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WO2007011827A2 - Agents cytostatiques g1 renforçant l'activite antivirale des medicaments et anticorps diriges contre l'enveloppe virale - Google Patents

Agents cytostatiques g1 renforçant l'activite antivirale des medicaments et anticorps diriges contre l'enveloppe virale Download PDF

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Publication number
WO2007011827A2
WO2007011827A2 PCT/US2006/027584 US2006027584W WO2007011827A2 WO 2007011827 A2 WO2007011827 A2 WO 2007011827A2 US 2006027584 W US2006027584 W US 2006027584W WO 2007011827 A2 WO2007011827 A2 WO 2007011827A2
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polypeptide
sequence
polypeptide sequence
viral
virus
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PCT/US2006/027584
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WO2007011827A3 (fr
Inventor
Alonso Heredia
George Lewis
Anthony L. Devico
Timothy Fouts
Robert C. Gallo
Robert R. Redfield
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University Of Maryland Biotechnology Institute
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Publication of WO2007011827A3 publication Critical patent/WO2007011827A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • This invention relates generally to antiviral therapy, more specifically to an antiviral combination therapy including a Gl cytostatic agent in combination with viral-envelope targeting drugs and/or antibodies, wherein the Gl cytostatic agent potentiates the antiviral activities of the viral-envelope targeting drugs and/or antibodies thereby reducing required antiviral therapeutic drug concentrations.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • the HIV viral particle comprises a viral core, composed in part of capsid proteins, together with the viral RNA genome and those enzymes required for early replicative events.
  • Myristylated gag protein forms an outer shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane.
  • the HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein, which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gpl20.
  • gp41 is a transmembrane glycoprotein
  • gpl20 is an extracellular glycoprotein, which remains non-covalently associated with gp41, recognized to be in a trimeric or multimeric form.
  • CD4 cell surface protein acts as the cellular receptor for the HIV virus. Viral entry into cells is dependent upon gpl20 binding the cellular CD4 receptor molecules, explaining HIV's tropism for CD4 cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane.
  • CCR5 serves as a co- receptor for the infection of CD4 cells by nonsyncytium-inducing (NSI) strains of HIV-I.
  • CCR5 receptor on T cells is dependent on the activation state of the cells. Resting lymphocytes do not express CCR5, however, upon activation, CCR5 is expressed. The importance of CCR5 for initial transmission of HIV-I is highlighted by the fact that individuals lacking expression of CCR5 (the CCR5- ⁇ 32 homozygous genotype) are usually resistant to infection. In addition, recent studies show that CCR5 cell-surface density correlates with disease progression in infected individuals.
  • humoral immunity can treat and/or prevent infection if an individual has high- titered neutralizing antibodies prior to exposure to the virus and provided the right antibodies are present in sufficient titers at the time of challenge or shortly thereafter.
  • the efficacy of antibodies to the coreceptor site is limited.
  • the neutralizing activity of IgG antibodies to the CD4 induced epitope cluster have limited activity against primary isolates of HIV-I.
  • Several studies suggest that their limited activity is due to kinetic and/or steric constraints.
  • the CD-4 induced epitope is formed only after gpl20 attaches to CD4 and it points down toward the cell membrane. It is thought that either there is not enough room for the IgG molecule to fit into the gap between CD4, the Env complex and the coreceptor or there is not enough time for efficient binding to take place.
  • the present invention relates to a combination therapy comprising viral-envelope targeting drugs and/or antibodies in combination with Gl cytostatic agents having the functional activity of reducing transcription of CCR5 thereby causing a reduced number of surface receptors for binding of HIV gp 120.
  • the invention relates to an antiviral therapy that comprises a Gl cytostatic agent in combination with chimeric polypeptides containing a virus coat polypeptide sequence and a viral receptor polypeptide sequence in which the coat polypeptide sequence and the receptor polypeptide sequence are linked by a spacer, wherein the Gl cytostatic agent potentiates the antiviral activities of the chimeric polypeptide or antibodies that are generated by an immunoresponse to the chimeric polypeptide, and wherein the combination reduces required therapeutic drug concentrations.
  • the virus coat polypeptide sequence of a chimeric polypeptide is an envelope polypeptide sequence (e.g., full-length gpl20 or a fragment thereof), a virus that binds a co-receptor polypeptide, an immunodeficiency virus, including HIV (e.g., HIV-I or fflV-2), SIV, FIV, FeLV, FPV, and a herpes virus.
  • HIV e.g., HIV-I or fflV-2
  • SIV FIV
  • FeLV FeLV
  • FPV FPV
  • the viral receptor polypeptide sequence is a CD4 polypeptide sequence or CD4 mimetic thereof, full-length sequence or a fragment thereof, such as the Dl, D2 domains and mutations thereof.
  • Introducing envelope genes derived from viruses that use alternative co-receptors could further expand the potential of these single chain molecules affording protection from viral infection of different cell types that express the different co-receptors.
  • heterologous domains impart a distinct functionality and include tags, adhesins and immunopotentiating agents.
  • heterologous domains can have an amino acid sequence, such as a c- myc polypeptide sequence or an immunoglobulin polypeptide sequence (e.g., a heavy chain polypeptide sequence).
  • the invention relates to an antiviral therapy comprising a Gl cytostatic agent in combination with antibodies.
  • compositions of the present invention may include any additional antiviral agent found to be effective against viral infections. 6 027584
  • polynucleotide sequences having a nucleic acid sequence encoding a chimeric polypeptide of the present invention in combination with a Gl phase arresting agent.
  • the polynucleotides can be included in an expression vector, are useful for expressing chimeric polypeptides, and can be used as a DNA vaccine that induces an immunomodulating effect.
  • antibodies and functional fragments thereof that bind to epitopes on the chimeric polypeptides of the present invention, CD4, gpl20 or epitopes that are induced with gpl20 and CD4 complex formation.
  • the antibodies are useful in treatment methods. Such antibodies can neutralize the immunodeficiency virus in vitro or in vivo, and can also be useful in inhibiting immunodeficiency virus infection, for example, by passive protection.
  • the combination Gl phase arresting agents and chimeric polypeptides; polynucleotides encoding such polypeptides; antibodies that recognize viral components; cell receptors that interact or bind with viral components; or epitopes that are hidden until viral components interact with the cell receptors; may be used as a therapeutic treatment.
  • the present invention relates to an antiviral combination therapy, the combination comprising;
  • the viral receptor polypeptide is CD4 or mimetic thereof, a full length polypeptide sequence or a fragment thereof, such as the Dl, D2 domains and mutations thereof.
  • the CD4 mimetic may include any molecule that interacts or binds with a HIV viral envelope protein and forms an interacting complex, including but not limited to miniproteins CD4M9, CD4M33, synthesized compounds such as BMS378806, BMS488043 or any molecule that recognizes and targets for binding with the "Phe43 cavity" of gpl20.
  • the Gl cytostatic agent may include any compound that arrests or prolongs the Gl phase in the cell cycle of mononuclear cells, for example, including but not limited to sodium butyrate, aphidicolin, hydroxyurea (HU), olomoucine, roscovitine, tocopherols, including alpha-tocopherol, beta-tocopherol, D-alpha-tocopherol, delta-tocopherol, gamma-tocopherol, tocotrienols, rapamycin (RAPA), indirubin derivatives including indirubin-3'-monoxime, 5- halogeno-indirubin, N-ethyl-indirubin and N-methylisoindigo and functional analogs or derivatives thereof.
  • HU hydroxyurea
  • olomoucine roscovitine
  • tocopherols including alpha-tocopherol, beta-tocopherol, D-alpha-tocopherol, delta-tocopherol,
  • the Gl cytostatic agent is RAPA, a bacterial macrolide that is currently approved for the treatment of renal transplantation rejection, which has been shown to exert cytostatic activity in T cells by disrupting molecular events resulting from the binding of IL-2 to the IL- 2 receptor. Further, it has been shown by the present inventors that RAPA inhibits CCR5- mediated viral entry by the downregulation of CCR5 protein expression.
  • Another aspect of the present invention relates to a method to enhance the efficacy of a chimeric polypeptide in antagonizing CCR5 receptors, wherein the chimeric polypeptide comprises a virus coat polypeptide sequence and a viral receptor polypeptide sequence wherein the coat polypeptide sequence and the receptor polypeptide sequence are linked by a spacer and exhibit receptor/ligand binding affinity, the method comprising: administering a composition comprising: a) the chimeric polypeptide or nucleotide sequences encoding for the chimeric polypeptide; and b) a Gl cytostatic agent in an amount effective to reduce expression of CCR5, whereby the inclusion of the Gl cytostatic agent in the composition increases the efficacy of the chimeric polypeptide.
  • the Gl cytostatic agent is RAPA and the efficacy of the combination is synergically increased.
  • the therapeutic dosage of the chimeric polypeptide or antibodies having affinity therefore is reduced due to the potentiating enhancement of the Gl cytostatic agent.
  • polyclonal and monoclonal antibodies and functional fragments thereof that are induced by and/or bind with the chimeric polypeptides of the present invention, gpl20, CD4 or CD4 induced hidden epitopes.
