptide sequence preferably includes between 2 and 15 amino acid residues, in which at least one amino acid residue is a hydrophobic amino acid residue.
  • hydrophobic amino acid residues include, without limitation, an alanine residue, a cysteine residue, an isoleucine residue, a leucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a methionine residue, a proline residue and a tryptophan residue, or any modification thereof, as is described hereinabove.
  • the hydrophobic peptide sequence can include a combination of naturally occurring and synthetic amino acids, which have been modified by incorporation of a hydrophobic moiety thereto.
  • the hydrophobic moiety or moieties of the present invention are preferably attached to the N-terminus and/or the C-terminus of the amino acid sequence of the peptide of the present invention.
  • the peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized. Cyclic peptides can either be synthesized in a cyclic form or configured so as to assume a cyclic form under desired conditions (e.g., physiological conditions).
  • a peptide according to the teachings of the present invention can include at least two cysteine residues flanking the core peptide sequence.
  • cyclization can be generated via formation of S-S bonds between the two Cys residues.
  • cyclization can be obtained, for example, through amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (-CO-NH or -NH-CO bonds).
  • the peptides according to the present invention can further include salts and chemical derivatives of the peptides.
  • chemical derivative describes a polypeptide of the invention having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • chemical derivatives include those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., SEQ ID NO.: 33 in which Leucine was replaced with Abu to promote association with Vif).
  • the chemical derivatization does not comprehend changes in functional groups which change one amino acid to another.
  • the peptides of the present invention may include useful modifications which are designed to increase the stability of the peptides in solution and, therefore, serve to prolong the half-life of the peptides in solutions, particularly biological fluids, such as blood, plasma or serum, by blocking proteolytic activity in the blood.
  • the peptides of the present invention can have stabilizing moiety such as N- ⁇ -trans L-4 hydroxyproline (Hyp), cis-4-amino-L-proline (Pam), PhOxalidine (Oxa), 4- thiazolidine carboxylic acid (Thiazole) or a stabilizing group at one or both termini.
  • stabilizing groups include amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the like end group modifications.
  • the peptides of the present invention preferably comprise at least one D- isomer of natural amino acids [i.e., inverso peptide analogues, Tjernberg (1997) J. Biol. Chem. 272:12601-5]. Additionally, the peptides of the present invention include retro, inverso and retro-inverso analogues thereof (see e.g., SEQ ID NOs.: 14 and 78-83). It will be appreciated that complete or extended partial retro-inverso analogues of hormones have generally been found to retain or enhance biological activity.
  • Retro-inversion has also found application in the area of rational design of enzyme inhibitors (see U.S. Pat. No. 6,261,569).
  • a "retro peptide” refers to peptides which are made up of L- amino acid residues which are assembled in opposite direction to the native peptide sequence. Retro-inverso modification of naturally occurring polypeptides involves the synthetic assembly of amino acids with ⁇ -carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse order to the native peptide sequence.
  • a rerto inverso analogue thus, has reversed termini and reversed direction of peptide bonds, while essentially maintaining the topology of the side chains as in the native peptide sequence.
  • the peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involve different chemistry.
  • Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide
  • Syntheses (2nd Ed., Pierce Chemical Company, 1984). Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles.
  • the composition of which can be confirmed via amino acid sequencing.
  • the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-
  • peptides of the present invention are preferably highly purified (e.g., medical grade; e.g., > 95 %) for use in therapy, as further described hereinebelow. Further generation of peptide mimetics, as described hereinabove, be effected using various approaches, including, for example, display techniques.
  • the present invention contemplates a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 2, at least 3, at least 4, at least 5, at least 7, or 9 consecutive amino acids derived from polypeptide sequences of the N-terminus of HIV PR (e.g., SEQ ID NOs: 3-4 and 10-23).
  • an "N-terminus of HIV PR” refers to an amino acid sequence encompassed by amino acid coordinates 1-100, preferably 1-50, more preferably, 1- 20, even more preferably, 1-10, even more preferably 1-5, even more preferably 1-4 of HIV-PR. Methods of constructing such display libraries are well known in the art.
  • Peptide mimetics generated according to the teachings of the present invention are preferably qualified using functional biochemical and/or cell biology/molecular biology assays which are well known in the art (e.g., binding inhibition and/orHIN propagation inhibition as described in details in the Examples section which follows).
  • functional biochemical and/or cell biology/molecular biology assays which are well known in the art (e.g., binding inhibition and/orHIN propagation inhibition as described in details in the Examples section which follows).
  • binding inhibition and/orHIN propagation inhibition as described in details in the Examples section which follows.
  • the antigen-binding site is capable of specifically recognizing an amino acid sequence encompassed by amino acids coordinates 1-10 of HIV-PR.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
  • animals may be immunized with free peptide (e.g., SEQ ID NO: 4); however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine may be coupled to a carrier using a linker such as m-maleirnidobenzoyl-N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde.
  • a linker such as m-maleirnidobenzoyl-N- hydroxysuccinimide ester (MBS)
  • MBS m-maleirnidobenzoyl-N- hydroxysuccinimide ester
  • booster injections may be needed, for example, at intervals of about two weeks, to provide a useful titer of anti- peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single- chain antigen binding proteins
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al, Science 242:423-426 (1988); Pack et al, Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR).
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity, hi some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al, Nature, 321:522-
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers [Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J.
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • these cells can be permissive cells, which were engineered to depend on Vif for virus propagation, such as, for example, 293 T cells which express exogenous APOBEC3G (GenBank Accession No. NPJ365854, Sheehy, Gaddis 2002, Nature 418:646-50).
  • Vif for virus propagation such as, for example, 293 T cells which express exogenous APOBEC3G (GenBank Accession No. NPJ365854, Sheehy, Gaddis 2002, Nature 418:646-50).
  • Vif activity e.g., interaction with PR, interaction with APOBEC family or protein, virion propagation, an effect which can also be achieved using PR-specific antibody or antibody fragment
  • peptides and antibodies generated according to the teachings of the present invention will also be useful in anti HIV therapy.
  • a method of treating HIV infection in a subject refers to a mammal, preferably a human which is infected with HIV, suspected of being infected with HIV or is at risk of being infected with HIV (e.g., a health worker).
  • treating refers to alleviating or diminishing a symptom associated with HIV infection.
  • treating cures, e.g., substantially eliminates, the symptoms associated with the infection and/or substantially decreases the viral load in the infected tissue.
  • the method according to this aspect of the present invention is effected by providing to a subject in need thereof, a therapeutically effective amount of the peptide and/or antibody of the present invention to thereby treat the HIV infection in the subject. Provision can be effected by administering the peptide and/or antibody of the present invention to the subject per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the polypeptide, polynucleotide or peptide, such as described hereinabove, which is accountable for the biological effect.
  • physiologically acceptable carrier and
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is mcluded under these phrases.
  • One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979)).
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichiorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichiorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose.
  • a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g.,
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • a pack or dispenser device such as an FDA approved kit
  • other additives such as stabilizers, buffers, blockers and the like may also be added.
  • the compositions of the present invention can be packaged in a one or more containers with appropriate buffers and preservatives and used for therapeutic treatment.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S.
  • the peptide of the present invention can also be provided to the subject by administering to the subject an expressible nucleic acid construct encoding the peptide or antibody (i.e., in-vivo gene therapy), or by administering cells transformed with the expressible nucleic acid construct (i.e., ex- vivo gene therapy).
  • T-cells precursors of the present invention may be ex- vivo or in-vivo treated to express the peptides of the present invention to develop resistance to HIV infection.
  • nucleic acid transfer techniques include transfection with plasmids, viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • plasmids such as adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • AAV adeno-associated virus
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, retroviruses such as lentiviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide or antibody from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • treatment of HIV infections according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy).
  • treatment of HIV infection according to the present invention can be combined with reverse-transcriptase-targeted drugs including 2', 3'-dideoxynucleoside analogs such as AZTTM, ddlTM, ddCTM, and d4TTM which were shown to be effective in partially halting HIN replication
  • the present invention also envisages identification of other anti HIV agents which are capable of interfering with HIV Vif-PR interaction and as such may be used as anti HIV drugs.
  • a method of identifying putative anti HIV agents e.g., chemicals, antibodies, aptamers, peptides and the like.
  • the method is effected by identifying agents which are capable of preventing formation of an HIV protease- Vif complex and/or HIN protease- APOBEC complex or dissociating the complex (i.e., pre-established) to thereby identify the putative anti HIN agents.
  • An HIN protease-Nif complex may be generated by incubating intact HIV-PR and Vif molecules (e.g., recombinant) under conditions which allow complex formation. Such conditions are well described in Example 1 of the Examples section.
  • peptides which encompass the interaction site of either of the proteins (e.g., a peptide encompassing amino acid coordinates 1-9 of PR and a peptide encompassing amino acids 78-98 of Vif) may be used to generate the complex of this aspect of the present invention.
  • Combinatorial chemical, nucleic acid or peptide libraries may be used to screen a plurality of agents. Screening according to this aspect of the present invention may be effected by contacting the agents with the pre-established complex described hereinabove, or with the-above described peptides.
  • HIV PR or Vif-PR complex are preferably bound to a solid support to monitor binding of the agent to HIV PR or to monitor dissociation of the pre-established complex, respectively.
  • the solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached, such as a test well in a microtiter plate, a nitrocellulose filter or another suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic such as polystyrene or polyvinylchloride.
  • Molecular immobilizing on a solid support is effected using a variety of techniques known to those in the art. A number of methods are known in the art for determining intermolecular interactions. Examples include, but are not limited to, ELISA, Biacore, Pull-down assay, immunoprecipitation and the like (see references in the Examples section which follows).
  • a competitive assay in which at least one of the assay component is labeled may also be employed. Labeling methods and tags are further described hereinbelow. It will be appreciated that when utilized along with automated equipment, the above-described method can be used to screen multiple agents both rapidly and easily. Agents identified using the above-described methodology can be further qualified by functional assays, such as by inhibiting HIV propagation in- vitro and in- vivo (see Examples 3-4 of the Examples section which follows). Because of the ability of the peptides, antibodies or other agents identified as described hereinabove to bind viral components (i.e., HIV-PR), it is conceivable that such peptides can also be used as potent detectors of HIV in biological samples.
  • functional assays such as by inhibiting HIV propagation in- vitro and in- vivo (see Examples 3-4 of the Examples section which follows). Because of the ability of the peptides, antibodies or other agents identified as described hereinabove to bind viral components (i.e., HIV-PR), it is
  • a method of detecting a presence or an absence of HIV in a biological sample is effected by incubating the biological sample with a peptide or antibody (or other agent as described hereinabove) which is capable of binding HIV- PR and detecting complexes which comprise the peptide or antibody, to thereby detect the presence or the absence of HIV in the biological sample.
  • the biological sample utilized for detection can be any body sample which contains HIV virions, such as blood (serum or plasma), sputum, ascites fluids, pleural effusions, urine, biopsy specimens and isolated cells.
  • blood serum or plasma
  • sputum sputum
  • ascites fluids pleural effusions
  • urine biopsy specimens and isolated cells.
  • blood Preferably used is blood.
  • tissue biopsies and body fluids from mammals are well known in the art.
  • the peptide of the present invention is contacted with the biological sample under conditions suitable for complex formation (i.e., buffer, temperature, incubation time etc.); suitable conditions are described in Example 1 of the Examples section.
  • Protein complexes within a biological sample can be detected via any one of several methods known in the art, which methods can employ biochemical and/or optical detection schemes.
  • the peptides of the present invention are labeled preferably by a tag or an antibody. It will be appreciated that labeling can be effected prior to, concomitant with or following complex formation, depending on the highlighting method.
  • tag refers to a molecule, which exhibits a quantifiable activity or characteristic.
  • a tag can be a fluorescent molecule including chemical fluorescers such as fluorescein or polypeptide fiuorescers such as the green fluorescent protein (GFP) or related proteins (www.clontech.com). In such case, the tag can be quantified via its fluorescence, which is generated upon the application of a suitable excitatory light.
  • a tag can be an epitope tag, a fairly unique polypeptide sequence to which a specific antibody can bind without substantially cross-reacting with other cellular epitopes.
  • Such epitope tags include a Myc tag, a Flag tag, a His tag, a leucine tag, an IgG tag, a streptavidin tag and the like. HIV diagnosis using the above-described methodology can be confirmed using prior art methods such as described hereinabove (e.g., anti HIV antibodies production).
