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WO1991013911A1 - Effet de blocage de l'inhibiteur peptidique de l'infection causee par le virus de l'immunodeficience humaine sur les interactions entre le virus et un nouveau recepteur cellulaire - Google Patents

Effet de blocage de l'inhibiteur peptidique de l'infection causee par le virus de l'immunodeficience humaine sur les interactions entre le virus et un nouveau recepteur cellulaire Download PDF

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Publication number
WO1991013911A1
WO1991013911A1 PCT/US1991/001549 US9101549W WO9113911A1 WO 1991013911 A1 WO1991013911 A1 WO 1991013911A1 US 9101549 W US9101549 W US 9101549W WO 9113911 A1 WO9113911 A1 WO 9113911A1
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cells
hiv
receptor
hsa
binding
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PCT/US1991/001549
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English (en)
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Lee Alan Henderson
David H. Coy
Robert Francis Garry, Jr.
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Administrators Of The Tulane Educational Fund
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to methods of inhibiting human immunodeficiency virus-mediated cell killing and infection which inhibit interaction between a viral protein and a novel specific cellular receptor.
  • HIV Human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • ARC AIDS related complex
  • the HIV virion or virus particle is a sphere that is roughly 1000 angstrom units across.
  • the particle is covered by a lipid bilayer membrane derived from the outer membrane of the infected host cell. Studding the viral membrane is an envelope glycoprotein which is synthesized as a precursor of 160 kd and subsequently processed into two glycoproteins: gp41 which spans the lipid bilayer, and gpl20 which extends beyond the lipid bilayer.
  • the envelope covers a core made up of proteins designated p24 and pl8.
  • the viral RNA is carried in the core, along with several copies of the enzyme, reverse transcrlptase, which catalyzes the assembly of viral DNA.
  • the HIV genome contains three genes that encode the components of retrovirus particles: env (which codes 5 for the envelope proteins) , gag (which codes for the core proteins) , and pol (which codes for reverse transcrlptase) . These three genes are flanked by stretches of nucleotides called long terminal repeats (LTRs) .
  • LTRs include sequences that have a role in 0 controlling the expression of viral genes.
  • the genome of HIV includes at least five additional genes, three of which have known regulatory functions, and the expression of which is thought to have an impact on the pathogenic mechanisms 5 exerted by the virus.
  • the tat gene encodes a protein that functions as a potent trans-activator of HIV gene expression, and, therefore, plays an important role in the amplification of virus replication.
  • the rev, or trs/art gene can upregulate HIV synthesis by a transacting antirepression mechanism; rev enables the integrated HIV virus to selectively produce either regulatory proteins or virion components.
  • the nef, or 3'-orf, gene appears to down-regulate virus expression by producing a cytoplasmic protein which, presumably via a second messenger, inhibits transcription of the HIV genome.
  • the vif, or sor gene is not essential for virion formation, but is critical to the efficient generation of infectious virions and influences virus transmission in vitro.
  • the pr, or R gene encodes an immunogenic protein of unknown function.
  • T lymphocytes which express the CD4 antigen
  • the envelope glycoprotein appears to play an important role in the entry of HIV into CD4 positive host cells.
  • the gpl20 portion has been shown to bind directly to the cellular CD4 receptor molecule, thereby producing HIV's tropism for host cells that express the CD4 receptor, e.g. , T helper cells (T4 cells) , macrophages, etc.
  • the virus After HIV binds to the CD4 molecule, the virus is internalized and uncoated. Once internalized, the genomic RNA is transcribed into DNA by the enzyme reverse transcriptase. The proviral DNA is then integrated into the host chromosomal DNA and the infection may assume a "dormant" or latent phase. However, once activation occurs, the proviral DNA is transcribed. Translation and post translational processing results in virus assembly and budding of mature virions from the cell surface.
  • CD4+ cell When active replication of virus occurs, the host CD4+ cell is usually killed, but some cells may persistently produce virus and are not killed.
  • CD4 cell killing has been observed with exposure to inactivated virus.