  • Antibodies may include, but not limited to, 17b, 48d, A32 and 211/c (induced epitope on gpl20/CD4 complex); IgGlbl2 (gpl20); 2G12 (gpl20); 2F5 (gp41); 4E10 (gp41); Z13 (gp41); CRA-3, CRA-4, 684-238, G3-136, G3-4, SC258, BAT-085 (V2 loop); G3-299 and G3-42 (Vp3/ CD4); 15e, 2Ih, 55, F19 (CD4).
  • the chimeric polypeptides, polynucleotides and antibodies of the present invention are useful for treating viral infection, or for inducing an immune response.
  • a pharmaceutically acceptable carrier in combination with a Gl cytostatic agent.
  • the present invention relates to antibodies and methods for producing such antibodies comprising: administering a chimeric polypeptide of the invention in an amount sufficient for the subject to produce antibodies to the chimeric polypeptide or administering a nucleotide sequence that encode for the chimeric polypeptides of the present invention in an amount sufficient for the subject to produce antibodies; and in combination with Gl phase arresting agent.
  • Figure 1 shows the three-dimensional interaction of RAPA and FLSC against HIV-I ADA. Synergy is shown as a rise from the additive surface. 95% confidence interval synergy plot.
  • Figure 2 shows the cell viability (MMT assays) in cultures exposed to FLSC in the presence and absence of RAPA.
  • Figure 3 shows the level that RAPA enhances the antiviral activity of neutralizing antibodies raised by the FLSC in the absence of cell toxicity. (Note the log scale used in panel a).
  • Figure 4 shows the level that RAPA enhances the neutralizing activity of CD4i monoclonal antibody 17b.
  • Figure 5 shows the level that RAPA enhances the neutralizing activity of CD4i monoclonal antibody X5.
  • FIG. 6 is a diagram of a polynucleotide construct that encodes exemplary chimeric polypeptides.
  • Full-length single chain (FLSC) chimeric polypeptide comprises an HW gpl20 (BaL strain), a 20 amino acid spacer polypeptide, a CD4 polypeptide sequence comprising the D 1 and D2 domains (Dl D2), and a myc peptide "tag.”
  • a truncated single chain (TsSC) chimera contains deletions in the Cl (constant region 1), Vl (variable region 1), V2, and C5. The deletions indicated for TcSC are numbered according to the BaL gpl20 sequence.
  • a FLSC R/T chimera has a single mutation in the furin cleavage site, an R is changed to a T, at the c-terminus of gpl20.
  • a FLSC R/T CD4M9 chimera has a single mutation in the furin cleavage site of gpl20, a 21 amino acid spacer polypeptide and a CD4M9 peptide sequence.
  • Figure 7 shows the Gl phase arresting activity of an Indirubin derivative.
  • the present invention is based on the discovery that a Gl cytostatic agent in combination with a chimeric polypeptide comprising an interacting receptor/ligand pair or antibodies induced by such chimeric polypeptide can increase the efficacy of the chimeric polypeptide whether by inducing an immune response and/or antagonizing coreceptor binding.
  • the chimeric polypeptide of the present invention preferably comprises an HIV envelope polypeptide and a CD4 receptor or CD4 mimetic to form an interacting complex capable of binding to a co-receptor as described in U. S. Patent Application Nos. 09/684,026 and
  • the chimeric polypeptides of the present invention mimic the envelope protein-CD4 transition state that occurs when HIV binds CD4 present on cells, wherein gpl20 displays conserved epitopes exposed upon complex formation that interact directly with co-receptor, CCR5. Formation of the envelope-CD4 transition state and subsequent binding to cell co-receptor is a critical step in HIV infection of cells. Therefore, agents that reduce the level of the co-receptor CCR5 can provide protection from HIV infection or at the least reduce the effects of HIV.
  • Chimeric polypeptides are also useful for producing antibodies specific for the interacting coat protein-receptor complex. Such specific antibodies can be used for passive protection against virus infection or proliferation, for diagnostic purposes and for identifying and characterizing epitopes exposed upon complex formation (e.g., a cryptic epitope). Even in the absence of intramolecular binding between virus coat protein and a receptor, a chimeric polypeptide may be more effective at eliciting an immune response than a virus coat polypeptide sequence alone. Accordingly, such non-interacting chimeric polypeptides also are valuable and are included herein.
  • Chimeric polypeptides containing a virus coat polypeptide that binds a receptor and co- receptor have the additional advantage of passively protecting against virus infection by inhibiting virus access to cell co-receptors in vivo.
  • virus binding to cell receptors is required for virus infection of any cell, chimeric polypeptides comprising a polypeptide sequence of any virus coat protein and a corresponding receptor are included in the invention compositions and methods.
  • chimeric polypeptides or nucleic acids encoding the chimeric polypeptides of the present invention in combination with a Gl phase arresting agent that reduces activation of CCR5 receptors can be used therapeutically for treating, inhibiting, preventing or ameliorating virus infection.
  • chimeric polypeptides are referred to herein as "full length single chain” (FLSC) molecules, are useful for binding to coreceptor binding sites and/or producing antibodies specific for the interacting coat protein-receptor complex.
  • FLSC full length single chain
  • Such specific antibodies can be used for passive protection against virus infection or proliferation, for diagnostic purposes and for identifying and characterizing epitopes exposed upon complex formation (e.g., a cryptic epitope).
  • a chimeric polypeptide may be effective at eliciting an immune response than a virus coat polypeptide sequence alone. Accordingly, such non- interacting chimeric polypeptides also are valuable and are included herein.
  • chimeric polypeptides comprising a virus coat polypeptide sequence and a viral receptor polypeptide sequence linked by a spacer, wherein the spacer is of sufficient length to provide for the two polypeptide sequences of the chimeric polypeptide to intramolecularly bind or interact.
  • the coat polypeptide sequence is an envelope polypeptide sequence of an immunodeficiency virus.
  • the coat polypeptide sequence is from a virus that binds a co-receptor polypeptide.
  • the coat polypeptide sequence and the receptor polypeptide sequence are active fragments of a corresponding full-length native sequence.
  • coat means a polypeptide sequence of virus origin that can bind to cells.
  • virus coat proteins are present near the exterior surface of the virus particle and allow binding and subsequent penetration into the cell membrane.
  • a coat polypeptide sequence includes any virus protein capable of binding to or interacting with a receptor polypeptide.
  • Coat polypeptide sequences as defined herein may be non-covalently or covalently associated with other molecular entities, such as carbohydrates, fatty acids, lipids and the like.
  • Coat polypeptide sequences may contain multiple virus polypeptide sequences.
  • a gag polypeptide sequence may also be included with an envelope polypeptide sequence in a chimeric polypeptide to maintain the envelope polypeptide sequence in a conformation that binds to a receptor polypeptide sequence.
  • Retroviridae e.g human immunodeficiency viruses, such as HTV
  • Picornaviridae e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses
  • Calciviridae e.g., strains that cause gastroenteritis
  • Togaviradae e.g., equine encephalitis viruses, rubella viruses
  • Flaviridae e.g., dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronaviridae e.g., coronaviruses
  • Rhabdoriridae e.g., vesicular stomatitis viruses, rabies viruses
  • the term "receptor” means any polypeptide expressed by a cell that a virus can bind. Generally, such receptors are naturally present on the surface of a cell, but can be engineered. Receptor polypeptides may be non-covalently or covalently associated with other molecular entities, such as carbohydrates, fatty acids, lipids and the like. A receptor polypeptide may comprise one or multiple contiguous polypeptide segments that are covalently or non-covalently attached. Such molecular entities or other polypeptide sequences may be important for receptor conformation, for example, for binding to a coat polypeptide sequence. Thus, additional elements including molecules important for receptor conformation may therefore be included in the chimeric polypeptides of the present invention.
  • the receptor polypeptide sequence can be either prokaryotic or eukaryotic in origin.
  • Preferred animal receptors are mammalian, including human and primates, for example, chimps, apes, macaques, gibbons, orangutans and the like, as well as other animal species, including domestic animals and livestock.
  • An example of a human receptor is CD4.
  • Other examples of receptors include glycosaminoglycan and CD2, CRl. Additional receptors are known and are applicable in the compositions and methods of the invention (see, for example, Table 1 J see also "Cellular Receptors For Animal Viruses" Eckard Wimmer, ed; Cold Spring Harbor Press (1994)). TABLE l
  • co-receptor means any receptor that is bound after or in conjunction with virus binding to receptor.
  • co-receptors include any polypeptide or molecular entity present on a cell that facilitates virus entry, directly or indirectly, by binding to virus polypeptide-receptor complex.
  • co-receptors that facilitate virus-entry into cells also included are co-receptors that mediate cell attachment or tropism without directly or indirectly facilitating virus entry.
  • co-receptors are the 7- transmembrane domain (7-TM) containing chemokine receptors, such as CCR5 and CXCR4, which can bind immunodeficiency virus.
  • Additional co-receptors include CCR-2b, CCR3, CCR8, V28/CXCR1 , US28, STRL 33/BOB/TYMSTR, GPR15/Bonzo and GPRl.
  • polypeptide As used herein, the terms "polypeptide,” “protein” and “peptide” are used interchangeably to denote a sequence polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). D- and L-amino acids, and mixtures of D- and L-amino acids are also included.
  • Chimeric polypeptide refers to an amino acid sequence having two or more parts which generally are not found together in an amino acid sequence in nature.
  • a chimeric polypeptide having a CD4 polypeptide sequence and an HIV envelope gpl20 polypeptide sequence that binds CD4 can bind to each other in the chimera when separated by an amino acid spacer sequence.