  • Peptides, antibodies and other agents of the present invention can be included in a diagnostic or therapeutic kit.
  • a peptide can be packaged in a container with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment.
  • the container includes a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the container may be formed from a variety of materials such as glass or plastic.
  • kits can also be attached to a solid support, such as beads, array substrate (e.g., chips) and the like and used for diagnostic purposes. Peptides included in kits or immobilized to substrates may be conjugated to a detectable label such as described hereinabove.
  • the kit can also include instructions for determining if the tested subject is suffering from, or is at risk of developing, a condition, disorder, or disease associated with HIN infection.
  • HeLa-CD4 4- ⁇ -gal cells were obtained from the NIH Reagent Program (Division of AIDS, NIAID, NIH, USA). The adherent cell lines were cultivated in Dolbecco's modified Eagle's medium (DMEM, Biological Industries, Beit Haemek, Israel) as a sub confluent monolayer. HeLa-CD4 + ⁇ -gal cells were maintained in the presence of 200 ⁇ g/ml G418 and lOO ⁇ g/ml Hygromycin B (Calbiochem, La Jolla, CA, USA).
  • DMEM Dolbecco's modified Eagle's medium
  • RPMI 1640 and DMEM media were supplemented with 10% fetal calf serum (FCS), lOOU/ml penicillin, lOOU/ml streptomycin and 2mM L-glutamine (Biological Industries, Beit Haemek, Israel).
  • FCS fetal calf serum
  • lOOU/ml penicillin lOOU/ml streptomycin
  • 2mM L-glutamine Biological Industries, Beit Haemek, Israel.
  • Viruses - Wild-type (wt) HIN-1 was generated by the transfection [Cullen (1987) Methods in Enzymology 152:693-694] of 293T cells with plasmid pSNC21 containing full length HIV-1HXB 2 viral D ⁇ A [Ratner, Haseltine et al. (1985) Nature 313:277-84].
  • 293T cells were transfected (Cullen 1987, supra) with pSVC21 ⁇ vif [Gabuzda, Lawrence et al. (1992) J. Virol. 66:6489-95]. Both wt and ⁇ vif virions were harvested 48 and 72 hours post transfection and viral titer was detected by MAGI assay, below. Infection of cultured cells - Cultured lymphocytes (lxlO 6 - 5xl0 6 ) were centrifuged for 5 minutes at 2000 rpm, supernatant was aspirated and the cells were resuspended in 0.2-0.5 ml of medium (RPMI 1640 Biological Industries, Beit Haemek, Israel) containing virus at m.o.i.
  • medium RPMI 1640 Biological Industries, Beit Haemek, Israel
  • HIV-1 titration - Titration of HIV-1 was effected by a multinuclear galactosidase (MAGI) activation assay, as described by Kimpton and Emerman
  • HeLa-CD4 + ⁇ -gaI cells were plated onto 96-well plates at a density of 15xl0 3 cells per well. On the following day, when the cultures were about 30% confluent, the medium was removed from the wells and the cells were infected with 50 ⁇ l of serially diluted virus in the presence of
  • P24 assay - The assay was effected using an HIV-1 p24 antigen capture assay kit (SAIC, Frederic, AIDS Vaccine Program), in accordance with the standards and instructions supplied by the manufacturers.
  • Bacterial cells - E.coli BL21 cells (DE3) were grown in Luria-Bertini (LB) medium in the presence of 100 ⁇ g/ml chloramphenicol (Sigma, Israel).
  • E.coli DH5 ⁇ strain was grown in antibiotic-free LB.
  • E.coli MC 1061 cells were grown in LB medium with 50 ⁇ g/ml tetracycline (Sigma, Israel).
  • chimeric PRs - Plasmids expressing chimeric HIV-1 PR and ASLV PR were constructed by replacing the DNA fragments encoding for the reciprocal 4 amino acids (aa) residing at the PRs termini.
  • pUC12N plasmid bearing the HIV-1 PR [Kotler, Katz et al. (1988a) J. Virol.
  • HIV-1 ASLV PR SEQ ID NO: 1
  • primers 5'- CCCGGGCCATGGTCCTCGCGATGACTCTTTGGCAACGACCC-3' SEQ ID NO: 5 and 5'-CCCGGGAAGCTTATAAATTTGTCAAGCAACCAATCTGAGTCAA-3' SEQ ID NO: 6) containing the sequences encoding for the ASLV-PR termini.
  • the amplified DNA fragment was cleaved by Nco I and Hind III and inserted into pUC12N.
  • the chimeric PR DNA fragment was rescued from the pUC12N by cleavage with Nco I and Hind III restriction enzymes and inserted into the pT5 vector for expression in E.coli BL21 strain (Baraz, Hutoran et al. 2002, supra). Primers (5'-
  • HIV-1 PR-derived peptides were as follows, PR1-9 PQITLWQRP-NH 2 (SEQ ID NO: 4), PR94-99 GCTLNF-NH 2 (SEQ ID NO: 9). Purification of viral proteases - ASLV PR was purified from E. coli MC 1061 bacterial cells, as previously described [Kotler, Katz et al. (1988b) PNAS 85:4185-9; Friedler, Blumenzweig et al. (1999) J. M. B. 287:93-101]. HIN-1 PR, HIV-l ⁇ 7 PR and ASLNTM 7"1 PR were purified as previously described [Baraz, Friedler et al. (1998) FEBS Lett. 441:419-26.
  • Tethered covalently linked dimer of HIN-1 PR [Krausslich, Traenckner et al. (1991) Adv. Exp. Med. Biol. 306:417-28] was expressed from pETl l PR-PR plasmid (kindly supplied by Dr. Konvalinka, Institute of Organic Chemistry and Biochemistry, Praha, Czech Republic) in E.coli BL21 and purified as described above for HIN-1 PR. HIV-1 Vif purification - The pDIO plasmid, which encodes for HIV-1 H X B2 Nif [Yang et al, (1996) J. Biol. Chem. 271:1012-19], was kindly provided by Dr.
  • Gabuzda (Dana-Farber Cancer Institute, Boston, MA). The Nif was expressed in E.coli MC-1061 and purified as previously described (Yang, Goncalves et al. 1996, supra). Vif/PR binding assay -Purified Nif and PR proteins were bound as previously described by (Baraz, Hutoran et al. 2002, supra). In brief, the wells of ELISA plate were covered with 200 ⁇ l of ELISA coating buffer containing 200 nM Nif, or 90 nM PR or 200nM BSA for 18 h at 4 °C.
  • Binding was determined by using alkaline phosphatase-conjugated goat anti-rabbit IgG diluted 1:1000 (Sigma) as a secondary antibody. Reactions were developed by addition of substrate p-r ⁇ tio phenyl phosphate (Sigma), and bound PR or Vif were quantified with an ELISA reader (Dynatech MR5000) at 405 nm. Antibodies - HIV-1 PR and ASLV PR polyclonal antibodies were generated by immunizing rabbits with 3 injections of purified recombinant proteases. Anti-CA and anti-Vif monoclonal antibodies were obtained from the ⁇ IH Reagent Program (Division of AIDS, ⁇ IAID, ⁇ IH, USA).
  • Vif polyclonal antibody was kindly provided by Dr. Gabuzda (Dana-Farber Cancer Institute, Boston, MA).
  • Alkaline phosphatase conjugated goat-anti-rabbit IgG and horseradish peroxidase conjugated goat anti- mouse sera were purchased from Sigma, St. Louis, USA.
  • Figure 3 shows that pre-incubation with PR HIV-1 and PR ASLVTM 7"1 , but not with PR HIV-l ⁇ 7 and PR ASLV, inhibits binding of Vif to PR HIV-1 and PR ASLVTM 7"1 .
  • the experiments described above suggest that Vif interacts with the N- terminus of HIV-1 PR.
  • two peptides were synthesized, PR1-9 (SEQ ID NO:4) and PR94-99 (SEQ ID NO:9), which were derived from the termini of HIV-1 PR.
  • PR1-9 SEQ ID NO:4
  • PR94-99 SEQ ID NO:9
  • H9 and Sup TI cells were infected with wt HIV-1 at m.o.i. 1.
  • One hour post infection the cells were suspended in media containing 50 ⁇ M of PRl-9 (SEQ ID NO:4) peptide or PR94-99 (SEQ ID NO:9) peptide.
  • the media were collected during 10 days post infection, and the amount of virus produced by these cells was estimated by measuring the amounts of p24 CA released by the cells.
  • the titer of the virus was determined by infection of HeLa CD4 cells.
  • PRl-9 peptide reduces virus production by permissive cells expressing APOBEC3G
  • Expression of APOBEC3G in the permissive 293 T cells turns them restrictive for ⁇ vif virus propagation.
  • the replication of HIV-1 in these cells becomes Vif- dependent (Sheehy, Gaddis et al. 2002, supra). Therefore, the question whether treatment with PRl-9 can reduce viral propagation in APOBEC3G transfected 293T cells was addressed. Results 293T cells were co-transfected with vectors expressing either wt or ⁇ vif HIN- ⁇ , 1 along with APOBEC3G.
  • control cells transfected with vectors expressing ⁇ vif virus and APOBEC3G vectors did not produce any infectious particles (data not shown). It is of interest, is that PRl-9 inhibited the production of infectious virus from 293T cells co-transfected with APOBEC3G and HIV-1 vectors more efficiently than in HIV-1 infected H9 cells.
  • PRl-9 Binds to Vif and interferes with the interaction of Vif/APOBEC3G.
  • the region 78-98 in Vif contains a motif similar to the consensus sequence of the PR C-terminus, which interacts with the PR N-terminus in establishing the 4- strand ⁇ -sheet structure.
  • PRl-9 blocks this active site in Vif.
  • PRl-9 competes with APOBEC3G on the same binding site to Vif.
  • binding of PRl-9 to Vif induces structural modification, which hampers the
  • PR1-3 peptide is sufficient to inhibit Vif binding to PR and arrest virus production in non-permissive cells
  • PRl-9 truncated peptides SEQ ID NOs: 3, 10, 11, 12, 13, 15, 16, 17, 18, 19 and 24
  • glycine substituted peptides SEQ ID NOs: 52, 53, 54, 55 and 56
  • PR1-5 amino acid sequence exhibited similar Vif-PR binding inhibitory activities.
  • PR1-5 modified to include degradation protective groups and/or hydrophobic residues exhibit similar activities to the non-modified peptide.
  • PR1-5 (SEQ ID NO: 13) was chemically modified to exhibit better bioavailability.
  • Results As is shown in Figures 12A-B, peptides modified to include a stabilizing moiety as set forth in N- ⁇ -trans L-4 hydroxyproline (Hyp, SEQ ID NO: 32), cis-4- amino-L-proline (Pam, SEQ ID NO: 35), 4-thiazolidine carboxylic acid (Thiazole, SEQ ID NO: 37), amino butyric acid (Abu, SEQ ID NO: 33) and PhOxalidine (SEQ ID NO: 36) as well as a double glycine residues at the C-termini of the peptides exhibited does dependent inhibitory activity, as determined by the inhibition of Vif binding to PR.
  • PR1-5 (SEQ ID NO: 13) was chemically modified to exhibit better association with Vif.
  • Results As is shown in Figures 13A-B, peptides modified to include negatively charged residues as set forth in SEQ ID NOs.: 39, 40, 43 were used to display increased affinity towards the RKKR positive sequence of Vif. Additionally or alternatively, the leucine residue and threonine residue were replaced by proline which introduces a turn in the peptide (SEQ ID NOs, 41, 42/43 respectively). As is shown in Figures 13A-B the above described modifications did not significantly alter PR1-5 binding to Vif.
  • PR1-8 (SI -2, SEQ ID NO: 10) was used as a positive control.
  • PR96-99 (Sl-12, SEQ ID NO: 65) was used as a negative control. It is suggested that such amino acid substitutions are featured by compact side chains thereby bringing the peptide in closer proximity to Vif and increasing their association. These peptides were also able to arrest viral production in non-permissive cells ( Figures 15A-B) and permissive cells transfected with ABOBEC-3G ( Figures 16A-B) in a dose dependent manner.

Abstract

A peptide useful as an anti HIV agent is provided. The peptide comprising at least two consecutive amino acids of the amino acid sequence X-Y-Z, wherein X is an amide derivative of an acidic amino acid, Y is an aliphatic amino acid and Z is a nucleophilic amino acid, the peptide being no more than 25 amino acids and is not as set forth in SEQ ID NOs.: 3, 12, 13 and 24.