  • a number of mechanisms for the im unopathogenesis and cytopathic effect of HIV infection have been proposed: the accumulation of large amounts of unintegrated viral DNA in the infected cells; massive increase in permeability of the cell membrane when large amounts of virus bud off the cell surface; speculations that HIV may induce terminal differentiation of infected T4 cells, leading to a shortened life span.
  • HIV inhibitory peptides currently being studied include peptides which interact with the HIV-1 protease (Bellich et al., 1988, J. Biol. Chem. 263:17905-
  • HIV-1 protease inhibitors HIV-1 protease inhibitors.
  • HIV transmembrane protein gp41 has been associated with immunosuppression.
  • Cauda et al. (1988, Cell 5 Immunol. 115:57-65) reports that natural killer cell activity appeared to be inhibited by synthetic peptides corresponding to amino acid residues 735-752 and 846-860 of
  • CS-3 derived from gp41 and having the amino acid sequences 0 LQARILAVERYLKDQQL
  • CS-3 derived from gp41 and having the amino acid sequences 0 LQARILAVERYLKDQQL
  • HIV infections contending that these peptides disrupt the interaction between gp41 and gpl20 envelope proteins of HIV particles.
  • Antibodies directed toward viral peptides are also being considered as therapeutic options.
  • Rusche et al. (1988, Proc. Natl. Acad. Sci. U.S.A. West:3198-3202) relates to antibodies which bind to a 24 amino acid sequence of gpl20 and thereby inhibit fusion of HIV-infected cells.
  • anti-idotype antibodies directed toward anti-CD4 antibodies, have been shown to bind to HIV virus in vitro, presumably by possessing protein configurations similar to CD4 determinants (Dalgleish et al., 1989, UCLA
  • the present invention relates to methods of inhibiting HIV-mediated cell killing and infection which comprises inhibiting the interaction between the CS3 region and viral gp41 and its cellular receptor.
  • the invention relates to the discovery that a 17 amino acid 0 region of HIV transmembrane glycoprotein gp41(TM), comprising amino acids 583-599 and denoted as CS3, binds to a unique cellular receptor.
  • the invention provides for methods which employ peptides, peptide derivatives, or ⁇ ntibodies to inhibit the CS3/CS3 receptor interaction.
  • the invention also relates to the CS3 receptor, which may be required for high affinity binding of HIV to cells.
  • the CS3 receptor or portions or derivatives thereof, or antibodies directed toward the receptor may be used to inhibit the virus/CS3 receptor interaction.
  • the CS3 receptor has been identified on the surface of neuroblastoma cells methods of blocking the CS3 receptor/HIV interaction may be used in the treatment of HIV-associated nervous system disorders.
  • the present invention also provides for an assay system to detect and/or quantitate HIV binding to cells.
  • the present invention is based in part on the discovery that a CS3 specific cellular receptor is widely distributed on human lymphocytes and forms a 108 kd complex with CS3-HSA peptide conjugate. It was further discovered that CS3 peptide effectively blocks HIV binding, infection, and virus-mediated cell killing. Therefore, the present invention provides for methods of treatment and prophylaxis of HIV infection as well as a means for better understanding the physiology of acquired immunodeficiency syndrome (AIDS) .
  • AIDS acquired immunodeficiency syndrome
  • CS3-HSA with RH9 cells A) HSA (EDC-treated) FITC control; (B) CS3-HSA-FITC 4 ⁇ g/10 6 cells; (C, D, & E)
  • CS3-HSA-FITC in the presence of 5 x CS3-HSA, 20 x CS3-HSA or 50 x DC7-HSA (DC7 [ASFDEREPYAH] coupled to HSA at similar ratios of peptide to HSA [11/1] respectively, as competing agents.
  • HIV virus and 20 ⁇ g/ml HSA (“HIV + HSA") ; (iv) HIV virus and CS3-HSA at 10 ⁇ g/ml (“HIV + CS3-HSA”) ; (v) HIV virus and 6.7 ⁇ g/ml antibody specific for CS3 ("HIV + anti- CS3"); or (vi) HIV virus and 6.7 ⁇ g/ml CS3 depleted anti-HIV IgG (“HIV + anti-HIV (-CS3)").