  • CD4 appears to be the target for entry of a variety of viruses associated with immunodeficiency.
  • cells of the immune system such as lymphocytes and macrophages express CD4, and are susceptible to infection by HIV, SIV, herpes virus 7 and many other viruses.
  • the term "immunodeficiency,” when used in reference to a virus means that the virus is capable of infecting cells of immune origin or cells that participate in immune responsiveness, and generally such infection can compromise an infected host's immune function.
  • the invention is applicable to any virus coat polypeptide of any virus or virus strain that can bind CD4.
  • the envelope polypeptide sequence is a polypeptide sequence of HIV, HTLV, SIV, FeLV, FPV and
  • the virus is a macrophage tropic or a lymphocyte tropic HIV.
  • the HIV is HIV-I or HIV-2.
  • the envelope polypeptide sequence is gpl20, g ⁇ l60 or gp41.
  • Receptor and virus coat polypeptide sequences of the chimeric polypeptide require a spacer region between them, for example, for forming an interacting complex between the two polypeptides.
  • the spacer allows the movement or flexibility between receptor and virus coat polypeptide sequences to form an intramolecular complex formed by binding affinity without the necessity of an additional crosslinking agent.
  • the term "spacer” refers to a physical or chemical moiety, or covalent or non- covalent bond of any size or nature that connects the virus coat polypeptide sequence to the receptor polypeptide sequence while affording the needed flexibility or movement for forming an interacting complex.
  • the spacer preferably links the two polypeptide sequences in an "end to end” orientation. "End to end” means that the amino or carboxyl terminal amino acid of the coat polypeptide is connected to the amino or carboxyl terminal amino acid of the receptor polypeptide sequence.
  • an amino acid spacer can connect the carboxyl terminal amino acid of the coat polypeptide sequence to the amino terminal amino acid of the receptor polypeptide sequence, as exemplified herein for HIV g ⁇ l20 and CD4, for example.
  • the spacer can connect the amino terminal amino acid of the coat polypeptide to the carboxyl terminal amino acid of the receptor polypeptide or the carboxyl terminal amino acids of the polypeptide sequences or the two amino terminal amino acids of the polypeptide sequences.
  • spacers include one or more amino acids or a peptidomimetic.
  • An amino acid spacer can essentially be any length, for example, as few as 5 or as many as 200 or more amino acids.
  • an amino acid spacer can have from about 10 to about 100 amino acids, or have from about 15 to about 50 amino acids.
  • the spacer has from about 20 to about 40 amino acids.
  • Other examples of spacers include a disulfide linkage between the termini of the polypeptide sequences.
  • a carbohydrate spacer also is contemplated. Those skilled in the art will know or can readily ascertain other moieties that can function to allow formation of an interacting complex between the virus coat polypeptide sequence and receptor polypeptide sequence.
  • Receptor and coat polypeptide sequences can be of any amino acid length. Preferably, they have a length that allows the polypeptide sequences to bind to each other when in a chimeric polypeptide.
  • receptor and coat polypeptide sequences include native full-length receptor and full-length coat polypeptide sequences as well as parts of the polypeptide sequences. For example, amino acid truncations, internal deletions or subunits of receptor, and coat polypeptide sequences are included. Preferably, such modified forms are capable of interacting with each other. For example, it is preferable that a truncated or deleted coat polypeptide sequence is capable of interacting with a receptor polypeptide sequence.
  • truncated receptor polypeptide sequence is the CD4 Dl and D2 domains, which are capable of interacting with HTV envelope polypeptide sequence.
  • An example of a truncated coat polypeptide sequence is a truncated HIV gpl20 lacking the amino terminal 60 amino acids and carboxy terminal 20 amino acids (e.g., in TcSC).
  • chimeric polypeptides including truncated or internally deleted sequences.
  • the virus coat polypeptide sequence or the receptor polypeptide sequence has one or more amino acids removed in comparison to their corresponding full-length polypeptide sequence.
  • the truncated virus coat polypeptide sequence is an HIV envelope polypeptide sequence and, in another aspect, the truncated receptor polypeptide sequence is a CD4 sequence.
  • the truncated HIV envelope polypeptide sequence is a gp 120 lacking the amino terminal 60 amino acids or the carboxy terminal 20 amino acids, and a truncated CD4 polypeptide comprising the D 1 and D2 domains.
  • the chimeric polypeptide comprises an internally deleted virus coat polypeptide sequence or an internally deleted CD4 polypeptide sequence.
  • a virus coat or receptor polypeptide sequence may have carbohydrates, fatty acids (palmitate, myristate), lipids, be phosphorylated or have other post-translational modifications typically associated with polypeptide sequences.
  • heterologous domain that imparts a distinct functionality upon either of the two polypeptides or the chimeric polypeptide.
  • a heterologous domain can be any small organic or inorganic molecule or macromolecule, so long as it imparts an additional function. Heterologous domains may or may not affect interaction or affinity between virus coat polypeptide and receptor polypeptide.
  • heterologous domains that impart a distinct function include an amino acid sequence that imparts targeting (e.g., receptor ligand, antibody, etc.), immunopotentiating function (e.g., immunoglobulin, an adjuvant), enable purification, isolation or detection (e.g., myc, T7 tag, polyhistidine, avidin, biotin, lectins, etc.).
  • targeting e.g., receptor ligand, antibody, etc.
  • immunopotentiating function e.g., immunoglobulin, an adjuvant
  • purification, isolation or detection e.g., myc, T7 tag, polyhistidine, avidin, biotin, lectins, etc.
  • heterologous domains may include a c-myc polypeptide sequence and an IgGl heavy chain polypeptide sequence.
  • a heterologous domain can have multiple functions.
  • IgGl can function as an immunopotentiator in vivo, as well as function as an adhesive molecule that can be purified, isolated, or detected (e.g., by reaction with a secondary antibody having an enzymatic activity, such as horseradish peroxidase or alkaline phosphatase).
  • a secondary antibody having an enzymatic activity such as horseradish peroxidase or alkaline phosphatase.
  • the heterologous domain is a c-myc polypeptide sequence glu-gln-lys-leu-ile-ser-glu-glu-asp-leu; (SEQ ID NO: 14).
  • the heterologous domain is an immunoglobulin polypeptide sequence comprising a heavy chain (SEQ ID NO: 32).
  • Receptor and coat polypeptide sequences can be of any amino acid length. Preferably, they have a length that allows the polypeptide sequences to bind to each other when in a chimeric polypeptide.
  • receptor and coat polypeptide sequences include native full-length receptor and full-length coat polypeptide sequences as well as parts of the polypeptide sequences.
  • the present invention includes full-length single chain (FLSC) chimeric polypeptide comprising a HW gpl20 (BaL strain), an amino acid spacer polypeptide, a CD4 polypeptide sequence comprising the D1D2 domain and a myc peptide "tag.”
  • the FLSC may further comprise a single mutation in the furin cleavage site, wherein an R is changed to a T, at the c-terminus of gpl20 (FLSC-R/T).
  • FLSC R/T contains an arginine to a threonine mutation.
  • the virus is an immunodeficiency virus, as described herein, such as HIV, HTLV, S ⁇ V, FeLV, FPV, or herpes virus.
  • the receptor polypeptide is a CCR5, CXCR4, CCR-2b, CCR3, CCR8, V28/CX3CR1, US28 (herpes virus
  • the present invention comprises a full-length single chain (FLSC) chimeric polypeptide comprising a HIV gpl20 (BaL strain), an amino acid spacer polypeptide, a CD4 polypeptide sequence comprising the D1D2 domain and a myc peptide "tag" (SEQ ID NO.: 2) or at least 95% sequence identity to SEQ ID NO: 2 that encodes the chimeric polypeptide.
  • FLSC full-length single chain
  • the prevention invention comprises a FLSC polypeptide having single mutation in a furin cleavage site of the FLSC polypeptide, wherein an R is changed to a T, at the c-terminus of gpl20 (FLSC-R/T) or at least 95% sequence identity to SEQ ID NO: 2 that encodes the chimeric polypeptide.
  • FLSC R/T contains an arginine to a threonine mutation at amino acid 506 (SEQ ID NO.: 4).
  • polypeptide sequence include substitutions, variations, or derivitizations of the amino acid sequence of one or both of the polypeptide sequences that comprise the chimeric polypeptide, so long as the modified chimeric polypeptide has substantially the same activity or function as the unmodified chimeric polypeptide.
  • a virus coat or receptor polypeptide sequence may have carbohydrates, fatty acids (palmitate, myristate), lipids, be phosphorylated or have other post-translational modifications typically associated with polypeptide sequences.
  • the virus coat polypeptide sequence or the receptor polypeptide sequence has one or more amino acid substitutions in comparison to their corresponding unmodified polypeptide sequences.
  • a nucleotide sequence (SEQ ID NO: 5) is provided that encodes for a polypeptide that includes a CD4 mimicking receptor that shows substantially the same activity or improved immune response.
  • the term "substantially the same activity or function," when used in reference to a chimeric polypeptide so modified, means that the polypeptide retains most, all or more of the activity associated with the unmodified polypeptide, as described herein or known in the art. Similarly, modifications that do not affect the ability of chimeric polypeptide to interact with co-receptor are included herein. Likewise, chimeric polypeptide modifications that do not affect the ability to induce a more potent immune response than administration of the virus coat protein alone are included. Modified chimeric polypeptides that are "active" or “functional” included herein can be identified through a routine functional assay.