Description

PEPTIDES , ANTIBODIES AND COMPOSITIONS CONTAINING SAME FOR TREATING HIV VIRUS INFECTION FIELD AND BACKGROUND OF THE INVENTION The present invention relates to peptides and antibodies and pharmaceutical composition containing same, which can be utilized for treating and detecting HIV virus infection. The human immunodeficiency virus (HIV) is the agent responsible for the slow degeneration of the immune system in patients suffering from acquired immune deficiency syndrome (AIDS) [Barre-Sinoussi, F., et al., (1983) Science 220:868-870; Gallo, R., et al., (1984) Science 224:500-503]. At least two distinct types of HIV are known to date, including HIV-1 [Barre-Sinoussi, F., et al., (1983), Science 220:868- 870; Gallo, R., et al., (1984), Science 224:500-503] and HIV-2 [Clavel, F., et al, (1986), Science 233:343-346; Guyader, M , et al, (1987), Nature 326:662-669]. Each of these types of viruses displays significant intra-population heterogeneity. In humans, HIV replication occurs predominantly in CD4+ T lymphocyte populations, and thus leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death. Since HIV infection is pandemic, HIN-associated diseases represent a major world health problem. Although considerable effort is being put into identifying or designing therapeutics effective in inhibiting HIN replication, current therapeutic approaches fail to eradicate the disease in infected individuals. HIN-1 encodes a number of accessory genes in addition to the canonical gag, pol, and env genes that are expressed by all replication-competent retroviruses. One of these accessory genes, Nif [viral infectivity factor, Ratner (1985) Nature 313:277-84], is expressed by all known lentiviruses except equine infectious anemia virus. The Nif protein of HIN-1 is a basic, 23-kDa protein composed of 192 amino acids. Sequence analysis of viral DΝA from HIN-1 -infected-individuals has revealed that the open reading frame of Nif remains intact. (Sova, P., et al., J. Nirol. 69:2557-2564, 1995; Wieland, U., et al., Virology 203:43-51, 1994; Wieland, U., et al., J. Gen. Nirol. 78:393- 400, 1997). Deletion of the vif gene dramatically decreases the replication of simian immunodeficiency virus (SIV) in macaques and HIN-1 replication in SCID-hu mice, indicating that the vif gene is essential for the production of infectious virus in vivo (Aldrovandi, G. M. & Zack, J. A., J. Nirol. 70:1505-1511, 1996; Desrosiers, R. C, et al.,
J. Nirol. 72:1431-1437, 1998). In cell culture systems, vif-deficient (Δvif) HIN-1 is incapable of infecting certain cells, such as H9 T cells, peripheral blood mononuclear cells, and monocyte- derived macrophages. This has led to classification of these cells as nonpermissive. However, in some cells, such as C8166, Jurkat, SupTl, and HeLa-T4 cells, the vif gene is not essential; these cells have been classified as permissive. (Gabuzda, D. H., et al., J. Virol. 66(ll):6489-95, 1992; von Schwedler, U., et al., J. Virol. 67(8):4945-55, 1993; Gabuzda, D. H., et al., J. AIDS 7(9):908-15, 1994). Vif requirements for HIV-1 replication in non-permissive cells but not by permissive cells can be explained by two alternative mechanisms. In permissive cells, there may be a cellular compensating factor (e.g., Nif homolog) that can replace Nif function in the virus-producing cells; alternatively, there may be an inhibitor(s) of viral replication in nonpermissive cells that requires Nif to counteract its effect (Trono, D., Cell 82:189-192, 1995). It has been suggested that Nif plays a role in viral assembly in virus-producing cells or cell-free virions (Blanc, D., et al., Virology 193:186-192, 1993; Gabuzda, D. H., et al., J. Virol. 66:6489-6495, 1992; von Schwedler, U., et al., J. Virol. 67:4945-4955, 1993). Essentially, in newly infected cells, Vif participates in the nuclear targeting of viral cores by acting as an adaptor, linking the pre-integration complex (PIC) to a cellular transport pathway and allowing viral transport to the nuclear membrane. In the absence of Vif, incoming virions diffuse, resulting in less efficient translocation of PIC to the nucleus [Karczewski and Strebel (1996) J. Virol. 70(1):494-507]. However, no co- localization of Vif and vimentin was detected in non-permissive T cells productively infected with HIV-1, suggesting that Vif may play a different function in the production of infectious particles [Simon, Fouchier et al. (1997) J. Virol. 71:5259-67]. Several cellular proteins were suggested as natural HIV-1 inhibitors [Madani and Kabat (1998) J. Virol. 72:10251-5; Zimmerman, Klein et al. (2002) Nature 415:88- 92]. Recently, it has been proposed that Vif neutralizes an intracellular virus inhibitor expressed in non-permissive cells. Essentially, the cellular factor, CEM 15, is currently suggested to be responsible for protecting cells against v/^negative HIV-1. It was shown that CEM 15 protein is intensively expressed in non-permissive, but not in permissive cells. Moreover, expression of CEM 15 in permissive cells renders them restrictive for replication of vz/^negative HIV-1. These results strongly suggest that Vif suppresses the anti- viral activity of CEM 15 protein in restrictive cells [Sheehy, Gaddis et al. (2002) Nat. Ned. 9:1404-7]. Sequence analysis of CEM 15 revealed its association with the APOBEC family of proteins, which are involved in RNA editing and function by deaminating cytosine in RNA [Teng, Burant et al. (1993) Science 260:1816-9;
Harris, Petersen-Mahrt et al. (2002) Mol. Cell 10:1247-53; Jarmuz, Chester et al.
(2002) Genomics 79:285-96]. However, APOBEC3G has been shown to act as a DNA mutator in E.coli, with the mutations attributed to dC→dU [Harris, Petersen-Mahrt et al. (2002) Mol. Cell 10:1247-53]. It has been shown that HIV-1 Vif expressed in cells binds APOBEC3G, leading to its ubiquitination and degradation via the proteosome pathway [Conticello, Harris et al. (2003) Curr. Biol. 13:2009-13; Kao, Khan et al.
(2003) J. Virol. 77:11398-407; Mariani, Chen et al. (2003) Cellll4:21-31; Marin, Rose et al. (2003) Nat. Med. 9:1398-403; Sheehy, Gaddis et al. (2003) Nat. Med. 9:1404-7; Stopak, de Noronha et al. (2003) Mol. Cell 12:591-601; Yu, Yu et al. (2003) Science 302: 1056-60; Mehle, Strack et al. (2004) J. Biol. Chem. 279:7792-8] . According to this model, Vif eliminates the APOBEC3G from cells, thus enabling the production of infectious virus. However, neutralization of APOBEC3G is not the sole mechanism envisaged for Vif. Gabuzda and her co-workers (Mehle, Strack et al. 2004, supra) showed that Vif function(s) is still required in cells expressing mutated inactive APOBEC3G, in order to produce infective virus. The present inventors and others have previously shown that Vif interacts with HIV protease [for review see Baraz and Kotler (2004) Curr. Med. Chem. 11:221-31]. These biochemical findings were later supported by cellular findings. Essentially, Vif- deficient particles produced by non-permissive cells differ from wild-type particles by several criteria: Vif-negative particles show non-homogenous packaging of the internal core, instead of the normal dense cone-shaped core structure. Vif-deficient virions are more susceptible to disruption by detergents, high salt concentration and buffers of various pH values [Ohagen and Gabuzda (2000) J. Virol. 74:11055-66; Khan, Akari et al. (2002) J. Virol. 76:9112-23]. These findings strongly suggest an interaction of Vif with PR [Baraz, Hutoran et al. (2002) FEBS Lett. 441:419-26; Blumenzweig, Baraz et al. (2002) 292:832-40]. It is well established that PR acts at specific time-window following or during budding. Unregulated cleavage causes production of "weak" particles, probably because the Gag-pol proteins are not in assembly equimolar ratio. Thus, it seems that Vif acts as a proteolytic regulator in non-permissive cells, directing the initiation of precursor processing to the correct location at the right time or stage, thus assuring production of mature virions. The retroviral protease, which is translated as part of a large polyprotein, processes Gag and Gag-Pol precursors, thereby contributing to virus maturation. The HIN-1 PR (GenBank Accession NO. AAL05141, HIV-PR) is a homodimeric aspartic protease composed of two 99 amino acid units. The active form of PR HIV-1 is a homodimer, in which its 4 termini form an anti-parallel β-sheet interface [Pearl and Taylor (1987) Nature 329:351-4; Meek, Dayton et al. (1989) PNAS 86:1841-5]. Dimerization of the enzyme occurs while PR is still a part of the Gag-Pol precursor, facilitating the autoprocessing of the polyproteins and resulting in virus maturation [Krausslich (1991) Adv. Exp. Med. Biol. 306:417-28. Expression of Vif with truncated Gag-Pol fusion polyproteins in bacterial cells inhibits processing of precursors [Kotler (1997) J. Virol. 71:5774-81]. The present inventors have previously found that recombinant Vif prevents degradation of synthetic peptide by PR, and inhibits PR-mediated Pr55Gag processing in vitro. This inhibitory activity of Vif was mapped to amino acid coordinates 78-98 [Baraz, Hutoran et al. (2002) J. Gen. Virol. 83:2225-30] pointing at the interaction between Vif and PR to the central region of Vif. While reducing the present invention to practice, the present inventors uncovered that peptides derived from the amino terminus of HIV PR are capable of inhibiting Vif binding to viral PR and thus are capable of inhibiting the production of infectious particles in cells conclusively showing that such peptides can be used as potent therapeutic agents against the HIV infection.
SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a peptide useful as an anti HIV agent comprising at least two consecutive amino acids of the amino acid sequence X-Y-Z, wherein X is an amide derivative of an acidic amino acid, Y is an aliphatic amino acid and Z is a nucleophilic amino acid, the peptide being no more than 25 amino acids and is not as set forth in SEQ ID NOs.: 3, 12, 13 and 24. According to another aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the peptide and a pharmaceutically acceptable carrier. According to yet another aspect of the present invention there is provided an article-of-manufacture comprising packaging material and a pharmaceutical composition identified for treating or preventing HIV infection, being contained within the packaging material, the pharmaceutical composition including, as an active ingredient, the peptide and a pharmaceutically acceptable carrier. According to still another aspect of the present invention there is provided use of the peptide as a pharmaceutical. According to an additional aspect of the present invention there is provided use of the peptide for the manufacture of a medicament identified for treating HIV infection. According to yet an additional aspect of the present invention there is provided a method of inhibiting HIV propagation in cells, the method comprising exposing the cells to the peptide, thereby inhibiting the HIV propagation in the cells. According to still an additional aspect of the present invention there is provided a method of treating HIV infection in a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the peptide, thereby treating the HIV infection in the subject. According to still further features in the described preferred embodiments the administering is effected by: (i) administering the peptide to the subject; and/or (ii) administering an expression construct encoding the peptide to the subject. According to still further features in the described preferred embodiments the amide derivative of the acidic amino acid is selected from the group consisting of asparagine and glutamine or functional mimetics thereof. According to still further features in the described preferred embodiments the aliphatic amino acid is selected from the group consisting of leucine, isoleucine and valine or functional mimetics thereof. According to still further features in the described preferred embodiments the nucleophilic amino acid is a hydroxylic amino acid or a functional mimetic thereof. According to still further features in the described preferred embodiments the hydroxylic amino acid is selected from the group consisting of threonine, serine and tyrosine. According to still further features in the described preferred embodiments the nucleophilic amino acid is selected from the group consisting of threonine, serine, proline and tyrosine or functional mimetics thereof. According to still further features in the described preferred embodiments at least one amino acid of the no more than 25 amino acids of the peptide is a D stereoisomer. According to still further features in the described preferred embodiments at least one amino acid of the no more than 25 amino acids of the peptide is an L stereoisomer. According to still further features in the described preferred embodiments the peptide is a linear or cyclic peptide. According to still further features in the described preferred embodiments the no more than 25 amino acids are in retro orientation. According to still further features in the described preferred embodiments the peptide is selected from the group consisting of SEQ ID NOs. 4, 10-11, 14-17, 19-23,
25-49, 52-57, 59-64, 67, 68, 71-75 and 76 or functional mimetics thereof. According to still further features in the described preferred embodiments the peptide is provided at medical grade purity. According to still further features in the described preferred embodiments the peptide further comprises a tripeptide sequence as set forth in SEQ ID NO: 50 or functional mimetics thereof attached to the Z. According to still further features in the described preferred embodiments the peptide further comprises a proline, glycine or tryptophan residue attached to the X. According to still further features in the described preferred embodiments the peptide is 3 amino acids in length. According to still further features in the described preferred embodiments the peptide is 2 amino acids in length. According to still further features in the described preferred embodiments at least one amino acid of the no more than 25 amino acids of the peptide is a synthetic amino acid. According to still further features in the described preferred embodiments at least one amino acid of the no more than 25 amino acids of the peptide is a natural amino acid. According to still further features in the described preferred embodiments the peptide further comprises a stabilizing moiety at an N- terminus and/or C-terminus thereof. According to still further features in the described preferred embodiments the stabilizing moiety is selected from the group consisting of N-α-trans L-4 hydroxyproline (Hyp), cis-4-amino-L-proline (Pam), PhOxalidine (Oxa), 4- thiazolidine carboxylic acid (Thiazole) and a stabilizing group. According to still further features in the described preferred embodiments the stabilizing group is selected from the group consisting of amido group, acetyl group, benzyl group, phenyl group, tosyl group, alkoxycarbonyl group, alkyl carbonyl group and benzyloxycarbonyl group. According to still further features in the described preferred embodiments the peptide further comprises a hydrophobic moiety at an N- terminus and/or C-terminus thereof. According to still further features in the described preferred embodiments the peptide further comprises at least one negatively charged ammo acid at an N- terminus and/or C-terminus thereof. According to a further aspect of the present invention there is provided an antibody or antibody fragment comprising an antigen binding site capable of specifically binding with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-4 and 10-23. According to yet a further aspect of the present invention there is provided a method of treating HIV infection in a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the antibody or antibody fragment, thereby treating the HIV infection in the subject. According to still a further aspect of the present invention there is provided a method of identifying putative anti HIV agents, the method comprising, identifying agents being capable of preventing formation of an HIV protease- Vif complex or capable of dissociating the complex, thereby identifying the putative anti HIV agents. According to still a further aspect of the present invention there is provided a method of identifying putative anti HIV agents, the method comprising, identifying agents being capable of preventing formation of an HIV protease- APOB EC complex or capable of dissociating the complex, thereby identifying the putative anti HIV agents. According to still further features in the described preferred embodiments the agents are selected from the group consisting of chemicals, antibodies, aptamers, peptides and combinations thereof. The present invention successfully addresses the shortcomings of the presently known configurations by providing peptides, antibodies and pharmaceutical compositions containing same which can be utilized to diagnose and treat HIV infection. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for puφoses of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIG. 1 is a schematic illustration showing mature wild-type HIV PR and
Avian sarcoma leukemia virus (ASLV) PR (Genebank Accession Nos. AAL05141 and AAK58475, respectively) and chimeric forms thereof (HIV-1 AS V and ASLV™7"1, SEQ ID NOs. 1 and 2, respectively) constructed as described herein below. FIGs. 2A-B are graphs depicting dose-dependent binding of wild-type and chimeric proteases to solid-phase bound Vif (Figure 2A) or binding of Vif to solid- phase bound proteases (Figure 2B). Figure 2A - Microwells of an ELIS A plate were coated with 0.2 μM of purified Vif, blocked with low fat milk for 2 h and incubated with 0, 25, 50, 100, and 200 nM of PR HIV-1 (closed circles), PR HIV-1ASLV (open circles), PR ASLV (closed squares) and PR ASLV™7"1 (open squares). Bound PRs were detected by using rabbit anti-PR and alkaline phosphatase conjugated goat anti- rabbit IgG and quantified in an ELISA reader at 405 nm. Figure 2B - Microwells of ELISA plate were coated with 90 nM of PR HIV-1 (closed circles), PR HIV-1ASLV (open circles), PR ASLV (closed squares), PR ASLVHIY"1 (open squares) and BSA (triangles), incubated with low fat milk for 2 h and incubated with 0, 20, 40, 80, and 160 nM of purified Vif protein. Bound Vif was detected by anti- Vif and alkaline phosphatase conjugated goat anti-rabbit IgG and quantified in an ELISA reader at 405 nm. FIG. 3 is a bar graph showing the effect of soluble PR on Vif binding to solid- phase bound PR chimeras, as determined by an ELISA assay. Wells were coated with 100 nM of PR HIV-1, PR HIV-1 ASLV, PR ASLV or PR ASLV™7"1, blocked with low fat milk. 80 nM of Vif protein was added, following pre-incubation with 100 nM of PRs for 18 h at 4 °C. Bound Vif was detected by anti- Vif and alkaline phosphatase conjugated goat anti-rabbit IgG and quantified in an ELISA reader at 405 nm. FIG. 4 is a bar graph showing the effect of PR1-9 (SEQ ID NO:4) on binding of Vif to PR HIV-1 and PR ASLV™7"1, as determined by ELISA. A microwell plate was coated with 90 nM of PR HIV-1, PR HIV-l^ PR ASLV or PR ASLV™7"1 and blocked with low fat milk. 150 nM of Vif protein were added following pre- incubation with the indicated amounts of PR1-9 (SEQ ID NO:4) and PR94-99 (SEQ ID NO:9) peptides for 18 h at 4°C. Vif was detected as in Figure 2B. FIGs. 5A-C are graphs depicting the effect of PR1-9 (SEQ ID NO:4) on HIV- 1 replication in restrictive and permissive cells. SupTl and H9 cells were incubated with 50μM PR1-9 and PR 94-99 for lh and then infected with HIV-1 wt (Figures 5A- i-ii) and Δvif (Figure 5A-iii) at multiplicity of infection moi 1 (Virus/cells = moi).
Cultured media were replaced with fresh one, containing peptides every 48 h.
Samples of media from days 1, 3, 6 and 10 were taken and analyzed for the presence of viral particles by p24 test (Figures 5A-i-iii) and for their infectivity by MAGI assay (Figure 5B-i-iv). Figure 5C shows the ratio of infectivity (a number of IU) to amount ofρ24 CA in day 10. FIGs. 6A-C are graphs showing the ability of PR1-9 (SEQ ID NO:4) to inhibit
FfiV-1 replication in 293T cells transfected with APOBEC3G. HIV-1 wt or HIV-1
Δvif containing plasmids were co-transfected with plasmids encoding APOBEC3G in 293T cells. 24 h post-transfection, culture media were removed and replaced with fresh medium or with a medium containing 50μM PR1-9 (SEQ ID NO:4) or PR94-99 (SEQ ID NO:9). Cultured media were replaced every 48 h. Samples of media from days 2,4 and 7 were taken and analyzed for viral infectivity by MAGI assay. Figure 6 A shows 293 T cells co-transfected with wild-type HIV-1 and APOBEC3G. Figure 6B shows 293T cells transfected with wild-type HIV-1. Figure 6C shows 293T cells transfected with HIV-1 Δvif. FIGs. 7A-B are bar graphs depicting binding of PR1-9 and deletions thereof (SEQ ID NOs: 3, 10, 11, 12, 13, 15-19 and 24) to Vif protein. Microwells were coated with Vif protein (Figure 7A) or PR protein(Figure 7B) and blocked with low fat milk. Figure 7A - Peptides were added to immobilized Vif and incubated for 2 hours at room temperature. Wells were washed and incubated with PR. Figure 7B - Vif was preincubated (for 18 hours at 4 °C) with PR-derived peptide. The solution was then added to immobilized PR for 2 and left to incubate for 2 hours at room temperature. Peptide complexes were determined by using anti-PR (Figure ' 7A) or anti- Vif (Figure 7b) and the interaction was determinated by ELISA as previously described befor. PR 93-99 (SEQ ID NO: 51) served as negative control. FIG. 8 is a bar graph depicting inhibition of HIV-1 production in H9 cells /infected with wild-type (wt) HIV by PR derived peptides. Samples of media from days 4, 7 and 10 were collected and analyzed for production of infectious virus by MAGI assay [Hutoran Virology. 2004 330(l):261-70]. FIGs. 9A-B are bar graphs depicting binding of glycine substituted PR-derived peptides (SEQ ID NOs: 52-56) to Vif protein, as described in Figures 7A-B. FIG. 10 is a bar graph depicting inhibition of HIV-1 production in H9 cells infected with HIV-1 by PR derived peptides. Assay was effected as described in
Figure 8. FIGs. 11A-B are bar graphs depicting binding of various PR-derived peptide configurations including amino acid sequence repeats and retro analogues thereof
(SEQ ID NOs: 25-30) to Vif protein, as described in Figures 7A-B. FIGs. 12A-B are bar graphs depicting binding of various PR-derived peptide configurations conferring bioavailability (SEQ ID NOs: 32-37) to Vif protein, as described in Figures 7A-B. FIGs. 13A-B are bar graphs depicting binding of various PR-derived peptide configurations (SEQ ID NOs: 38-43) to Vif protein, as described in Figures 7A-B. FIGs. 14A-B are bar graphs depicting binding of various PR-derived peptide configurations including conservative amino acid substitutions (SEQ ID NOs: 44-49) to Vif protein, as described in Figures 7A-B. FIGs. 15A-B are bar graphs depicting inhibition of HIV-1 production in H9 cells infected with HIV-1 wild type by the PR derived peptides of Figures 14A-B.
The assay was effected as described in Figure 8 only samples of media were collected on days 3, 6 and 8. FIGs. 16A-B are bar graphs depicting inhibition of HIV-1 production in 293 T cells trancfected with Apo3G by PR-derived peptides of Figures 14A-B provided at
500 μM (Figure 16A) or at 20 μM (Figure 16B).
DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of peptides, antibodies, and pharmaceutical compositions, which can be used for treating and detecting HIV virus infection in mammals, such as humans. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. The human immunodeficiency virus (HIV) is the primary cause of acquired immune deficiency syndrome (AIDS) [Barre-Sinoussi, F., et al., (1983) Science 220:868-870; Gallo, R., et al., (1984) Science 224:500-503]. HIV infection leads to depletion of T cells, which serve as hosts for viral replication and as such, the disease eventually leads to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death. Although considerable effort is invested into identifying or designing therapeutics effective in inhibiting HIV replication, current therapeutic approaches fail to eradicate the disease in infected individuals. In general, two therapeutic targets are considered when designing an anti- viral drug, cellular proteins, which participate in virion production and viral proteins; the latter serve as a far more attractive target for therapy since they are specific to the virus. One of the recently more investigated viral targets is Vif, a basic, 23 -kDa protein composed of 192 amino acids which is expressed by most Lentiviruses (for further detail see, for example, U.S. Pat. No. 6,653,443). The present inventors and others have previously shown that Vif interacts with HIV protease (PR, for review see (Baraz and Kotler 2004), thereby regulating processing of the Gag-Pol precursor and subsequently viral replication. The present inventors have also mapped a central site of Vif (i.e., Vif 35-50 and Vif 78-98), which interacts with the protease [Baraz, Hutoran et al. (2002) J. Gen. Virol. 83:2225-30;
Volsky (1998) Proc. Natl. Acad. Sci. 95(23): 13865-8] and designed Vif inhibitory peptides which abolish protease activity. While reducing the present invention to practice, the present inventors uncovered an amino acid sequence at the N-terminus of the protease, which interacts with Vif. These findings enabled the design and synthesis of PR-derived peptides, which abolish Vif-PR interaction, thereby inhibiting Vif-related activities such as without being bound by theory interaction thereof with APOBEC family of proteins and consequently inhibiting infectious virion production. As is illustrated in the Examples section which follows, the present inventors have shown that (i) the four amino acids residing at the N-terminus of HIV PR are essential for Vif/PR interaction; (ii) synthetic peptides derived from the N terminus of
HIV PR inhibit Vif/PR binding; (iii) such synthetic peptides inhibit the propagation of
HIV-1 in restrictive cells. These findings strongly support the use of PR-derived peptides of the present invention inhibit Vif in anti HIV therapy. It will be appreciated that HIV-PR derived peptides were previously described, such as be Zutshi and Chmielewski [(2000) Bioorganic & Midicinal Chemistry Letters 10:1901-1903]. However, in sharp contrast to the present invention, prior art peptides consist of C-terminus derived PR peptides or such peptides linked to N-terminus derived PR peptides which are aimed at inhibiting HIV-PR rather than Vif, as they are directed at the dimerization interface of HIV-PR (i.e., by targeting a Cys residue present at the dimerization interface). Furthermore, it is suggested that these peptide inhibitors exhibit poor HIV inhibitory activity. Thus, according to one aspect of the present invention there is provided a peptide useful as an anti HIV agent. As used herein an "anti HIV agent" refers to an agent which is capable of preventing HIV infection and/or at least alleviating symptoms associated with HIV infection. As used herein "HIV" refers to type I and type II human immunodeficiency virus (HIV). It will be appreciated that anti HIV agents of the present invention may also be effective in combating HIV-related viruses such as simian immunodeficiency virus (SrV) and feline immunodeficiency virus (FIV). The peptide according to this aspect of the present invention comprises at least two consecutive amino acids of the amino acid sequence X-Y-Z, wherein X is an amide derivative of an acidic amino acid, Y is an aliphatic amino acid and Z is a nucleophilic amino acid, is not as set forth in SEQ ID NOs.: 3, 12, 13 and 24. The peptide of this aspect of the present invention is preferably no more than 50 amino acids in length, preferably no more than 40 amino acids in length, preferably no more than 30 amino acids in length, preferably no more than 25 amino acids in length, preferably no more than 20 amino acids in length, preferably no more than 15 amino acids in length, preferably no more than 12 amino acids in length, preferably no more than 10 amino acids in length, preferably no more than 9 amino acids in length, preferably no more than 8 amino acids in length, preferably no more than 7 amino acids in length, preferably no more than 6 amino acids in length, preferably no more than 5 amino acids in length, preferably no more than 4 amino acids in length, preferably no more than 3 amino acids in length, preferably no more than 2 amino acids in length. As used herein "an amide derivative of an acidic amino acid" refers to the uncharged derivatives of glutamate and aspartate, that is glutamine and asparagine, which contain a terminal amide group in place of a carboxylate. The term refers also to functional mimetics (i.e., synthetic) of these amino acids, such as provided in Table
2 below. As used herein "an aliphatic amino acid" refers to amino acids having straight or branched side chain structures. Examples of aliphatic amino acids which can be used in accordance with this aspect of the present invention include, but are not limited to, leucine, isoleucine and valine or functional mimetics thereof, such as provided in Table 2 below. As used herein "a nucleophilic amino acid" refers to amino acids having side chains capable of donating electrons to a species known as an electrophile in order to form a chemical bond. Examples of nucleophilic amino acids which can be used in accordance with this aspect of the present invention include, but are not limited to, hydroxylic amino acids (e.g., threonine, serine and tyrosine) and proline or functional mimetics thereof, such as provided in Table 2 below. It will be appreciated that the peptide of this aspect of the present invention may be modified or further comprise amino acids which enhance its interaction with Vif. Thus, for example, the peptide may comprise a peptide sequence, such as set forth in SEQ ID NO: 50 or functional mimetics thereof attached to the above described nucleophilic amino acid. Inclusion of such a peptide sequence was shown by the present inventors to promote interaction with Vif and inhibit HIV propagation (see Example 4 of the Examples section which follows). Alternatively or additionally, the peptide may further comprise at least one negatively charged amino acid residue which promotes association with a positively charged amino acid sequence of Vif (i.e., RKKR). Alternatively or additionally, the peptide may comprise an amino acid residue N-terminally of the above-described amide derivative of an acidic amino acid. Examples of such amino acids include, but are not limited to, glycine, tryptophan or proline. It will be appreciated that each amino acid sequence of the peptide (or a portion thereof) may be presented at least once (e.g., twice, see SEQ ID NOs.: 25-30) in the peptide of this aspect of the present invention. Chemical (e.g., caproic acid) or peptide linkers (e.g., Ala-Ala) and/or spacers may be included in the peptide sequence. Typically a spacer is inserted in a papeitde sequence to generate a space between two moieties (e.g., peptide moieties). Typical linkers which may be used in accordance with this aspect of the present invention include, but are not limited to, alpha, omega- bifunctional moiety, e.g., hydrocarbon such as alkyl, cycloalkyl, aryl, alkenyl, alkynyl, substituted by two functional groups that can form a bond with the N-terminus and/or the C-terminus of the peptide. Typical spacers which may be used in accordance with this aspect of the present invention include, but are not limited to, alkyl, cycloalkyl, aryl and O-alkyl. According to a preferred embodiment of this aspect of the present invention, the peptide includes the amino acid sequence of SEQ ID NO: 4, 10-11, 14-17, 19-23, 25-49, 52-57, 59-64, 67, 68, 71-75 or 76. As used herein the term "mimetics" when made in reference to peptides refers to molecular structures, which serve as substitutes for the peptides of the present invention in interaction with HIV- Vif (Morgan et al. (1989) Ann. Reports Med. Chem. 24:243-252 for a review of peptide mimetics). Peptide mimetics, as used herein, include synthetic structures (known and yet unknown), which may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of a peptide ligand. Types of amino acids which can be utilized to generate mimetics are further described hereinbelow. The term, "peptide mimetics" also includes peptoids and oligopeptoids, which are peptides or oligomers of N-substituted amino acids [Simon et al. (1972) Proc. Natl. Acad. Sci. USA 89:9367-9371]. Further included as peptide mimetics are peptide libraries, which are collections of peptides designed to be of a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto. Methods for the production of peptide mimetics are described hereinbelow. As used herein the term "functional" refers to the ability to interfere with HIV infection. Hence "functional mimetic" refers to the above-described mimetics which function to interfere with HIV infection. The term "peptide" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and as mentioned hereinabove, peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2- CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder. Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), α-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2- ), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time. Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc). As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids. Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non- conventional or modified amino acids (Table 2) which can be used with the present invention.
Table 1
Figure imgf000018_0001
Table 2
Figure imgf000020_0001
Figure imgf000021_0001
Since the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain. To improve bioavailability peptides of this aspect of the present invention may include a hydrophobic moiety which promotes cell penetration. As used herein the phrase "hydrophobic moiety" refers to any substance which is nonpolar and generally immiscible with water. A hydrophobic moiety according to the present invention is preferably a hydrophobic residue (portion) of a hydrophobic substance. The hydrophobic moiety of the present invention can be attached to the amino acid sequence via covalent interactions. Representative examples of hydrophobic substances from which the hydrophobic moiety of the present invention can be derived include, but are not limited to, substituted and unsubstituted, saturated and unsaturated hydrocarbons, where the hydrocarbon can be an aliphatic, an alicyclic or an aromatic compound. Preferably, the hydrocarbon bears a functional group which enables attachment thereof to an amino acid residue. Representative examples of such functional groups include, without limitation, a free carboxylic acid (C(=O)0H), a free amino group (NH2), an ester group (C(=O)OR, where R is alkyl, cycloalkyl or aryl), an acyl halide group (C(=O)A, where A is fluoride, chloride, bromide or iodide), a halide (fluoride, chloride, bromide or iodide), a hydroxyl group (OH), a thiol group (SH), a nitrile group (C≡N), a free C-carbamic group (NR"-C(=O)-0R', where each of R' and R" is independently hydrogen, alkyl, cycloalkyl or aryl), a free N-carbamic group (OC(=O)-NR'-5 where R' is as defined above), a thionyl group (S(=O)2A, where A is halide as defined above) and the like. For example, the hydrophobic moiety of the present invention may be a fatty acid, such as myristic acid, lauric acid, palmitic acid, stearic acid (C18), oleic acid, linolenic acid and arachidonic acid Alternatively, the hydrophobic moiety can be an amino acid residue that is modified to include a fatty acid residue, or any other residue of a hydrophobic substance as described hereinabove, such that this modified amino acid residue is attached to the amino acid sequence via a peptide bond or a substituted peptide bond, as is described hereinabove. Still alternatively, the hydrophobic moiety can be a short peptide in which one or more amino acid residues are modified to include a fatty acid residue or any other residue of a hydrophobic substance as described hereinabove. As an alternative to, or in combination with the hydrophobic moiety described above, the peptide of the present invention, can also include a hydrophobic peptide sequence attached to the amino acid sequence described above. This hydrophobic peptide sequence preferably includes between 2 and 15 amino acid residues, in which at least one amino acid residue is a hydrophobic amino acid residue. Representative examples of hydrophobic amino acid residues include, without limitation, an alanine residue, a cysteine residue, an isoleucine residue, a leucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a methionine residue, a proline residue and a tryptophan residue, or any modification thereof, as is described hereinabove. Alternatively, the hydrophobic peptide sequence can include a combination of naturally occurring and synthetic amino acids, which have been modified by incorporation of a hydrophobic moiety thereto. The hydrophobic moiety or moieties of the present invention are preferably attached to the N-terminus and/or the C-terminus of the amino acid sequence of the peptide of the present invention. The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized. Cyclic peptides can either be synthesized in a cyclic form or configured so as to assume a cyclic form under desired conditions (e.g., physiological conditions). For example, a peptide according to the teachings of the present invention can include at least two cysteine residues flanking the core peptide sequence. In this case, cyclization can be generated via formation of S-S bonds between the two Cys residues. Side chain to side chain cyclization can also be generated via formation of an interaction bond of the formula -(-CH2-)n-S-CH2-C-, wherein n = 1 or 2, which is possible, for example, through incorporation of Cys or homoCys and reaction of its free SH group with, e.g., bromoacetylated Lys, Orn, Dab or Dap. Furthermore, cyclization can be obtained, for example, through amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (-CO-NH or -NH-CO bonds). Backbone to backbone cyclization can also be obtained through incorporation of modified amino acids of the formulas H-N((CH2)n-COOH)-C(R)H-COOH or H-N((CH2)n-COOH)- C(R)H-NH2, wherein n = 1-4, and further wherein R is any natural or non-natural side chain of an amino acid. The peptides according to the present invention can further include salts and chemical derivatives of the peptides. As used herein, the phrase "chemical derivative" describes a polypeptide of the invention having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., SEQ ID NO.: 33 in which Leucine was replaced with Abu to promote association with Vif). The chemical derivatization does not comprehend changes in functional groups which change one amino acid to another. The peptides of the present invention may include useful modifications which are designed to increase the stability of the peptides in solution and, therefore, serve to prolong the half-life of the peptides in solutions, particularly biological fluids, such as blood, plasma or serum, by blocking proteolytic activity in the blood. Hence, the peptides of the present invention can have stabilizing moiety such as N-α-trans L-4 hydroxyproline (Hyp), cis-4-amino-L-proline (Pam), PhOxalidine (Oxa), 4- thiazolidine carboxylic acid (Thiazole) or a stabilizing group at one or both termini. Typical stabilizing groups include amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the like end group modifications. It will be appreciated that since one of the main obstacles in using short peptide fragments in therapy is their proteolytic degradation by stereospecific cellular proteases, the peptides of the present invention preferably comprise at least one D- isomer of natural amino acids [i.e., inverso peptide analogues, Tjernberg (1997) J. Biol. Chem. 272:12601-5]. Additionally, the peptides of the present invention include retro, inverso and retro-inverso analogues thereof (see e.g., SEQ ID NOs.: 14 and 78-83). It will be appreciated that complete or extended partial retro-inverso analogues of hormones have generally been found to retain or enhance biological activity. Retro-inversion has also found application in the area of rational design of enzyme inhibitors (see U.S. Pat. No. 6,261,569). As used herein a "retro peptide" refers to peptides which are made up of L- amino acid residues which are assembled in opposite direction to the native peptide sequence. Retro-inverso modification of naturally occurring polypeptides involves the synthetic assembly of amino acids with α-carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse order to the native peptide sequence. A rerto inverso analogue, thus, has reversed termini and reversed direction of peptide bonds, while essentially maintaining the topology of the side chains as in the native peptide sequence. The peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involve different chemistry. Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide
Syntheses (2nd Ed., Pierce Chemical Company, 1984). Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles.
WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing. In cases where large amounts of the peptides of the present invention are desired, the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-
544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature
310:511-514, Taka atsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984)
EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.