  • HIV + HSA HIV virus and CS3-HSA at 10 ⁇ g/ml
  • HIV virus and 6.7 ⁇ g/ml antibody specific for CS3 (“HIV + anti- CS3”
  • HIV virus and 6.7 ⁇ g/ml CS3 depleted anti-HIV IgG (“HIV + anti-HIV (-CS3)").
  • Figure 6 HIV virus bound to MOLT cells in the presence (A) or absence (B) of CS3-HSA.
  • C represents control cells, exposed to neither virus nor peptide. .
  • Figure 7 Expression of CS3 receptor, HLA-DR, T4, T8, T3, and Til on control cells, and cells treated with c-CS3- HSA or the irrelevant peptide DC7-HSA, as measured by fluorescence flow cytometry.
  • FIG. 1 Expression of CS3 receptor on PBMC activated with PHA or TPA.
  • Figure 9 Inhibition of HIV binding to CD4 cells by sCD4 and CS3-HSA.
  • sCD4 was added to aliquots of HIV, in 100 ⁇ l, for 30 minutes on ice prior to addition of 10 MT4 cells for an additional 30 minutes on ice.
  • CS3-HSA was added to cells for 30 minutes on ice prior to addition of HIV for an additional 30 minutes. Following this the incubations were layered over 6:1 mixture of silicon oil to fluid. Following icrofuge centrifugation for 30 5 seconds the tube was frozen in liquid N,. The cell pellet was snipped off and solubilized for the Abbott antigen capture assay.
  • Results represent an average of duplicate flasks from 5 which four fields were counted on a hemocytometer. The number of cells in duplicate flasks varied by less than
  • PBMC peripheral blood mononuclear cells
  • TPA mitogenic phorbol ester, 10 ng/ml
  • 5 cultured in medium without mitogen for 48 hours before harvesting and analyzing for expression of CS3 receptor l ⁇ g/ml or TPA (a mitogenic phorbol ester, 10 ng/ml) or 5 cultured in medium without mitogen for 48 hours before harvesting and analyzing for expression of CS3 receptor.
  • CS3 binding (right panel) or CS3 binding (right panel). .50% of RH9 35 cells bound CS3-HSA-FITC. Relative to control cultures treatment with anti-CD3 increased CS3 domain expressing cells by 71% at 48 hours and 56% at 72 hours, while TPA increased CS3-binding by 32% at 72 hours. CS3-HSA-FITC fluorescence intensity, reflecting the number of receptors per cell, increased while CD4 expression decreased in intensity and number of positive cells (8% at 24 hours) .
  • Figure 13 Schematic diagram of HIV cell binding assay.
  • the present invention relates to methods of inhibiting HIV-mediated cell killing and infection which comprise inhibiting the interaction between the CS3 region of viral gp41 and a novel cellular receptor.
  • methods of inhibiting HIV-mediated cell killing and infection comprise inhibiting the interaction between the CS3 region of viral gp41 and a novel cellular receptor.
  • any peptide or protein which inhibits the interaction between the CS3 region of viral gp41 (or its homologue in other retroviruses, including those which infect human as well as nonhuman hosts, may be used according to the invention.
  • these inhibitors may include peptides related to the CS3 region of gp41, or derivatives thereof, as well as peptides or proteins which are identical or homologous to the CS3 receptor, or portions thereof.
  • Antibodies may also be used, and are discussed in section 5.2, infra.
  • Peptides related to the CS3 region of gp41 are, according to the invention, identical or homologous to the amino acid sequence LQARILAVERYLKDQQL, or a portion thereof or, alternatively, to a homologous peptide sequence associated with another virus, including, but not limited to, HIV-2, in which the corresponding amino acid sequence is substantially LQARVTAIEKYLQD A.
  • Peptides related to the CS3 region may comprise at least three sequential residues of LQARILAVERYLKDQQL, or a homologous peptide, but preferably comprise at least 8 residues, and most preferably all 17 residues of this sequence.
  • the term CS3-related peptides should be construed to mean peptides in which amino acids are substituted by functionally equivalent amino acids (see infra) as well as derivatives of these peptides, including but not limited to benzylated derivatives, glycosylated derivatives, and peptides which include enantiomers of naturally occurring amino acids.