  • Chimeric polypeptides that induce a more potent immune response can be identified by measuring antibody titers following administration of the chimera to a subject, for example. Modifications that destroy the interaction between the virus coat polypeptide sequence and the receptor polypeptide sequence, or the ability of a chimeric polypeptide having a virus coat polypeptide sequence and receptor sequence which do not interact to induce a more potent immune response, do not have substantially the same activity or function as the corresponding, unmodified chimeric polypeptide and, as such, are not included. ⁇
  • homologous refers to amino acid sequence similarity between two polypeptides. When an amino acid position in both of the polypeptides is occupied by identical amino acids, they are homologous at that position. Thus, by “substantially homologous” means an amino acid sequence that is largely, but not entirely, homologous, and which retains most or all of the activity as the sequence to which it is homologous.
  • modified chimeric polypeptides will retain activity or function associated with unmodified chimeric polypeptide
  • modified chimeric polypeptides will generally have an amino acid sequence "substantially identical” or “substantially homologous" with the amino acid sequence of the unmodified polypeptide.
  • the term “substantially identical” or “substantially homologous,” when used in reference to a polypeptide sequence means that a sequence of the polypeptide is at least 50% identical to a reference sequence.
  • Modified polypeptides and substantially identical polypeptides will typically have at least 70%, alternatively 85%, more likely 90%, and most likely 95% homology to a reference polypeptide.
  • the length of comparison to obtain the above-described percent homologies between sequences will generally be at least 25 amino acids, alternatively at least 50 amino acids, more likely at least 100 amino acids, and most likely 200 amino acids or more.
  • substantially identical or homologous polypeptides include additions, truncations, internal deletions or insertions, conservative and non-conservative substitutions, or other modifications located at positions of the amino acid sequence which do not destroy the function of the chimeric polypeptide (as determined by functional assays, e.g., as described herein).
  • a particular example of a substitution is where one or more amino acids is replaced by another, chemically or biologically similar residue.
  • the term "conservative substitution” refers to a substitution of one residue with a chemically or biologically similar residue.
  • conservative substitutions include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • a hydrophobic residue such as isoleucine, valine, leucine, or methionine for another
  • a polar residue for another such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • Substantially identical or homologous polypeptides also include those having modifications that improve or confer an additional function or activity.
  • FLSC R/T has a mutated furin site which increases stability of the modified FLSC (see, e.g., FIG. 6).
  • Modified polypeptides further include "chemical derivatives," in which one or more of the amino acids therein have a side chain chemically altered or derivatized.
  • derivatized polypeptides include, for example, amino acids in which free amino groups form amine hydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the free carboxy groups form salts, methyl and ethyl esters; free hydroxyl groups that form O-acyl or O-alkyl derivatives, as well as naturally occurring amino acid derivatives, for example, 4- hydroxyproline, for proline, 5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine, and so forth. Also included are D-amino acids and amino acid derivatives that can alter covalent bonding, for example, the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide.
  • the terms "isolated” or “substantially pure,” when used as a modifier of invention chimeric polypeptides, sequence fragments thereof, and polynucleotides, means that they are produced by human intervention and are separated from their native in vivo - cellular environment. Generally, polypeptides and polynucleotides so separated are substantially free of other proteins, nucleic acids, lipids, carbohydrates or other materials with which they are naturally associated.
  • a polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and other molecules with which it is naturally associated.
  • the preparation is likely at least 75%, more likely at least 90%, and most likely at least 95%, by weight pure.
  • Substantially pure chimeric polypeptide can be obtained, for example, by expressing a polynucleotide encoding the polypeptide in cells and isolating the polypeptide produced.
  • expression of a recombinant polynucleotide encoding a gpl20-CD4 polypeptide in mammalian cells allows isolating the chimerical polypeptide from the culture media using an immunoaffinity column.
  • the chimeric polypeptide can be chemically synthesized. Purity can be measured by any appropriate method, e.g., polyacrylamide gel electrophoresis, and subsequent staining of the gel (e.g., silver stain) or by HPLC analysis.
  • the chimeric polypeptides of the present invention and modifications thereof can be prepared by a variety of methods known in the art.
  • the polypeptide modifications can be introduced by site-directed (e.g., PCR based) or random mutagenesis (e.g., EMS) by exonuclease deletion, by chemical modification, or by fusion of polynucleotide sequences encoding heterologous domain, for example.
  • Chimeric polypeptides can be obtained by expression of a polynucleotide encoding the polypeptide in a host cell, such as a bacteria, yeast or mammalian cell, and purifying the expressed chimeric polypeptide by purification using typical biochemical methods (e.g., immunoaffinity purification, gel purification, expression screening etc). Other well-known methods are described in Deutscher et al., (Guide to Protein Purification: Methods in Enzymology, Vol. 182, Academic Press (1990), which is incorporated herein by reference).
  • the present invention further provides polynucleotide sequences encoding chimeric polypeptides, fragments thereof, and complementary sequences.
  • nucleic acids encode the chimeric gpl20-CD4 polypeptide exemplified herein.
  • SEQ ID NO.: 1 defines the sequence encoding FLSC described hereinabove comprising a nucleotide sequence encoding gpl20 (SEQ ID 23) and CD4 D1D2 (SEQ ID NO: 25).
  • SEQ. ID NO: 3 defines a sequence encoding FLSC R/T wherein an arginine amino acid is substituted for a threonine at the c-terminal of the gpl20, a suspect furin cleavage site in gpl20, thereby improving the stability of the FLSC-R/T over FLSC.
  • the nucleotide sequence of FLSC-RT comprises a modified gpl20 encoded by SEQ ID NO: 29 and CD4D1D2 (SEQ ID NO: 25).
  • the present invention provides for polynucleotide sequence SEQ ID NO.: 5 that encodes for a chimeric polypeptide FLSC R/T CD4M9 comprising a substituted furin cleavage site and further provides for replacement of gene sequence encoding the CD4 D1D2 region with a sequence that encodes for an amino acid sequence that mimics a CD4 receptor, thereby providing for an improved immune response and additional stability relative to FLSC or FLSC-R/T.
  • the FLSC R/T CD4M9 is encoded by nucleotide sequences comprising SEQ ID .NO: 29 that encodes for a modified gpl20 and SEQ ID NO: 19 encoding for CD4M9.
  • the FLSC R/T CD4M9 chimeric polypeptide may additionally comprise SEQ E) NOs: 23 and 19.
  • TsSC (SEQ ID NO: 12) encode a gpl20-CD4 polypeptide (SEQ ID NO: 13) in which the gpl20 has amino acid sequences truncated from the amino and carboxy terminus.
  • the nucleotide sequence of TsSC comprises a sequence (SEQ ID NO: 27) that encodes for a truncated gpl20 and CD4D1D2 (SEQ ID NO: 25).
  • a chimeric polypeptide gpl20-CD4-IgGl is encoded by nucleotide SEQ ID NO: 1 with an additional tag (SEQ ID NO: 31)
  • nucleic acid As used herein, the terms “nucleic acid,” “polynucleotide,” “oligonucleotide,” and “primer” are used interchangeably to refer to deoxyribonucleic acid (DNA) or ribonucleic (RNA), either double- or single-stranded, linear or circular.
  • RNA can be unspliced or spliced mRNA, rRNA, tRNA, or antisense RNAi.
  • DNA can be complementary DNA (cDNA), genomic DNA, or an antisense.
  • nucleotide analogues and derivatives such as those that are resistant to nuclease degradation, which can function to encode an invention chimeric polypeptide. Nuclease resistant oligonucleotides and polynucleotides are particularly useful for the present nucleic acid vaccines described herein.
  • an "isolated" or “substantially pure” polynucleotide means that the nucleic acid is not immediately contiguous with the coding sequences with either the 5' end or the 3' end with which it is immediately contiguous in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment produced during cloning), as well as a recombinant DNA incorporated into a vector, an autonomously replicating plasmid or virus, or a genomic DNA of a prokaryote or eukaryote.
  • the term therefore does not include nucleic acids present but uncharacterized among millions of sequences in a genomic or cDNA library, or in a restriction digest of a library fractionated on a gel.
  • the polynucleotides of the invention also include nucleic acids that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. All degenerate polynucleotide sequences are included that encode invention chimeric polypeptides.
  • polynucleotides sequences of the present invention can be obtained using standard techniques known in the art (e.g., molecular cloning, chemical synthesis) and the purity can be determined by polyacrylamide or agarose gel electrophoresis, sequencing analysis, and the like. Polynucleotides also can be isolated using hybridization or computer-based techniques that are well known in the art.
  • Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening of polypeptides expressed by DNA sequences (e.g., using an expression library); (3) polymerase chain reaction (PCR) of genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.
  • PCR polymerase chain reaction
  • the invention also includes substantially homologous polynucleotides.
  • the term "homologous,” when used in reference to nucleic acid molecule, refers to similarity between two nucleotide sequences. When a nucleotide position in both of the molecules is occupied by identical nucleotides, then they are homologous at that position. "Substantially homologous" nucleic acid sequences are at least 50% homologous, more likely at least 75% homologous, and most likely 90% or more homologous.
  • polynucleotides substantially homologous to invention polynucleotides encoding chimeric polypeptides encode polypeptides that retain most or all of the activity or function associated with the sequence to which it is homologous.