(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463. Regardless of the synthesis method employed, peptides of the present invention are preferably highly purified (e.g., medical grade; e.g., > 95 %) for use in therapy, as further described hereinebelow. Further generation of peptide mimetics, as described hereinabove, be effected using various approaches, including, for example, display techniques. Thus, the present invention contemplates a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 2, at least 3, at least 4, at least 5, at least 7, or 9 consecutive amino acids derived from polypeptide sequences of the N-terminus of HIV PR (e.g., SEQ ID NOs: 3-4 and 10-23). As used herein an "N-terminus of HIV PR" refers to an amino acid sequence encompassed by amino acid coordinates 1-100, preferably 1-50, more preferably, 1- 20, even more preferably, 1-10, even more preferably 1-5, even more preferably 1-4 of HIV-PR. Methods of constructing such display libraries are well known in the art. Such methods are described in, for example, Young AC, et al., "The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes" J Mol Biol 1997 Dec 12;274(4):622-34; Giebel LB et al. "Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities" Biochemistry 1995 Nov 28;34(47): 15430-5; Davies EL et al., "Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes" J Immunol Methods 1995 Oct 12;186(l):125-35; Jones C RT al. "Current trends in molecular recognition and bioseparation" J Chromatogr A 1995 Jul 14;707(l):3-22; Deng SJ et al. "Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries" Proc Natl Acad Sci U S A 1995 May 23;92(11):4992-6; and Deng SJ et al. "Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display" J Biol Chem 1994 Apr l;269(13):9533-8, which are incorporated herein by reference. Peptide mimetics can also be uncovered using computational biology. For example, various compounds can be computationally analyzed for an ability to bind Vif using a variety of three-dimensional computational tools. Software programs useful for displaying three-dimensional structural models, such as RIBBONS (Carson, M., 1997. Methods in Enzymology 277, 25), O (Jones, TA. et al, 1991. Acta Crystallogr. A47, 110), DINO (DINO: Visualizing Structural Biology (2001) http://www.dino3d.org); and QUANTA, INSIGHT, SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J., 1991. Appl Crystallogr. 24, 946) can be utilized to model interactions between Vif and prospective peptide mimetics to thereby identify peptides which display the highest probability of binding to a specific Vif region. Computational modeling of protein-peptide interactions has been successfully used in rational drug design, for further detail, see Lam et al., 1994. Science 263, 380; Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993. Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993. Perspectives in Drug Discovery and Design 1, 109, and Mauro MJ. et al., 2002. J Clin Oncol. 20, 325-34. Peptide mimetics generated according to the teachings of the present invention are preferably qualified using functional biochemical and/or cell biology/molecular biology assays which are well known in the art (e.g., binding inhibition and/orHIN propagation inhibition as described in details in the Examples section which follows). The availability of sequence information on HIV-PR that is important for Vif binding allows the generation of antibodies, which may interfere with this interaction.
Such antibodies are expected to find utility in anti HIV therapy. Thus, according to another aspect of the present invention there is provided an antibody or an antibody fragment having an antigen-binding site capable of binding
HIV-PR and interfering with binding of the PR to Vif. According to a preferred embodiment of this aspect of the present invention the antigen-binding site is capable of specifically recognizing an amino acid sequence encompassed by amino acids coordinates 1-10 of HIV-PR. As used herein the term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule contaimng the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference). Generally, animals may be immunized with free peptide (e.g., SEQ ID NO: 4); however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine may be coupled to a carrier using a linker such as m-maleirnidobenzoyl-N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 mg peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for example, at intervals of about two weeks, to provide a useful titer of anti- peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art. Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659- 62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single- chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al, Science 242:423-426 (1988); Pack et al, Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety. Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)]. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity, hi some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al, Nature, 321:522-
525 (1986); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10,: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern.
Rev. Immunol. 13, 65-93 (1995). As is illustrated in Example 3 of the Examples section, which follows, PR- derived peptides arrest virus production in non-permissive cells and in permissive cells expressing APOBEC3G. Based on these results it is suggested that the peptides and/or antibodies of the present invention can be used for inhibiting HIV propagation in cells. To inhibit HIV propagation in cells, a peptide or antibody molecule generated according to the teachings of the present invention, is contacted with the cells under conditions, which allow inhibition of HIV propagation in these cells. It will be appreciated that the cells used herein can be restrictive cells such as peripheral blood lymphocytes, macrophages and cell lines such as H9 and Hut78. Alternatively, these cells can be permissive cells, which were engineered to depend on Vif for virus propagation, such as, for example, 293 T cells which express exogenous APOBEC3G (GenBank Accession No. NPJ365854, Sheehy, Gaddis 2002, Nature 418:646-50). A more detailed description of such an approach is provided in Example 3 of the Examples section. Since the peptides of the present invention are capable of preventing HIV propagation in host cells by inhibiting Vif activity (e.g., interaction with PR, interaction with APOBEC family or protein, virion propagation, an effect which can also be achieved using PR-specific antibody or antibody fragment), it is clear that peptides and antibodies generated according to the teachings of the present invention will also be useful in anti HIV therapy. Thus, according to yet another aspect of the present Invention there is provided a method of treating HIV infection in a subject. As used herein the term "subject" refers to a mammal, preferably a human which is infected with HIV, suspected of being infected with HIV or is at risk of being infected with HIV (e.g., a health worker). As used herein "treating" refers to alleviating or diminishing a symptom associated with HIV infection. Preferably, treating cures, e.g., substantially eliminates, the symptoms associated with the infection and/or substantially decreases the viral load in the infected tissue. The method according to this aspect of the present invention is effected by providing to a subject in need thereof, a therapeutically effective amount of the peptide and/or antibody of the present invention to thereby treat the HIV infection in the subject. Provision can be effected by administering the peptide and/or antibody of the present invention to the subject per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term "active ingredient" refers to the polypeptide, polynucleotide or peptide, such as described hereinabove, which is accountable for the biological effect. Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is mcluded under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979)). Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichiorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g.,
Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l]. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. In addition, other additives such as stabilizers, buffers, blockers and the like may also be added. It will be appreciated that the compositions of the present invention can be packaged in a one or more containers with appropriate buffers and preservatives and used for therapeutic treatment. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S.
Food and Drug Administration for prescription drugs or of an approved product insert. It will be appreciated that the peptide of the present invention can also be provided to the subject by administering to the subject an expressible nucleic acid construct encoding the peptide or antibody (i.e., in-vivo gene therapy), or by administering cells transformed with the expressible nucleic acid construct (i.e., ex- vivo gene therapy). It will be appreciated that T-cells precursors of the present invention may be ex- vivo or in-vivo treated to express the peptides of the present invention to develop resistance to HIV infection. Currently preferred in vivo nucleic acid transfer techniques include transfection with plasmids, viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example,
DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14 (1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, retroviruses such as lentiviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide or antibody from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. For example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers. It will be appreciated that treatment of HIV infections according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). Thus, for example, treatment of HIV infection according to the present invention can be combined with reverse-transcriptase-targeted drugs including 2', 3'-dideoxynucleoside analogs such as AZT™, ddl™, ddC™, and d4T™ which were shown to be effective in partially halting HIN replication The present invention also envisages identification of other anti HIV agents which are capable of interfering with HIV Vif-PR interaction and as such may be used as anti HIV drugs. Thus, according to another aspect of the present invention there is provided a method of identifying putative anti HIV agents (e.g., chemicals, antibodies, aptamers, peptides and the like). The method is effected by identifying agents which are capable of preventing formation of an HIV protease- Vif complex and/or HIN protease- APOBEC complex or dissociating the complex (i.e., pre-established) to thereby identify the putative anti HIN agents. An HIN protease-Nif complex may be generated by incubating intact HIV-PR and Vif molecules (e.g., recombinant) under conditions which allow complex formation. Such conditions are well described in Example 1 of the Examples section. Alternatively, peptides, which encompass the interaction site of either of the proteins (e.g., a peptide encompassing amino acid coordinates 1-9 of PR and a peptide encompassing amino acids 78-98 of Vif) may be used to generate the complex of this aspect of the present invention. Combinatorial chemical, nucleic acid or peptide libraries may be used to screen a plurality of agents. Screening according to this aspect of the present invention may be effected by contacting the agents with the pre-established complex described hereinabove, or with the-above described peptides. HIV PR or Vif-PR complex are preferably bound to a solid support to monitor binding of the agent to HIV PR or to monitor dissociation of the pre-established complex, respectively. The solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached, such as a test well in a microtiter plate, a nitrocellulose filter or another suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic such as polystyrene or polyvinylchloride. Molecular immobilizing on a solid support is effected using a variety of techniques known to those in the art. A number of methods are known in the art for determining intermolecular interactions. Examples include, but are not limited to, ELISA, Biacore, Pull-down assay, immunoprecipitation and the like (see references in the Examples section which follows). A competitive assay in which at least one of the assay component is labeled may also be employed. Labeling methods and tags are further described hereinbelow. It will be appreciated that when utilized along with automated equipment, the above-described method can be used to screen multiple agents both rapidly and easily. Agents identified using the above-described methodology can be further qualified by functional assays, such as by inhibiting HIV propagation in- vitro and in- vivo (see Examples 3-4 of the Examples section which follows). Because of the ability of the peptides, antibodies or other agents identified as described hereinabove to bind viral components (i.e., HIV-PR), it is conceivable that such peptides can also be used as potent detectors of HIV in biological samples. This is of a special significance since to date primary detection of HIV is based on the presence of HIV antibodies, via an ELISA assay, a test which is often non-specific and produces false positive results. Thus, according to yet another aspect of the present invention there is provided a method of detecting a presence or an absence of HIV in a biological sample. The method is effected by incubating the biological sample with a peptide or antibody (or other agent as described hereinabove) which is capable of binding HIV- PR and detecting complexes which comprise the peptide or antibody, to thereby detect the presence or the absence of HIV in the biological sample. The biological sample utilized for detection can be any body sample which contains HIV virions, such as blood (serum or plasma), sputum, ascites fluids, pleural effusions, urine, biopsy specimens and isolated cells. Preferably used is blood.