  • the CS3 peptides, related peptides or derivatives are linked to a carrier molecule such as a protein, including but not limited to,
  • CS-3 related peptides comprising additional amino acids may also be used according to the invention.
  • a cysteine residue may be added to the N-terminal portion of the peptide.
  • Peptides may be produced from naturally occurring or recombinant viral proteins, or may be produced using standard recombinant DNA techniques (e.g. the expression of peptide by a microorganism which contains recombinant nucleic acid molecule encoding the desired peptide, under the control of a suitable transcriptional promoter, and the harvesting of desired peptide from said microorganism) .
  • the peptides of the invention may be synthesized using any methodology known in the art, including but not limited to Merrifield solid phase synthesis (Clark-Lewis et al., 1986, Science
  • CS3 receptor may be isolated and characterized using any method known in the art based on the discovery of the present invention that the CS3 region of gp41 binds to a specific cellular receptor.
  • CS3 receptor may be isolated from extracts of lymphocyte cell membranes either by affinity chromatography, in which CS3 peptide is bound to a solid support, or by preparative SDS-polyacrylamide gel electrophoresis, in which gel slices containing the CS3 receptor are identified by allowing labeled CS3 peptide to bind to the receptor.
  • the CS3 receptor forms a complex with 125I labelled CS3-HSA that has an apparent molecular weight of 108 kd.
  • CS3 receptor may be freed from cross-linked peptide, or may be separated from other proteins by polyacrylamide gel electrophoresis and then identified by subsequent binding to labelled CS3.
  • CS3 may require further purification before it may either be used according to the methods of the invention, or, preferably, be subjected to amino acid sequencing (e.g. Hewick, 1981, J. Biol. Chem. 256:7990- 7997) .
  • Oligonucleotide probes corresponding to amino acid sequence thus obtained may then be generated by standard techniques, and then used to identify cDNA or genomi ⁇ clones encoding the CS3 receptor using standard techniques including polymerase chain reaction (Saiki et al., 1985, Science 230:1350-1354) .
  • CS3 receptor may then be produced in quantity using standard expression systems, including, but not limited to, vaccinia virus and baculovirus expression vector systems.
  • anti-CS3 receptor antibody may be generated as described below and then used to clone the CS3 receptor gene by polysome precipitation.
  • the CS3 receptor gene may be cloned by a
  • CS3 receptor or derivatives or portions thereof, may be utilized according to the invention.
  • Useful CS3 related peptides and useful portions or derivatives of the CS3 receptor may be preferably identified using an assay system which can detect inhibition of the CS3 peptide/CS3 cellular receptor interaction.
  • an assay system which can detect inhibition of the CS3 peptide/CS3 cellular receptor interaction.
  • potentially useful CS3 related peptides and CS3 receptor related peptides may be tested for their ability to block HIV mediated cytopathicity of RH9 cells as described in Section 7, infra.
  • CS3 peptides may be tested for their ability to block HIV binding to cells.
  • the CS3 peptides or proteins, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence LQARILAVERYLKDQQL (or a homologous peptide, for example, the homologous peptide from HIV-2) including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged
  • (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • CS3 peptide is capable of partially activating lymphocytes (see Section 11, infra) , and upregulates expression of its own receptor, it may be useful to utilize CS3 related peptides which are not associated with partial lymphocyte action but which continue to block the virus/CS3 receptor interaction.
  • CS3 related peptides may comprise D-amino acids, or may comprise an inefficient carrier protein, or no carrier protein at all. It has been observed that CS3 unconjugated to HSA fails to partially activate lymphocytes.
  • CS3 and CS3 receptor proteins or fragments or derivatives thereof which are differentially modified during or after translation, e.g. , by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • the present invention also relates to antibodies which inhibit the interaction between CS3 and its cellular receptor.
  • Such antibodies may be directed toward, for example, epitopes in or near the CS3 peptide region of gp41, or in or near the binding site of the CS3 peptide region on the CS3 receptor.
  • the antibodies may be produced using, as immunogen, all or portions of gp41, the CS3 peptide region (LQARILAVERYLKDQQL or a homologous peptide) , or the CS3 receptor.