  • the length of comparison between sequences will generally be at least 30 nucleotides, alternatively at least 50 nucleotides, more likely at least 75 nucleotides, and most likely 110 nucleotides or more.
  • BLAST Altschul et al, J. MoI. Biol. 15:403-10 (1990)
  • the polynucleotides of the present invention can, if desired: be naked or be in a carrier suitable for passing through a cell membrane (e.g., polynucleotide-liposome complex or a colloidal dispersion system), contained in a vector (e.g., retrovirus vector, adenoviral vectors, and the like), linked to inert beads or other heterologous domains (e.g., antibodies, ligands, biotin, streptavidin, lectins, and the like), or other appropriate compositions disclosed herein or known in the art.
  • a cell membrane e.g., polynucleotide-liposome complex or a colloidal dispersion system
  • a vector e.g., retrovirus vector, adenoviral vectors, and the like
  • inert beads or other heterologous domains e.g., antibodies, ligands, biotin, streptavidin, lectins, and the like
  • polynucleotides of the present invention can also contain additional nucleic acid sequences linked thereto that encode a polypeptide having a distinct functionality, such as the various heterologous domains set forth herein.
  • the polynucleotides of the present invention can also be modified, for example, to be resistant to nucleases to enhance their stability in a pharmaceutical formulation.
  • the described polynucleotides are useful for encoding chimeric polypeptides of the present invention, especially when such polynucleotides are incorporated into expression systems disclosed herein or known in the art. Accordingly, polynucleotides including an expression vector are also included.
  • vectors described herein can be inserted into a vector.
  • vector refers to a plasmid, virus, or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid.
  • vectors can be used for genetic manipulation (i.e., "cloning vectors") or can be used to transcribe or translate the inserted polynucleotide (i.e., "expression vectors").
  • a vector generally contains at least an origin of replication for propagation in a cell and a promoter.
  • Control elements including promoters present within an expression vector, are included to facilitate proper transcription and translation (e.g., splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and stop codons).
  • a promoter operably linked to the nucleic acid refers to a minimal nucleic acid sequence sufficient to direct transcription of the nucleic acid to which the promoter is operably linked (see, e.g., Bitter et ah, Methods in Enzymology, 153:5 16-544 (1987)). Promoters can constitutively direct transcription, can be tissue-specific, or can render inducible or repressible transcription; such elements are generally located in the 5' or 3' regions of the gene so regulated.
  • a tissue-specific promoter can be operably linked to the polynucleotide sequence to confer expression of the chimeric polypeptide in an appropriate target cell.
  • tissue-specific promoter means a promoter that is active in particular cells or tissues that confers expression of the operably linked polynucleotide in the particular cells, e.g., liver cells, hematopoietic cells, or cells of a specific tissue within an animal.
  • the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in one or more other tissues as well.
  • inducible promoter can also be used to modulate expression in cells.
  • “Inducible promoter” means a promoter whose activity level increases in response to treatment with an external signal or agent (e.g., metallothionein HA promoter, heat shock promoter).
  • a “repressible promoter” or “conditional promoter” means a promoter whose activity level decreases in response to a repressor or an equivalent compound. When the repressor is no longer present, transcription is activated or derepressed.
  • Such promoters may be used in combination and also may include additional DNA sequences that are necessary for transcription and expression, such as introns and enhancer sequences.
  • operably linked means that a selected polynucleotide (e.g., encoding a chimeric polypeptide) and regulatory sequence(s) are connected in such a way as to permit transcription when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
  • a promoter is located at the 5' end of the polynucleotide and may be in close proximity of the transcription initiation site to allow the promoter to regulate expression of the polynucleotide.
  • indirect operable linkage is also possible when a promoter on a first vector controls expression of a protein that, in turn, regulates a promoter controlling expression of the polynucleotide on a second vector.
  • constitutive promoters such as T7 and the like, as well as inducible promoters, such as pL of bacteriophage gamma, plac, ptrp, ptac, may be used.
  • constitutive promoters such as SV40, RSV and the like, or inducible promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the mouse mammary tumor virus long terminal repeat, the adenovirus late promoter), may be used.
  • promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.
  • Mammalian expression systems that utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the nucleic acid sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • the vaccinia virus 7.5K promoter may be used (see, e.g., Mackett et ah, Proc. Natl. Acad. ScL USA, 79:7415-7419 (1982); Mackett et al, J. Virol, 49:857-864 (1984); Panicali et al, Proc. Natl. Acad. ScL USA, 79:4927-4931 (1982)).
  • Mammalian expression systems further include vectors specifically designed for "gene therapy" methods, including adenoviral vectors (U.S. Patent Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Patent No. 5,604,090), herpes simplex virus vectors (U.S. Patent No. 5,501,979), and retroviral vectors (U.S. Patent Nos. 5,624,820, 5,693,508 and 5,674,703 and WIPO publications WO92/05266 and WO92/14829).
  • the chimeric polypeptide encoding gene can be introduced into vaccine delivery vehicles, such as attenuated vaccinia (M.
  • Vectors based on bovine papilloma virus have the ability to replicate as extra-chromosomal elements (Sarver et al, MoI. Cell. Biol, 1:486 (1981)). Shortly after entry of an extra-chromosomal vector into mouse cells, the vector replicates to about 100 to 200 copies per cell. Because transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, a high level of expression occurs.
  • Such vectors also have been employed in gene therapy (U.S. Patent No. 5,719,054). CMV-based vectors also are included (U.S. Patent No. 5,561,063).
  • yeast expression a number of vectors containing constitutive or inducible promoters may be used (see, e.g., Current Protocols in Molecular Biology, Vol. 2, Ch. 13, ed. Ausubel et al, Greene Publish. Assoc. & Wiley Interscience (1988); Grant et al, "Expression and Secretion Vectors for Yeast," in Methods in Enzvmology, Vol. 153, pp. 516-544, eds. Wu & Grossman, 3 1987, Acad. Press, N.Y. (1987); Glover, DNA Cloning. Vol. ⁇ . Ch. 3, IRL Press, Wash., D.C.
  • yeast artificial chromosomes are typically used when the inserted polynucleotides are too large for more conventional yeast expression vectors (e.g., greater than about 12 kb).
  • the polynucleotides may be inserted into an expression vector for expression in vitro (e.g., using in vitro transcription/translation kits, which are available commercially), or may be inserted into an expression vector that contains a promoter sequence that facilitates expression in either prokaryotes or eukaryotes by transfer of an appropriate nucleic acid into a suitable cell, organ, tissue, or organism in vivo.
  • transgene is any piece of a polynucleotide inserted by artifice into a host cell, and becomes part of the organism that develops from that cell.
  • a transgene can include one or more promoters and any other DNA, such as introns, necessary for expression of the selected DNA, all operably linked to the selected DNA, and may include an enhancer sequence.
  • a transgene may include a polynucleotide that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Transgenes may integrate into the host cell's genome or be maintained as a self-replicating plasmid.
  • a "host cell” is a cell into which a polynucleotide is introduced that can be propagated, transcribed, or encoded polypeptide expressed.
  • the term also includes any progeny of the subject host cell. It is understood that not all progeny may be identical to the parental cell, since there may be mutations that occur during replication.
  • Host cells include but are not limited to bacteria, yeast, insect, and mammalian cells.
  • bacteria transformed with recombinant bacteriophage polynucleotide, plasmid nucleic acid, or cosmid nucleic acid expression vectors for example, bacteria transformed with recombinant bacteriophage polynucleotide, plasmid nucleic acid, or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid), insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus), or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for stable expression.
  • recombinant virus expression vectors e
  • expression vectors containing viral origins of replication cells can be transformed with a nucleic acid controlled by appropriate control elements (e.g.,promoter/enhancer sequences, transcription terminators, polyadenylation sites, etc.).
  • control elements e.g.,promoter/enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • stable maintenance of expression vectors in mammalian cells is believed to occur by integration of the vector into a chromosome of the host cell.
  • the expression vector also can contain a nucleic acid encoding a selectable marker conferring resistance to a selective pressure or reporter indicating the cells into which the gene has been introduced, thereby allowing cells having the vector to be identified, grown, and expanded.
  • reporter gene means a gene whose expression may be assayed; such genes include, without limitation, lacZ, amino acid biosynthetic genes, e.g. the yeast LEI2 gene, luciferase, or the mammalian chloramphenicol transacetylase (CAT) gene. Reporter genes may be integrated into the chromosome or may be carried on autonomously replicating plasmids (e.g., yeast 2 micron plasmids). Alternatively, the selectable marker can be on a second vector cotransfected into a host cell with a first vector containing an invention polynucleotide.
  • CAT mammalian chloramphenicol transacetylase
  • a number of selection systems may be used, including, but not limited to the neomycin gene, which confers resistance to the aminoglycoside G418 (Colberre-Garapin et al, J MoI. Biol, 150: 1 (1981)) and the hygromycin gene, which confers resistance to hygromycin (Santerre et al, Gene, 30: 147 (1984)).
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine (Hartman et al., Proc. Natl. Acad. Sci.
  • transformation means a genetic change in a cell following incorporation of a polynucleotide (e.g., a transgene) exogenous to the cell.