Methods of obtaining tissue biopsies and body fluids from mammals are well known in the art. The peptide of the present invention is contacted with the biological sample under conditions suitable for complex formation (i.e., buffer, temperature, incubation time etc.); suitable conditions are described in Example 1 of the Examples section. Protein complexes within a biological sample can be detected via any one of several methods known in the art, which methods can employ biochemical and/or optical detection schemes. To facilitate complex detection, the peptides of the present invention are labeled preferably by a tag or an antibody. It will be appreciated that labeling can be effected prior to, concomitant with or following complex formation, depending on the highlighting method. As used herein the term "tag" refers to a molecule, which exhibits a quantifiable activity or characteristic. A tag can be a fluorescent molecule including chemical fluorescers such as fluorescein or polypeptide fiuorescers such as the green fluorescent protein (GFP) or related proteins (www.clontech.com). In such case, the tag can be quantified via its fluorescence, which is generated upon the application of a suitable excitatory light. Alternatively, a tag can be an epitope tag, a fairly unique polypeptide sequence to which a specific antibody can bind without substantially cross-reacting with other cellular epitopes. Such epitope tags include a Myc tag, a Flag tag, a His tag, a leucine tag, an IgG tag, a streptavidin tag and the like. HIV diagnosis using the above-described methodology can be confirmed using prior art methods such as described hereinabove (e.g., anti HIV antibodies production). Peptides, antibodies and other agents of the present invention can be included in a diagnostic or therapeutic kit. For example, a peptide can be packaged in a container with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment. Preferably, the container includes a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials such as glass or plastic. In addition, other additives such as stabilizers, buffers, blockers and the like may also be added. The peptides of such kits can also be attached to a solid support, such as beads, array substrate (e.g., chips) and the like and used for diagnostic purposes. Peptides included in kits or immobilized to substrates may be conjugated to a detectable label such as described hereinabove. The kit can also include instructions for determining if the tested subject is suffering from, or is at risk of developing, a condition, disorder, or disease associated with HIN infection.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Nolumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Nols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Nolumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Nolumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Νorwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al.,
"Strategies for Protein Purification and Characterization - A Laboratory Course
Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. Experimental Procedures Cells - The cell lines Sup TI and H9 were provided by the NIH Reagent Program (Division of AIDS, NIAID, NIH, USA). Cells were grown in RPMI 1640 medium (Biological Industries, Beit Haemek, Israel). The adherent cell line 293T was kindly supplied by Dr. E. Bacharach (Tel Aviv University, Israel). HeLa-CD4 4- β-gal cells were obtained from the NIH Reagent Program (Division of AIDS, NIAID, NIH, USA). The adherent cell lines were cultivated in Dolbecco's modified Eagle's medium (DMEM, Biological Industries, Beit Haemek, Israel) as a sub confluent monolayer. HeLa-CD4+β-gal cells were maintained in the presence of 200 μg/ml G418 and lOOμg/ml Hygromycin B (Calbiochem, La Jolla, CA, USA). RPMI 1640 and DMEM media were supplemented with 10% fetal calf serum (FCS), lOOU/ml penicillin, lOOU/ml streptomycin and 2mM L-glutamine (Biological Industries, Beit Haemek, Israel). Viruses - Wild-type (wt) HIN-1 was generated by the transfection [Cullen (1987) Methods in Enzymology 152:693-694] of 293T cells with plasmid pSNC21 containing full length HIV-1HXB2 viral DΝA [Ratner, Haseltine et al. (1985) Nature 313:277-84]. 293T cells were transfected (Cullen 1987, supra) with pSVC21Δvif [Gabuzda, Lawrence et al. (1992) J. Virol. 66:6489-95]. Both wt and Δvif virions were harvested 48 and 72 hours post transfection and viral titer was detected by MAGI assay, below. Infection of cultured cells - Cultured lymphocytes (lxlO6 - 5xl06) were centrifuged for 5 minutes at 2000 rpm, supernatant was aspirated and the cells were resuspended in 0.2-0.5 ml of medium (RPMI 1640 Biological Industries, Beit Haemek, Israel) containing virus at m.o.i. 0.1 to 5. Following absorption for 1 hour (h) at 37 °C, the cells were washed to discard unbound virus and incubated for an additional 1-12 days. HIV-1 titration - Titration of HIV-1 was effected by a multinuclear galactosidase (MAGI) activation assay, as described by Kimpton and Emerman
[Kimpton and Emerman (1992) J. Virol 66: 223209]. HeLa-CD4+β-gaI cells were plated onto 96-well plates at a density of 15xl03 cells per well. On the following day, when the cultures were about 30% confluent, the medium was removed from the wells and the cells were infected with 50 μl of serially diluted virus in the presence of
20 μg/ml of DEAE-dextran (Pharmacia, Sweden). Following absorption for 2 h at 37
°C, 150 μl of medium was added. Two days post infection, the medium was removed and the cultured cells were fixed with 100 μl of 1 % formaldehyde solution and a subsequent incubation in 0.2 % glutaraldehyde in PBS for 5 min. The cells were then washed 3 times with PBS and incubated for 50 min at 37 °C in 100 μl of a solution including 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl2 and 0.4 mg/ml of X-Gal (Ornat, Israel). When the staining solution was removed and the cells washed twice in PBS, the reactions were terminated. Blue cells were counted under a light microscope at a magnification of x 100. P24 assay - The assay was effected using an HIV-1 p24 antigen capture assay kit (SAIC, Frederic, AIDS Vaccine Program), in accordance with the standards and instructions supplied by the manufacturers. Bacterial cells - E.coli BL21 cells (DE3) were grown in Luria-Bertini (LB) medium in the presence of 100 μg/ml chloramphenicol (Sigma, Israel). E.coli DH5ά strain was grown in antibiotic-free LB. E.coli MC 1061 cells were grown in LB medium with 50 μg/ml tetracycline (Sigma, Israel). Construction of chimeric PRs - Plasmids expressing chimeric HIV-1 PR and ASLV PR were constructed by replacing the DNA fragments encoding for the reciprocal 4 amino acids (aa) residing at the PRs termini. pUC12N plasmid bearing the HIV-1 PR [Kotler, Katz et al. (1988a) J. Virol. 62:2696-700] was used as a template to amplify the HIV-1ASLV PR (SEQ ID NO: 1) by using primers (5'- CCCGGGCCATGGTCCTCGCGATGACTCTTTGGCAACGACCC-3' SEQ ID NO: 5 and 5'-CCCGGGAAGCTTATAAATTTGTCAAGCAACCAATCTGAGTCAA-3' SEQ ID NO: 6) containing the sequences encoding for the ASLV-PR termini. The amplified DNA fragment was cleaved by Nco I and Hind III and inserted into pUC12N. The chimeric PR DNA fragment was rescued from the pUC12N by cleavage with Nco I and Hind III restriction enzymes and inserted into the pT5 vector for expression in E.coli BL21 strain (Baraz, Hutoran et al. 2002, supra). Primers (5'-
CCCGGGCCATGGTCCCTCAGATCACAATGGAACATAAAGAT-3' SEQ ID NO: 7 and 5'-CCCGGGAAGCTTAAAAATTTAATGTGCGGAGCCCTAGGCCC-
3' SEQ ID NO: 8) containing the DNA sequences encoding for the 4 aa residing in the HIV-1 PR (Figure 1) were used to achieve ASLV™7"1 (SEQ ID NO: 2) by amplification of the pEV containing the DNA sequence of ASLV PR [Kotler, Arad et al. (1992) J. Virol 66:6781-3]. The amplified DNA fragment was cleaved by Bam HI and Hind III and inserted into pUC12N and then into pT5 plasmid as described above.
The expression of PRs inserted into pT5 was induced by lmM of IPTG (Isopropyl β-o galactopyranoside), while the ASLV PR expressed by pEV was produced following a temperature shift to 42 °C (Kotler, Katz et al. 1988b; Kotler, Arad et al. 1992, supra). Peptides - Peptides were synthesized using the solid phase peptide synthesis (SPPS) method, on an ABI peptide synthesizer model 433A on Rink amide resin (loading 0.5 nmol/g) by standard Fmoc chemistry. HIV-1 PR-derived peptides were as follows, PR1-9 PQITLWQRP-NH2 (SEQ ID NO: 4), PR94-99 GCTLNF-NH2 (SEQ ID NO: 9). Purification of viral proteases - ASLV PR was purified from E. coli MC 1061 bacterial cells, as previously described [Kotler, Katz et al. (1988b) PNAS 85:4185-9; Friedler, Blumenzweig et al. (1999) J. M. B. 287:93-101]. HIN-1 PR, HIV-l^7 PR and ASLN™7"1 PR were purified as previously described [Baraz, Friedler et al. (1998) FEBS Lett. 441:419-26. Tethered covalently linked dimer of HIN-1 PR [Krausslich, Traenckner et al. (1991) Adv. Exp. Med. Biol. 306:417-28] was expressed from pETl l PR-PR plasmid (kindly supplied by Dr. Konvalinka, Institute of Organic Chemistry and Biochemistry, Praha, Czech Republic) in E.coli BL21 and purified as described above for HIN-1 PR. HIV-1 Vif purification - The pDIO plasmid, which encodes for HIV-1HXB2 Nif [Yang et al, (1996) J. Biol. Chem. 271:1012-19], was kindly provided by Dr. Gabuzda (Dana-Farber Cancer Institute, Boston, MA). The Nif was expressed in E.coli MC-1061 and purified as previously described (Yang, Goncalves et al. 1996, supra). Vif/PR binding assay -Purified Nif and PR proteins were bound as previously described by (Baraz, Hutoran et al. 2002, supra). In brief, the wells of ELISA plate were covered with 200 μl of ELISA coating buffer containing 200 nM Nif, or 90 nM PR or 200nM BSA for 18 h at 4 °C. Following removal of the coating buffer and blocking for 1 h with low-fat milk, increasing concentrations of PR (20 - 250 nM) or Nif (20-150 nM) in PBS were loaded into the wells and incubated for 1 h. In the competitive binding experiments, increasing concentrations of PR- derived peptides were pre-incubated for 18 h at 4 °C with Nif or PR proteins respectively, prior to loading onto the coated wells. The plates were extensively washed and the amounts of Nif or PR bound to the coated wells were determined by using rabbit anti-Vif (diluted 1:10,000) or anti-PR sera (diluted 1:1,000), respectively. Binding was determined by using alkaline phosphatase-conjugated goat anti-rabbit IgG diluted 1:1000 (Sigma) as a secondary antibody. Reactions were developed by addition of substrate p-rήtio phenyl phosphate (Sigma), and bound PR or Vif were quantified with an ELISA reader (Dynatech MR5000) at 405 nm. Antibodies - HIV-1 PR and ASLV PR polyclonal antibodies were generated by immunizing rabbits with 3 injections of purified recombinant proteases. Anti-CA and anti-Vif monoclonal antibodies were obtained from the ΝIH Reagent Program (Division of AIDS, ΝIAID, ΝIH, USA). Vif polyclonal antibody was kindly provided by Dr. Gabuzda (Dana-Farber Cancer Institute, Boston, MA). Alkaline phosphatase conjugated goat-anti-rabbit IgG and horseradish peroxidase conjugated goat anti- mouse sera were purchased from Sigma, St. Louis, USA.
EXAMPLE 1 Binding of ASLV or HIV-1 PRs to Vif The present inventors have previously suggested that Vif hampers the β-sheet structure formed by the termini regions of the 4 strands of the dimeric HIV-1 PR (Blumenzweig, Baraz et al. 2002, supra). The present inventors have now addressed the mechanism of interaction between the two proteins. Results To investigate further the interaction of Vif and PR N termini, chimeric proteases of HIV-1 and Avian Sarcoma and Leukemia Virus (ASLV) PRs were constructed, in which the termini of HIV-1 PR and ASLV PR were replaced by the 4 amino acid residues residing at the N and C termini of ASLV and HIV-1 PRs, respectively (Figure 1). Recombinant wild type and the chimeric PRs were expressed in bacteria cells, and were purified. Vif-coated micro wells of ELISA plate were used to determine whether the wild type and the chimeric PRs bind to Vif. Figure 2A clearly shows that HIV-1 PR and ASLV™7"1 PR bound Vif in a dose-dependent manner, as determined by ELISA with anti HIV-1 or anti ASLV PRs polyclonal sera, respectively. On the other hand, ASLV PR and fflV-lASLV PR did not bind to the same Vif-coated wells. Similar results were obtained by a reciprocal experiment, where increasing amounts of Vif were added to wild type and chimera PR coated wells. Figure 2B shows that Vif molecules bound to HIV-1 and ASLV™7"1
PRs, but did not bind ASLV and HIV-1 ASLV PRs. Note, Vif did not bind BSA, which was used as a negative control (Figure 2B). The results of these experiments strongly suggest that Vif interacts with the 4 amino acids residing at the termini of HIV-1 PR. In order to ensure that the binding of Vif to PRs is specific, competition experiments were preformed, in which PRs were incubated with Vif prior to addition thereof into the wells. ELISA plate coated with 90nM PRs were covered with 80 nM Vif pre-incubated with 100 nM of PRs. Figure 3 shows that pre-incubation with PR HIV-1 and PR ASLV™7"1, but not with PR HIV-l^7 and PR ASLV, inhibits binding of Vif to PR HIV-1 and PR ASLV™7"1. The experiments described above suggest that Vif interacts with the N- terminus of HIV-1 PR. To resolve this structural issue, two peptides were synthesized, PR1-9 (SEQ ID NO:4) and PR94-99 (SEQ ID NO:9), which were derived from the termini of HIV-1 PR. Incubation of Vif with these peptides prior to addition to PR- coated wells showed that PRl-9 blocks the binding of Vif to HIV-1 PR and ASLV™7"
!-PR in a dose-responsive manner, while pre-incubation with PR94-99 did not affect the binding (Figure 4). Again, Vif did not bind to ASLV and HIV-1ASLV in the presence or absence of these 2 peptides. These results suggest that Vif interacts with the conserved N-terminal region of PR.
EXAMPLE 2 PR-derived peptide arrests virus production in non-permissive cells Vif is essential for production of infectious virus in non-permissive cells [Lee,
Coligan et al. (1986) Science 231:1546-9; Sodroski, Goh et al. (1986) Science 231:1549-53; Fisher, Ensoli et al. (1987) Science 237:888-93; Strebel, Daugherty et al. (1987) Nature 328:728-30; Kishi, Nisliino et al. (1992) J. Gen. Virol. 73:77-87]. The experiments described above demonstrate that PRl-9 inhibit Vif function since it inhibits virus production only in non permissive cells. Results To examine the ability of PRl-9 (SEQ ID NO:4) to halt HIV infection in restrictive cells, H9 and Sup TI cells were infected with wt HIV-1 at m.o.i. 1. One hour post infection the cells were suspended in media containing 50μM of PRl-9 (SEQ ID NO:4) peptide or PR94-99 (SEQ ID NO:9) peptide. The media were collected during 10 days post infection, and the amount of virus produced by these cells was estimated by measuring the amounts of p24 CA released by the cells. The titer of the virus was determined by infection of HeLa CD4 cells. The results clearly show that PRl-9 peptide but not PR 94-99 reduced viral propagation in H9 cells (restrictive cells) but not in Sup TI cells (permissive cells, Figures 5Ai-ii). As is shown in Figures 5Aϋ-iii, the production of p24 by Sup TI cells infected with wild type or Δvif viruses was not affected by the peptides derived from the HIN-1 PR termini, suggesting that the PRl-9 blocks the Nif function(s). Titration of virus released from H9 and Sup TI cells treated with peptides derived from the PR termini verifies, that only PRl-9 inhibits the production of infectious particles from H9 but not from Sup TI cells (Figures 5Bi-iv). The ratio between the amounts of p24 and infectious units produced by the permissive and restrictive cells following treatment with PRl-9 (SEQ ID NO:4) and PR94-99 (SEQ
ID NO:9) are presented in Figure 5C. Altogether these results indicate that treatment of restrictive H9 cells infected with HIN-1, with PRl-9 does not merely reduce the amount of particles released from these cells, but that the proportional amount of infectious particles to the viral proteins in this preparation is low.