  • antiidiotype antibodies directed toward antibodies which interact with gp41 the CS3 peptide region (LQARILAVERYLKDQQL or a homologous peptide)
  • CS3 peptide, or the CS3 receptor may be inhibitory to the CS3 peptide region/CS3 receptor interaction.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature £56:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today :72), a method such as that described in Huse et al., 1989, (Science 246:1275-1281) and the EBV-hybridoma technique to produce human monoclonal antibodies Colde et al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp. 77-96) and the like are within the scope of the present invention.
  • the monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g. , Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
  • Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.
  • mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille
  • a molecular clone of an antibody to a gp41, CS3 or CS3 receptor epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
  • Antibody molecules may be purified by known techniques, e.g. , immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography) , or a combination thereof, etc.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include, but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab / )_ fragment, and the 2 Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • the CS3 related peptides, CS3 receptor proteins or peptides, or antibodies of the invention may be utilized to inhibit retrovirus mediated cell killing and may, accordingly, be used in the treatment of HIV infection and also in prophylaxis against HIV infection.
  • the peptides or antibodies of the invention may be administered to patients in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal.
  • the invention also provides for pharmaceutical compositions comprising CS3 related or CS3 receptor peptides, peptide fragments, or derivatives administered via liposomes, microparticles, or microcapsules.
  • compositions comprising CS3 related or CS3 receptor peptides, peptide fragments, or derivatives administered via liposomes, microparticles, or microcapsules.
  • it may be useful to utilize such compositions to achieve sustained release of CS3 related peptides or CS3 receptor peptides.
  • CS3-HSA may be used to inhibit HIV-mediated cell killing in a person or animal in need of such treatment.
  • CS3 may also be linked to another suitable molecular carrier other than HSA, or may be administered free of carrier. By blocking the cellular receptor for virus, infection of the target cells may be prevented.
  • the CS3 receptor, or portions or derivatives thereof relevant to the virus-CS3 receptor interaction may be used as decoys for viral attachment.
  • the CS3 receptor, portions or derivatives thereof may be administered to a person or animal in need of such treatment. It may be desirable to administer the CS3 receptor, or peptides or derivatives thereof, linked to a molecular carrier, for example, but not limited to, HSA.
  • the antibodies of the invention, directed toward CS3 or its receptor may be administered to persons or animals in need of such treatment.
  • the CS3 peptides, CS3 receptor, or antibodies may be administered to subjects who suffer from retroviral infection (e.g. acquired immunodeficiency syndrome or AIDS-related complex (ARC)) as well as to those at risk for retroviral infection.
  • retroviral infection e.g. acquired immunodeficiency syndrome or AIDS-related complex (ARC)
  • ARC AIDS-related complex
  • CS3 receptor As the CS3 receptor has been identified on the surface of neuroblast ⁇ ma cells methods of blocking the CS3 receptor/HIV interaction may be used in the treatment of HIV-associated nervous system disorders.
  • the CS3 peptides, CS3 receptor, and antibodies of the invention may also be used to study the mechanisms of retroviral infection and, additionally, lymphocyte activation. Defining the tissue distribution of the CS3 receptor may also be useful in identifying portals for virus entry in non-lymphocyte tissues. As shown in Example Section 12, a variety of cell lines have been observed to express the CS3 receptor indicating that the CS3 receptor may be expressed in multiple tissue types and human and nonhuman species. CS3 receptor has been observed on macrophages. The CS3 receptor may be the entrance for HIV into the central nervous system, via macrophages or via neurons themselves. The present invention also provides for an assay system to measure or detect HIV binding as exemplified in
  • a binding assay has been developed for viruses, particularly HIV, which has broad application for screening for antivirals (drugs, peptides and antibodies) that are potentially neutralizing by virtue of the ability to prevent binding of virus to target cells (See Figure 13) .
  • the binding assay comprises exposing cells to virus (e.g. HIV) in medium for a period of time sufficient to allow binding of virus to cells to occur, centrifuging the cells and virus through a high specific gravity liquid such as, preferably, silicon oil, freezing the media and cell pellet, separating the cell pellet from the frozen medium, and assaying the cell pellet for viral core protein.