  • a polynucleotide e.g., a transgene
  • transformed cell is a cell into which, or a progeny of which, a polynucleotide has been introduced by means of recombinant techniques. Transformed cells do not include an entire human being. Transformation of a host cell may be carried out by conventional techniques known to those skilled in the art. When the host cell is a eukaryote, methods of DNA transformation include, for example, calcium phosphate, microinjection, electroporation, liposomes, and viral vectors. Eukaryotic cells also can be co-transformed with invention polynucleotide sequences or fragments thereof, and a second DNA molecule encoding a selectable marker, as described herein or otherwise known in the art.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells, and express the protein (see, e.g., Eukaryotic SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells, and express the protein (see, e.g., Eukaryotic
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • prokaryotic e.g., E. coli
  • competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well-known in the art. Transformation of prokaryotes also can be performed by protoplast fusion of the host cell.
  • the viral receptor molecule can include CD4 mimetics such as CD4M9, CD4M33, synthesized compounds such as BMS378806, BMS488043 or any molecule that recognizes and targets for binding with the "Phe43 cavity" of gpl20.
  • CD4 mimetics such as CD4M9, CD4M33, synthesized compounds such as BMS378806, BMS488043 or any molecule that recognizes and targets for binding with the "Phe43 cavity" of gpl20.
  • polynucleotides sequences of the present invention can be obtained using standard techniques known in the art (e.g., molecular cloning, chemical synthesis) and the purity can be determined by polyacrylamide or agarose gel electrophoresis, sequencing analysis, and the like. Polynucleotides also can be isolated using hybridization or computer-based techniques that are well known in the art.
  • Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening of polypeptides expressed by DNA sequences (e.g., using an expression library); (3) polymerase chain reaction (PCR) of genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.
  • PCR polymerase chain reaction
  • Chimeric polypeptides, polynucleotides, and expression vectors containing same of the present invention can be encapsulated within liposomes using standard techniques and introduced into cells or whole organisms. Cationic liposomes are preferred for delivery of polynucleotides.
  • the use of liposomes for introducing various compositions in vitro or in vivo, including proteins and polynucleotides, is known to those of skill in the art (see, for example, U.S. Patent Nos. 4,844,904, 5,000,959, 4,863,740 and 4,975,282).
  • Liposomes can be targeted to a cell type or tissue of interest by the addition to the liposome preparation of a ligand, such as a polypeptide, for which a corresponding cellular receptor has been identified.
  • a ligand such as a polypeptide
  • CD4+ cells are an appropriate target and HIV gpl20 could be an appropriate ligand for intracellular introduction of a liposome containing a chimeric polypeptide or polynucleotide sequence as described herein.
  • Monoclonal antibodies can also be used for targeting; many such antibodies specific for a wide variety of cell surface proteins are known to those skilled in the art and are available.
  • the selected ligand is covalently conjugated to a lipid anchor in either preformed liposomes or are incorporated during liposome preparation (see Lee & Low, J Biol. Chem., 269:3 198 (1994); Lee & Low Biochem. Biophys. Actu, 1233: 134 (1995)).
  • the chimeric polypeptides described herein can be used to generate additional reagents, such as antibodies.
  • Invention antibodies are useful in the various treatment methods set forth herein.
  • the antibody produced in an immunized subject can protect the subject against virus infection or, alternatively, be transferred to a recipient subject, thereby passively protecting the second subject against infection.
  • Antibodies that bind to an epitope exposed upon complex formation between a virus coat polypeptide sequence and a receptor polypeptide sequence also can be generated.
  • antibodies that bind to chimeric polypeptides including antibodies specific for cryptic epitopes exposed upon complex formation as set forth herein, are provided.
  • the antibody neutralizes multiple viral isolates and viruses from different geographic clades (termed “broadly neutralizing") in vitro.
  • the antibody inhibits, prevents, or blocks virus infection in vitro or in vivo.
  • the virus neutralized is an imnmnodeficiency virus, including the HIV-I and HIV-2 immunodeficiency viruses set forth herein.
  • Antibody comprising polyclonal antibodies, pooled monoclonal antibodies with different epitopic specificities, and distinct monoclonal antibody preparations, also are provided.
  • Antibodies to chimeric polypeptide are produced by administering a chimeric polypeptide to an animal.
  • the antibodies can be produced, isolated, and purified using methods well-known in the art.
  • the invention provides methods for producing an antibody to a chimeric polypeptide of the present invention.
  • a method of the invention includes administering a chimeric polypeptide to a subject and isolating the antibodies that bind to the chimeric polypeptide.
  • the generated antibody binds to a cryptic epitope exposed upon the binding between a virus coat polypeptide sequence and a receptor polypeptide sequence.
  • antibodies bind to cryptic epitopes exposed when the virus coat polypeptide sequence (e.g., envelope polypeptide sequence) and the receptor polypeptide sequence bind to each other.
  • virus coat polypeptide sequence e.g., envelope polypeptide sequence
  • the HIV envelope polypeptide sequence gpl20 exposes a cryptic epitope upon binding to CD4 receptor polypeptide sequence, and antibodies to the exposed epitope can lead to broad neutralization of HIV.
  • Such epitopes may be shared among different viral isolates and geographic clades accounting for broad-spectrum neutralizing activity of the antibodies directed to these epitopes.
  • epitopic determinants refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the term “cryptic” refers to a property or feature that requires a structural or conformational change for the feature or property to become apparent; in the absence of the change, the feature or property is "hidden.” Cryptic epitopes may be present on either virus coat protein or receptor polypeptide sequences.
  • antibody includes intact molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding to an epitopic determinant present in a chimeric polypeptide described herein. Other antibody fragments are included, so long as the fragment retains the ability to selectively bind with its antigen.
  • Antibody fragments (e.g., Fab, F(ab') 2 , and Fv) of the present invention can be prepared by proteolytic hydrolysis of the antibody, for example, by pepsin or papain digestion of whole antibodies.
  • Antibodies that bind to disclosed chimeric polypeptides can be prepared using intact chimeric polypeptide or fragments thereof as the immunizing antigen.
  • the virus coat polypeptide sequence and the receptor polypeptide sequence maintain the ability to bind each other so that any cryptic epitopes present will be exposed.
  • the chimeric polypeptide used to immunize an animal is derived from translated polynucleotide or is chemically synthesized and, if desired, can be conjugated to a carrier.
  • Such commonly used carriers chemically coupled to the immunizing peptide include, for example, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • Monoclonal antibodies are made by methods well-known to those skilled in the art (Kohler et al, Nature, 256:495 (1975); and Harlow et al, Antibodies: A Laboratory Manual, p. 726, eds. Cold Spring Harbor Pub. (1988), which are incorporated herein by reference).
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by analyzing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques, which include, for example, affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan et al, "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," In: Current Protocols in Immunology. ⁇ 2.7.1-2.7.12 and ⁇ 2.9.1-2.9.3; and Barnes et al, "Purification of Immunoglobulin G (IgG),” In: Methods in Molecular Biology, Vol. 10, pp. 79-104, Humana Press (1992)).
  • affinity chromatography with Protein-A Sepharose size-exclusion chromatography
  • ion-exchange chromatography see, e.g., Coligan et al, "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamster
  • antibodies to a chimeric polypeptide produced in one species can be humanized so that the antibody does not induce an immune response when administered to the host, for example, for passive immunization.
  • humanized antibodies are produced by replacing a non-human constant region with a human constant region.
  • Such antibody humanization methods are known in the art and are particularly useful in the methods of the invention (Morrsion et al, Proc. Natl. Acad. Sd. USA, 81:685 1 (1984); Takeda et al., Nature, 314:452 (1985); Singer et al, J. Immunol, 150:2844 (1993)).
  • Antibodies that bind a chimeric polypeptide can neutralize the virus in vitro or in vivo (i.e., in a subject). Such antibodies can therefore prevent or inhibit virus infection in vitro or in vivo, and may ameliorate some or all of the symptoms associated with the infection. Such antibodies can be produced in one subject and then introduced into another, i.e., for passive immunotherapy. Alternatively, antibodies that bind chimeric polypeptides, when produced in a subject, can protect that subject from infection or ameliorate some or all of the symptoms associated with the infection.
  • a method of the invention includes administering an effective amount a Gl phase arresting agent that reduces expression or activation of CCR5 in combination with an antibody that binds to gpl20, CD4 or a complex formed between gpl20 and CD4 to a subject in need of such treatment, thereby preventing or inhibiting virus infection in the subject.
  • the "effective amount” will be sufficient to inhibit, prevent, or ameliorate a viral infection in a subject, or will be sufficient to produce an immune response in a subject.
  • an effective amount of chimeric polypeptide can be that which elicits an immune response to the polypeptide or a virus upon which the coat protein is based.
  • An effective amount administered to a subject already infected with the virus can also be that which decreases viral load, or increases the number of CD4 + cells.
  • An effective amount can be that which inhibits transmission of the virus from an infected subject to another (uninfected or infected).
  • a method of the invention includes administering an effective amount of a Gl phase arresting agent in combination with a chimeric polypeptide of the present invention to a subject, thereby producing an immune response sufficient for preventing or inhibiting virus infection in the subject.
  • a method of the invention includes administering to a subject an effective amount of a polynucleotide encoding an invention chimeric polypeptide in combination with a Gl phase arresting agent.
  • compositions of the present invention include any Gl cytostatic agent that arrests, delays or prolongs cell-cycle activity in the Gl phase and/or Gl-S interface of mononuclear cells and reduces expression of CCR5.