EXAMPLE 3 PRl-9 peptide reduces virus production by permissive cells expressing APOBEC3G Expression of APOBEC3G in the permissive 293 T cells turns them restrictive for Δvif virus propagation. Thus, the replication of HIV-1 in these cells becomes Vif- dependent (Sheehy, Gaddis et al. 2002, supra). Therefore, the question whether treatment with PRl-9 can reduce viral propagation in APOBEC3G transfected 293T cells was addressed. Results 293T cells were co-transfected with vectors expressing either wt or Δvif HIN- ■, 1 along with APOBEC3G. Transfected cultures were treated or untreated with the peptides derived from the HIV-1 PR termini and the titer of the viruses produced by these cells was determined by MAGI assay. As is shown in Figure 6A, treatment of cells with PRl-9 peptide (SEQ ID ΝO:4) reduced the titer of infectious virus by 3 times, while the same cells treated with PR94-99 (SEQ ID NO:9) peptide produced large amounts of infectious virus similarly to untreated cells. Accordingly, PR- derived peptides did not affect the production of infectious particles from cells transfected with vectors expressing wild type, or Δvif viruses (Figures 6B and 6C). Notably, control cells transfected with vectors expressing Δvif virus and APOBEC3G vectors did not produce any infectious particles (data not shown). It is of interest, is that PRl-9 inhibited the production of infectious virus from 293T cells co-transfected with APOBEC3G and HIV-1 vectors more efficiently than in HIV-1 infected H9 cells. Previously it was demonstrated that Vif/APOBEC3G interaction neutralizes the natural HIV-1 inhibitor APOBEC3G, enabling the production of infectious particles in restrictive cells. Based on these and the present results it is suggested that PRl-9 Binds to Vif and interferes with the interaction of Vif/APOBEC3G. The region 78-98 in Vif contains a motif similar to the consensus sequence of the PR C-terminus, which interacts with the PR N-terminus in establishing the 4- strand β-sheet structure. In line with these findings it is suggested that PRl-9 blocks this active site in Vif. Without being bound by theory, it is currently suggested that PRl-9 competes with APOBEC3G on the same binding site to Vif. Alternatively, binding of PRl-9 to Vif induces structural modification, which hampers the
VIF/APOBEC3G interaction.
EXAMPLE 4 PR1-3 peptide is sufficient to inhibit Vif binding to PR and arrest virus production in non-permissive cells In order to elucidate which are the critical residues in PRl-9 which are sufficient to inhibit Vif binding to PR and to arrest HIV production in H9 cells a series of PRl-9 truncated peptides (SEQ ID NOs: 3, 10, 11, 12, 13, 15, 16, 17, 18, 19 and 24) and glycine substituted peptides (SEQ ID NOs: 52, 53, 54, 55 and 56) was synthesized and analyzed. Results As is shown in Figures 7 A-B, a PR derived peptide as short as 3 amino acids in length was able to inhibit Vif binding to PR (SE ID NO: 15). Interestingly the crucial residues included the isoleucine, glutamine or threonine since PR2-7 (SEQ ID NO: 16) and PR3-7 (SEQ ID NO: 17) exhibited similar binding inhibition (see Figure 7B). These results were further substantiated in the HIV production assay illustrated in Figure 8. Note, that all deletion peptides tested were able to arrest viral production in the non-permissive H9 cells. A glycine scan analysis was then effected to further validate the results of the truncation peptides. Interestingly, all four amino acids of PR1-4 are necessary for inhibiting Vif binding to PR, as four glycine substitution peptide (SEQ ID NO: 55, Figures 9A-B) could not inhibit Vif binding to PR. However, from the HIV production assay illustrated in Figure 10 it is evident that the four glycine substitution peptide was able to arrest virus production in H9 cells indicating that glutamine and isoleucine are sufficient to mediate the inhibitory activity. Altogether, these results suggest that a two amino acid peptide including Ql or
IT (SEQ ID NO: 23 or 14), can be used as a potent inhibitor of virus production in non-permissive cells. EXAMPLE S PR1-5 and retro analogues thereof exhibit viral inhibitory activity In order to increase the anti- viral activity of the PR1-5 peptide (SEQ ID NO:
13) a series of analogues thereof was synthesized (SEQ ID NOs. 25-30). These included amino acid sequence repeats, retro configurations and combinations thereof. As is shown in Figures 11A-B, all the above described peptide configurations of the
PR1-5 amino acid sequence exhibited similar Vif-PR binding inhibitory activities.
EXAMPLE 6 PR1-5 modified to include degradation protective groups and/or hydrophobic residues exhibit similar activities to the non-modified peptide. PR1-5 (SEQ ID NO: 13) was chemically modified to exhibit better bioavailability. Results As is shown in Figures 12A-B, peptides modified to include a stabilizing moiety as set forth in N-α-trans L-4 hydroxyproline (Hyp, SEQ ID NO: 32), cis-4- amino-L-proline (Pam, SEQ ID NO: 35), 4-thiazolidine carboxylic acid (Thiazole, SEQ ID NO: 37), amino butyric acid (Abu, SEQ ID NO: 33) and PhOxalidine (SEQ ID NO: 36) as well as a double glycine residues at the C-termini of the peptides exhibited does dependent inhibitory activity, as determined by the inhibition of Vif binding to PR.
EXAMPLE 7 PR1-5 (SEQ ID NO: 13) was chemically modified to exhibit better association with Vif. Results As is shown in Figures 13A-B, peptides modified to include negatively charged residues as set forth in SEQ ID NOs.: 39, 40, 43 were used to display increased affinity towards the RKKR positive sequence of Vif. Additionally or alternatively, the leucine residue and threonine residue were replaced by proline which introduces a turn in the peptide (SEQ ID NOs, 41, 42/43 respectively). As is shown in Figures 13A-B the above described modifications did not significantly alter PR1-5 binding to Vif.
EXAMPLE 8 Conservative substitution in PR1-5 significantly increase peptide inhibitory activity Results As is shown in Figures 14A-B conservative substitutions replacing glutamine of position 2 to asparagine (S3-3, SEQ ID 46) completely inhibited Vif binding to PR.
Similarly, replacement of leucine of position 5 to another hydrophobic residue, valine
(SEQ ID NO: 48, S3-5) completely inhibited Vif binding to PR. Other conservative substitutions were able to increase peptide activity as well, as illustrated for S3-1
(Q7N), S3-2 (I3L, L5I) and S3-4 (T4S). PR1-8 (SI -2, SEQ ID NO: 10) was used as a positive control. PR96-99 (Sl-12, SEQ ID NO: 65) was used as a negative control. It is suggested that such amino acid substitutions are featured by compact side chains thereby bringing the peptide in closer proximity to Vif and increasing their association. These peptides were also able to arrest viral production in non-permissive cells (Figures 15A-B) and permissive cells transfected with ABOBEC-3G (Figures 16A-B) in a dose dependent manner.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:
1. A peptide useful as an anti HIV agent comprising at least two consecutive amino acids of the amino acid sequence X-Y-Z, wherein X is an amide derivative of an acidic amino acid, Y is an aliphatic amino acid and Z is a nucleophilic amino acid, the peptide being no more than 25 amino acids and is not as set forth in SEQ ID NOs.: 3, 12, 13 and 24.
2. A pharmaceutical composition comprising as an active ingredient the peptide of claim 1 and a pharmaceutically acceptable carrier.
3. An article-of-manufacture comprising packaging material and a pharmaceutical composition identified for treating or preventing HIV infection, being contained within said packaging material, said pharmaceutical composition including, as an active ingredient, the peptide of claim 1 and a pharmaceutically acceptable carrier.
4. Use of the peptide of claim 1 as a pharmaceutical.
5. Use of the peptide of claim 1 for the manufacture of a medicament identified for treating HIV infection.
6. A method of inhibiting HIV propagation in cells, the method comprising exposing the cells to the peptide of claim 1, thereby inhibiting the HIV propagation in the cells.
7. A method of treating HIV infection in a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the peptide of claim 1, thereby treating the HIV infection in the subject.
8. The method of claim 7, wherein said administering is effected by: (i) administering the peptide to the subject; and/or (ii) administering an expression construct encoding the peptide to the subject.
9. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein said amide derivative of said acidic amino acid is selected from the group consisting of asparagine and glutamine or functional mimetics thereof.
10. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein said aliphatic amino acid is selected from the group consisting of leucine, isoleucine and valine or functional mimetics thereof.
11. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein said nucleophilic amino acid is a hydroxylic amino acid or a functional mimetic thereof.
12. The peptide, pharmaceutical composition, article of manufacture, use, and method of claim 11, wherein said hydroxylic amino acid is selected from the group consisting of threonine, serine and tyrosine.
13. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein said nucleophilic amino acid is selected from the group consisting of threonine, serine, proline and tyrosine or functional mimetics thereof.
14. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein at least one amino acid of said no more than 25 amino acids of the peptide is a D stereoisomer.
15. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein at least one amino acid of said no more than 25 amino acids of the peptide is an L stereoisomer.
16. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide is a linear or cyclic peptide.
17. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein said no more than 25 amino acids are in retro orientation.
18. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide is selected from the group consisting of SEQ ID NOs. 4, 10-11, 14-17, 19-23, 25-49, 52-57, 59-64, 67, 68, 71-75 and 76 or functional mimetics thereof.
19. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide is provided at medical grade purity.
20. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide further comprises a tripeptide sequence as set forth in SEQ ID NO: 50 or functional mimetics thereof attached to said Z.
21. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide further comprises a proline, glycine or tryptophan residue attached to said X.
22. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide is 3 amino acids in length.
23. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide is 2 amino acids in length.
24. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein at least one amino acid of said no more than 25 amino acids of the peptide is a synthetic amino acid.
25. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein at least one amino acid of said no more than 25 amino acids of the peptide is a natural amino acid.
26. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide further comprises a stabilizing moiety at anN- terminus and/or C-terminus thereof.
27. The peptide, pharmaceutical composition, article of manufacture, use, and method of claim 26, wherein said stabilizing moiety is selected from the group consisting of N-α-trans L-4 hydroxyproline (Hyp), PhOxalidine, cis-4-amino-L- proline (Pam), 4-thiazolidine carboxylic acid (Thiazole) and a stabilizing group.
28. The peptide, pharmaceutical composition, article of manufacture, use, and method of claim 27, wherein said stabilizing group is selected from the group consisting of amido group, acetyl group, benzyl group, phenyl group, tosyl group, alkoxycarbonyl group, alkyl carbonyl group and benzyloxycarbonyl group.
29. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide further comprises a hydrophobic moiety at an N- terminus and/or C-terminus thereof.
30. The peptide, pharmaceutical composition, article of manufacture, use, and method of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the peptide further comprises at least one negatively charged amino acid at an N- terminus and/or C- terminus thereof.
31. An antibody or antibody fragment comprising an antigen binding site capable of specifically binding with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-4 and 10-23.
32. A method of treating HIV infection in a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the antibody or antibody fragment of claim 31, thereby treating the HIV infection in the subject.
33. A method of identifying putative anti HIV agents, the method comprising, identifying agents being capable of preventing formation of an HIV protease- Vif complex or capable of dissociating said complex, thereby identifying the putative anti HIV agents.
34. A method of identifying putative anti HIV agents, the method comprising, identifying agents being capable of preventing formation of an HIV protease-APOBEC complex or capable of dissociating said complex, thereby identifying the putative anti HIV agents.
35. The method of any of claims 33 and 34, wherein said agents are selected from the group consisting of chemicals, antibodies, aptamers, peptides and combinations thereof.
PCT/IL2005/000531 2004-05-24 2005-05-24 Peptides, antibodies and compositions containing same useful for treating hiv virus infection WO2005116055A1 (en)

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