  • virus e.g. HIV
  • a high specific gravity liquid such as, preferably, silicon oil
  • freezing the media and cell pellet separating the cell pellet from the frozen medium, and assaying the cell pellet for viral core protein.
  • This assay may be used to test the ability of a compound (e.g. a peptide) to block virus biding.
  • CS3-HSA may be incubated with
  • MT4 cells prior to the addition of HIV (5-6 infectious particles per cell) . Cells may then be centrifuged
  • the tube may then be frozen (for example in liquid N_) then the pellet snipped off to separate it from the frozen medium.
  • the cell pellet may then be assayed for p24 core antigen content using, for example, the Abbott assay or any suitable HIV assay system known in the art. For its use with HIV, freezing the tube is essential to prevent any
  • HIV from the medium from contaminating the cell pellet.
  • This assay may be used to identify compounds which block
  • HIV/cell binding such as compounds which block HIV binding to the CS3 receptor. 6.
  • EXAMPLE EXPRESSION OF CS3 RECEPTORS ON LYMPHOCYTES
  • Cysteine-CS3 which has the sequence CLQARILAVERYLKDQQL was coupled to human serum albumin as described (Ciancialo et al., 1988, Immunology Letters
  • C-CS3-HSA-FITC labelled 98% of RH9 cells at 4 ⁇ g/10 cells and further addition did not increase the fluorescence intensity.
  • C-CS3-HSA-FITC at 1 ⁇ g/10 RH9 showed minimal binding.
  • Jurkat cells and normal human peripheral blood T cells, B cells and mononuclear cells were also positive by flow cytometry.
  • PBMC subset analysis was performed by dual staining with C-CS3-HSA- Rho and fluorescein labelled monoclonal antibodies (Coulter Cytometry) to CD4, CD8, CD2 for T cells (T4, T8 and Til, respectively) , HLA-DR for B (12) and CDllb or CD14 for monocytes (M01 and M02, respectively).
  • a putative cell surface receptor which is specific for C-CS3 is broadly distributed on human lymphocytes and is represented on CD4 cell lines. Similar experiments with CS3-HSA yielded lower binding on RH9 cells (20-25%) , although as seen below, both are effective in inhibiting HIV mediated killing of RH9 and in cross-linking to receptor. The reason for this difference is unknown, but may be related to the ability of cysteine residues to stabilize peptide binding to receptors. Importantly, cys-CS3-HSA bound in a saturable manner and was specific as shown by competition assays
  • HIV stocks were prepared by
  • Results represent average of duplicate flasks from which 4 fields were counted on a hemacytometer. Difference in cell numbers in duplicate flasks was less than 5%.
  • the density of the putative receptor for cys-CS3 increased approximately 5 fold when PBMC were incubated for 48 hrs in the presence of PHA (1 ⁇ g/ml) or TPA (10 ⁇ g/ml) . This increase in surface density is less than increases in molecules such as CD4 and CD8 (10 fold) when stimulated in a similar manner, although the comparison is based upon fluorescence. Klutzman and Gluckman (1986,
  • Immunol. Today 1_: 2 1. have suggested that HIV binding occurs with both high and low affinity and that the high affinity binding, which represents perhaps 10% of HIV binding, leads to productive infection.
  • the high affinity binding may require the interaction of HIV with CD4 in addition to binding to a receptor for CS3. Thus, cell killing or infection may be blocked if this interaction is prevented.
  • CS3-HSA completely prevented HIV- mediated cell killing of RH9 cells in a dose range of 0.5 to 5 ⁇ g/ml.
  • Some enhancement of RH9 cell growth was noted in some experiments when CS3-HSA was incubated with RH9 cells in the absence of HIV, although this was not a reproducible observation.
  • An irrelevant peptide conjugate, DC7-HSA did not protect against HIV mediated cell killing at similar doses.
  • DSS disuccinimidyl suberate
  • DC7-HSA was also used for crosslinking (FIG. 3D) .
  • DC7-HSA did not crosslink to an detectable cell surface molecule in the presence of DSS (FIG. 3D) nor did it compete for binding in flow cytometry experiments (FIG. 1) .
  • DC7-HSA on SDS gels was similar to that of CS3-HSA.