  • Gl cytostatic agent disrupts the response of a lymphocyte to EL-2 (through the IL-2R) which governs the transition from Gl to S phase, as well as the progression through S phase.
  • Gl cytostatic agents may include, but are not limited to, sodium butyrate, aphidicolin, hydroxyurea (HU), olomoucine, roscovitine, tocopherols, tocotrienols, rapamycin (RAPA) and/or functional analogs thereof.
  • the composition comprises rapamycin, which inhibits the T cell response to IL-2, the substance which triggers T cells already activated by the TCR to progress through Gl. Rapamycin therefore stops the cell at the Gl-S transition.
  • the composition comprises an effective amount of RAPA to disrupt the response of a lymphocyte to IL-2 (through the IL-2R) which governs the transition from Gl to S phase thereby causing a reduction of CCR5 expression and concomitantly reducing receptor sites for entry of HIV.
  • the present invention employs one of the above-identified Gl cytostatic agent for administration to a subject in combination with a chimeric polypeptide comprising a ligand/spacer/receptor binding complex, wherein the Gl cytostatic agent down-regulates the expression of CCR5 receptors and/or slows the fusion kinetics of HIV gpl20 to receptor binding sites thereby allowing for more time or spatial area for effective binding of the neutralizing antibodies to the CD4i epitope.
  • the present invention provides compositions comprising at least one Gl cytostatic agent in combination with a chimeric polypeptide of the present invention, nucleotide sequence encoding such chimeric polypeptides or antibodies raised by such chimeric polypeptides.
  • the two compounds can be administered, separately, simultaneously, concurrently or sequentially.
  • Doses to be administered are variable according to the Gl cytostatic agent, the chimeric polypeptide, the treatment period, frequency of administration, the host, and the nature and severity of the infection. The dose can be determined by one skilled in the art without an undue amount of experimentation.
  • compositions of the invention are administered in substantially non-toxic dosage concentrations sufficient to ensure the release of a sufficient dosage unit of the present combination into the patient to provide the desired inhibition of the HIV virus.
  • the actual dosage administered will be determined by physical and physiological factors such as age, body weight, severity of condition, and/or clinical history of the patient.
  • the active components are ideally administered to achieve in vivo plasma concentrations of about 0.001 wM to about 100 «M, more preferably about 0.01 to 10 «M.
  • the methods of the present invention may use compositions to provide from about 0.0001-500 mg/kg body weight/day of the chimeric polypeptide, more preferably from about 0.001-200 mg/kg/day, and most preferably .01-50 mg/kg/day; and from about 0.0001-1000 mg/kg body weight/day of a Gl cytostatic agent (Gl phase arresting), more preferably from about 0.001-1000 mg/kg/day, or most preferably from about 0.5-50 mg/kg/day.
  • Gl cytostatic agent Gl phase arresting
  • Particular unit dosages of a Gl cytostatic agent and an antiviral agent of the present invention include lmg, 5mg, 25mg, 50 mg, 100 mg, 200 mg, 500 mg, and 1000 mg amounts, for example, formulated separately, or together as discussed infra. It will be understood, however, that dosage levels that deviate from the ranges provided may also be suitable in the treatment of a given viral infection.
  • Therapeutic efficacy of the combination of the Gl cytostatic agent and chimeric polypeptides of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The LD50 (The Dose Lethal To 50%
  • the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds, which exhibit large therapeutic indexes, are preferred.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions of the present invention may comprise both the above-discussed components of the chimeric peptides and Gl cytostatic agent, together with one or more acceptable carriers thereof and optionally other therapeutic agents.
  • Each carrier must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • the terms "pharmaceutically acceptable” and “physiologically acceptable” refer to carriers, diluents, excipients, and the like that can be administered to a subject, preferably without excessive adverse side effects (e.g., nausea, headaches, etc.).
  • Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media.
  • Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial, anti-oxidants, chelating agents, and inert gases and the like.
  • Various pharmaceutical formulations appropriate for administration to a subject known in the art are applicable in the methods of the invention (e.g., Remington's Pharmaceutical Sciences. 18 th ed., Mack Publishing Co., Easton, PA (1990); and The Merck Index. 12 th ed., Merck Publishing Group, Whitehouse, NJ (1996)).
  • Controlling the duration of action or controlled delivery of an administered composition can be achieved by incorporating the composition into particles or a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers.
  • the rate of release of the composition may be controlled by altering the concentration or composition of such macromolecules.
  • Colloidal dispersion systems include macromolecule complexes, nano- capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the present invention provides a method for the treatment or prophylaxis of a viral infection such as retroviral infections which may be treated or prevented in accordance with the invention including human retroviral infections such as human immunodeficiency virus.
  • a viral infection such as retroviral infections
  • human retroviral infections such as human immunodeficiency virus.
  • the combination of Gl cytostatic agent and chimeric polypeptides compounds, compositions and methods according to the invention are especially useful for the treatment of ADDS and related HIV-positive conditions.
  • compositions according to the present invention may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the infection and the chosen active ingredient.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, caches or tablets, each containing a predetermined amount of the ingredients; as a powder or granules; as a solution or a suspension in an aqueous or nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the one or more of the compounds of the present invention, such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multidose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the drug combination of the present invention may be, for example, administered approximately after 30 weeks of pregnancy and continued through delivery. Interventions around the time of late gestation and delivery (when the majority of transmissions are thought to occur) are most efficacious.
  • the present invention relates to use of the combination of the Gl cytostatic agent and a chimeric polypeptide, nucleotide sequence encoding such polypeptide or antibodies generated in response to such polypeptide in the manufacture of a medicament or pharmaceutical for the treatment of a viral disease such as HIV, SFV, FIV, FeLV, FPV, and a herpes virus.
  • a viral disease such as HIV, SFV, FIV, FeLV, FPV, and a herpes virus.
  • Rapamycin (RAPA) and the CD4-gpl20 complex (FLSC) have been previously described as having anti-HIV-1 activity. Potential side effects that may arise upon administration of RAPA or FLSC might be avoided or reduced by lowering drug concentrations.
  • RAPA potentiates the antiviral activities (both coreceptor antagonist and antibody neutralization activities) of the FLSC. This enhancing effect allows for in vivo use of lower concentrations of RAPA and the FLSC, thereby overcoming possible toxicities that could arise at higher drug concentrations.
  • the RAPA/FLSC combination targets a step of the virus life cycle different than the ones targeted with currently approved antiretrovirals.
  • the RAPA/FLSC combination provides a new kind of antiviral treatment in HIV-I infection.
  • the CD4-gpl20 full-length single chain vaccine complex is a HIV-I vaccine candidate that elicits production of neutralizing antibodies.
  • the FLSC comprises a gpl20 polypeptide linked to a CD4 receptor polypeptide through a spacer of amino acid residues.
  • the FLSC is a CCR5 antagonist that inhibits virus entry by binding to the CCR5 coreceptor.
  • Rapamycin Rapamycin
  • infectivity assays were performed in the presence of RAPA and FLSC, alone and in combination.
  • the infectivity assays were performed using PBMCs from healthy donors. PBMCs were cultured for 7 days in the presence of IL-2 (100 U/ml) in the presence or absence of RAPA. On day 7, cells were infected by exposure to the R5 strain HIV-I ADA (m.o.i of 0.001) for 2 h. Non-adsorbed virus was removed by 3 washes with PBS. Infected cells were then plated in RPMI-10 supplemented with IL-2 (100 U/ml) in the presence of different concentrations of RAPA and FLSC.
  • the computer calculates the values of the dose required for 50 % inhibition of viral replication (IC50 or Dm), the coefficient of the sigmoidicity of the dose-effect curve (m), the linear correlation coefficient of the median- effect plot (r), and the combination index (CI).
  • Table 2
  • Fa Fractional inhibition
  • m slope coefficient of the curve
  • Dm dose at 50 % inhibition (equivalent to IC50 value)
  • r linear correlation coefficient of the median effect plot.
  • the combination index (CI) value was calculated.
  • the CI value determines the degree and nature of the drug 5 interaction. CI values were calculated at 50, 75, 90 and 95 % inhibition levels of viral replication, and indicated that the combination is synergistic (Table 3).
  • values for the dose reduction index (DRT) were calculated (Table 3). The DRI helps to determine dose reduction, which may lead to less toxicity and improvement of therapeutic efficacy.
  • CI Combination Index at combination ratio of RAPA + FLSC-Ig of 1 : 1.
  • CI ⁇ 1 indicates synergy
  • CI 1 indicates additivity
  • CI >1 indicates antagonism.
  • DRI Dose Reduction Index. Indicates fold of dose reduction for each drug in combination, for a given degree of inhibition, when compared with the dose of each drug alone for the same degree of inhibition.
  • MacSynergy II analysis of the antiviral data obtained using the RAPA and FLSC combination is depicted on Fig 1. Synergistic interactions were observed across the entire concentration grid with a calculated synergy volume of 251.68 (95 % CL, 142-362).
  • Figure 4 shows the results of RAPA enhancement of the neutralizing activity of the CD4i monoclonal antibody 17b.
  • PBMCs were cultured for 7 days in the presence of IL-2 and
  • Figure 5 shows the results of RAPA enhancement of the neutralizing activity of the CD4i monoclonal antibody X5.
  • PBMCs were cultured for 7 days in the presence of IL-2 and RAPA and were exposed to HTV-I ADA (moi of 0.001) for 2 hours in the presence of the indicated concentrations of mAb X5.
  • Infected cells were cultured in the presence of RAPA and X5.
  • culture medium was replaced with fresh IL-2 medium containing RAPA and X5 at the same concentration as before.
  • virus production was measured by p24. Note the log scale used on the Y axis. It is very evident that with the inclusion of RAPA there was significant reduction in HIV infection because of the reduction in the p24.
  • This Example describes the construction of polynucleotides encoding a single chain gpl20- CD4 chimeric polypeptide FLSC, TsSC, FLSC-R/T and RLSC-R/T CD4M9, as shown in Figure 6.
  • the strategy for building a single chain complex is based on the placement of a 20 to 30 amino acid linker sequence between the C terminus of gpl20 and the N terminus of CD4. Analyses of the crystal structure of modified gpl20 bound to soluble CD4 and 17b Fab (Dwong, P.D.
  • a single chain nucleic acid encoding a gpl20-CD4 chimeric polypeptide was constructed by arranging the respective coding sequences in the following order: (1) at the 5' end, a synthetic, codon encoding gpl20 of the macrophage-tropic HIVs, BaL; (2) a sequence encoding a 20 amino acid linker consisting of glycines, alanine, and serines; (3) sequences for soluble CD4 domains 1 and 2 (D1D2); and (4) at the 3' end, sequences encoding a short polypeptide derived from the c-myc oncogene for FLSC.
  • FLSC-R/T nucleotide sequence (SEQ ID NO: 3) encodes for a protein having a mutation at the c-terminal end of gpl20 wherein the arginine is replaced with a threonine (SEQ TD NO: 4).
  • FLSC-R/T CD4M9 (SEQ E) NO: 5) includes further changes in the nucleotide sequence of a chimera polypeptide (SEQ ID NO: 6) of the present invention wherein the CD4 D1D2 region is replaced with a sequence coding for CD4M9 that encodes for a peptide that mimics the functional activity of the CD4 D1D2 region.
  • the codon optimized gpl20 sequence was used as it permits high-level expression in a rev-independent manner (Haas, J., et at, Curr. Biol, 6:3 15-24 (1996)).
  • the human CD4 sequence used was derived from T4-pMV7 (Maddon, P. J., et al, Cell, 47:333-48 (1986); NlH AIDS Reagent Repository, Bethesda, MD).
  • the myc polypeptide sequence allows convenient analyses, purification, and other manipulation of the chimeric polypeptide.
  • FLSC molecule was constructed via PCR using the plasmids pMRlWl-9 and T4- pMV7 as templates.
  • the gpl20 forward primer was GGG-GGT-ACC-ATG-CCC-ATG- GGG-TCT-CTG-CAA-CCG-CTG-GCC (SEQ ID NO:7) and the reverse primer was GGG- TCC-GGA-GCC-CGA-GCC-ACC-GCC-ACC-AGA-GGA-TCC-ACG-CTT-CTC-GCG-
  • the CD4 forward primer was GGG- TCC-GGA-GGA-GGT-GGG-TCG-GGT-GGC-GGC-GCG-GCC-GCT-AAG-AAA-GTG- GTG-CTG-GGC-AAA-AAA-GGG-GAT (SEQ ID NO:9) and the reverse primer was GGG- GTT-TAA-ACT-TAT-TAC-AGA-TCC-TCT-TCT-GAG-ATG-AGT-TTT-GTT-CAG-CTA- GCA-CCA-CGA-TGT-CTA-TTT-TGA-ACT-C (SEQ ID NO: 10).
  • the PCR product was subcloned into pEF6 (Invitrogen, Carlsbad, CA) using Kpn ⁇ and Pmel restriction sites.
  • the full-length gpl20 expressing sequence in pEF6- FLSC was exchanged for a truncated version of the gp 120 sequence (DC1DC5DV1V2).
  • the truncated gpl20 was generated using GGG-GGT-ACC-ATG-CCC-ATG-GGG-TCT-CTG- CAA-CCG-CTG-GCC-ACC-TTG-TAC-CTG-CTG-GGG-ATG-CTG-GTC-GCT-TCC- TGC-CTC-GGA-AAG-AAC-GTG-ACC-GAG-AAC-TTC-AAC-ATG-TGG (SEQ ID NO: 15) as a forward primer and GGG-GGA-TCC-GAT-CTT-CAC-CAC-CTT-GAT-CTT- GTA-CAG-CTC (SEQ ID NO: 16) as a reverse primer.
  • Vl and V2 regions were deleted using CTG-TGC-GTG-ACC-CTG-GGC-GCG-GCC-GAG-ATG-AAG-AAC-TGC-AGC- TTC-AAC-ATC-GGC-GCG-GGC-CGC-CTG-ATC-AGC-TGC (SEQ ID NO: 17) as a forward primer and GCA-GCT-GAT-CAG-GCG-GCC-CGC-GCC-GAT-GTT-GAA-GCT- GCA-GTT-CTT-CAT-CTC-GCC-CGC-GCC-CAG-GGT-CAC-GCA-CAG (SEQ ID NO: 18) as a reverse primer.
  • the CD4M9 sequence (SEQ E) NO: 19) used to clone into FLSC R/T CD4M9 was generated by using the 5' to 3 ' primers GCG-GCC-GCT-TGC-AAC-CTG-GCC-CGC-TGC- CAG-CTG-CGC-TGC-AAG-AGC-CTG-GGC-CTG-CTG-GGC-AAG-TGC-GCC-GGC- AGC-TTC-TGC-GCC-TGC-GGC-CCC-TAA-GAA-TTC (SEQ ID NO: 21) as a forward primer and GAA-TTC-TTA-GGG-GCC-GCA-GGC-GCA-GAA-GCT-GCC-GGC-GCA- CTT-GCC-CAG-CAG-GCC-CAG-GCT-CTT-GCA-GCG-CAG-CTG-GCA-GCG-GGC- CAG-GTT-GCA-AGC
  • Fragments were cut with Notl & BamHl, then subcloned into pEF6-FLSC R/T that had been prepared by cutting with Notl & BamHl and gel purified to remove the relieved hDlD2 from the FLSC R/T sequence. Clones were confirmed by sequencing.
  • the recombinant constructs are shown in FIG. 1.
  • the chimeric recombinant which contained the BaL gpl20 (SEQ ID NO: 24) sequence with a spacer region (SEQ ID NO: 11) and CD4D1D2 region (SEQ ID NO: 26) was designated full-length single chain (FLSC).
  • FLSC full-length single chain
  • a second construct was designed to produce complexes more closely resembling the molecules used to solve the gpl20 crystal structure. This construct was designated truncated single chain (TcSC) and constructed as with FLSC except that a sequence encoding ⁇ C1 ⁇ C5 ⁇ V1V2 gpl20 was used in place of the full length coding sequence (SEQ ID NO: 28).
  • constructs designated FLSC-R/T wherein the BaL gpl20 is mutated at amino acid 506 (SEQ ID NO: 30) and FLSC-R/T CD4M9 comprising sequences SEQ ID NO: 30 and 20.
  • the amino acid sequence of the spacer region shown in this example is GSSGGGGSGSGGGGSGGGAAA (SEQ ID NO: 11) (encoded by SEQ ID NO: 33).
  • Figure 7 shows the results that indicate that an indirubin derivative acts as a Gl phase arresting agent.
  • PBMCs from 2 normal donors were activated in absence and presence of 4 ⁇ M Indirubin-3'-momoxime (IM) for 24 hours.
  • IM Indirubin-3'-momoxime
  • Cells were then fixed with 70 % Ethanol, stained with Propidium Iodide, and analyzed by Flow cytometry. Percentages of cells in each phase of the cell cycle are shown. In the presence of IM, the % of cells in Gl increases and this cycle arrest leads to lower pecentages of cells in S and G2 phases as compared to the untreated control.
  • IM Indirubin-3'-monoxime

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Abstract

L'invention se rapporte à un traitement combiné comprenant des médicaments et/ou des anticorps dirigés contre l'enveloppe virale, combinés à des agents cytostatiques G1 ayant pour activité fonctionnelle de réduire la transcription de CCR5 et entraînant ainsi une réduction du nombre de récepteurs de surface permettant de fixer la glycoprotéine gpl20 du VIH.
PCT/US2006/027584 2005-07-15 2006-07-17 Agents cytostatiques g1 renforçant l'activite antivirale des medicaments et anticorps diriges contre l'enveloppe virale WO2007011827A2 (fr)

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Citations (2)

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US20020155121A1 (en) * 1999-10-08 2002-10-24 Devico Anthony Louis Virus coat protein/receptor chimeras and methods of use
US20050124645A1 (en) * 1998-04-20 2005-06-09 Finkel Terri H. Methods and compositions for increasing CD4lymphocyte immune responsiveness

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050124645A1 (en) * 1998-04-20 2005-06-09 Finkel Terri H. Methods and compositions for increasing CD4lymphocyte immune responsiveness
US20020155121A1 (en) * 1999-10-08 2002-10-24 Devico Anthony Louis Virus coat protein/receptor chimeras and methods of use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DE ROSNY ET AL.: 'Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes.' J. VIROL. vol. 78, no. 5, March 2004, pages 2627 - 2631 *

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