  • the putative receptor for CS3 is a molecule with a minimum subunit size of approximately 44 Kd, determined by subtraction of the apparent molecular weight of the complex from CS3-HSA (108-64 Kd) .
  • HIV appears to utilize a number of mechanisms to kill cells (H. Temin, Rev. Inf. Dis., 10, 399 (1988); R.F. Garry, AIDS, in press) .
  • a TM carboxyl terminus mutant eliminating 177-200 base pairs from the gp41 gene, retained the ability to induce cell-cell fusion, but was attenuated in its ability to kill single cells (A.G. Fisher, et al., Science 233, 655, 1986), Thus, regions of TM other than those encoding CS3 may be important in cytopathology.
  • Our results suggest that HIV interaction with the CS3 receptor may be required for cell killing.
  • CS3 peptide competes with HIV for binding to CD4+ cells.
  • CS3 receptor of the elicitation of neutralizing antibody to prevent HIV interaction with the CS3 receptor may be important therapeutic modalities for the treatment of
  • Antibodies directed toward CS3 were prepared by passing anti-HIV immunoglobulin (plasma from an HIV- seropositive patient) through A protein G affinity column
  • RH9 cells at a concentration of 2 x 10 cells/ml to either (i) medium; (ii) HIV virus alone; (iii) HIV virus in the presence of HSA at 20 ⁇ g/ml; (iv) HIV virus and CS3-HSA at 10 ⁇ g/ml; (v) HIV virus and 6.7 ⁇ g/ml antibody specific for CS3; or (vi) HIV virus and 6.7 ⁇ g/ml CS3 depleted anti-HIV IgG; for 4 hours, then washed three times in a 100X volume of HBSS. The cells were then incubated in RPMI containing 10% serum and 20 mM
  • HIV infection was measured in terms of expression of p24 protein, as quantitated by a standard assay (e.g. the F.D.A. approved Abbott Laboratory Assay) .
  • CS3-HSA was observed to significantly inhibit HIV infection of RH9 cells, as measured by p24 production (FIG. 5) . This inhibition was not observed when cells were exposed to virus and HSA alone. Therefore, not only is cell killing inhibited by CS3, but infection of virus is inhibited by CS3 as well. Furthermore, inhibition was achieved using 1 ⁇ g/ml CS3-HSA as well as 10 ⁇ g/ml CS3- HSA.
  • anti-CS3 antibody also inhibits production of p24 (FIG. 5) . This correlates with clinical data which indicates that .antibody specific to CS3 in HIV seropositive patients is associated with the absence of AIDS-related disease.
  • FIG. 6 A four-fold decrease in mean fluorescence was observed by incubation of MQLT-4 cells with 20 ⁇ g CS3- HSA/10 6 cells prior to addition of HIV. It is possible that binding of g41 to the CS3 receptor is necessary for high affinity binding of HIV and consequent infection.
  • PBMC peripheral blood mononuclear cells exposed to CS3-HSA
  • PBMC peripheral blood mononuclear cells exposed to CS3-HSA
  • C-CS3-HSA 2.5 ⁇ g/1- cells
  • DC7-HSA 5 ⁇ g/10 cells
  • the cells were washed, and then incubated for 20 minutes at 4 ⁇ C then incubated with FITC labelled anti-CD4 (T4), FITC anti-CD3 (T3), or FITC anti-CD2 (Til), and rhoda ine C-CS3-HSA, or phycoerythrin labelled anti-CD8 (T8) , and FITC C-CS3-HSA, or phycoerythrin anti-HLA-DR and FITC C-C53-HSA.
  • T4 FITC labelled anti-CD4
  • T3 FITC anti-CD3
  • T6 FITC anti-CD2
  • PBMC phytohemagglutinin
  • TPA tetraphorbol 13-myristyl acetate
  • the CS3 receptor has been identified, by flow cytometric analysis using FITC-C-CS3-HSA on a variety of cell lines, as shown in Table I. TABLE I
  • CTLL murine T lymphocyte clone
  • YAC-1 murine hematopoietic tumor
  • the present invention also provides for an assay system to measure or detect HIV binding.
  • a binding assay has been developed for viruses, particularly HIV, which has broad application for screening for antivirals (drugs, peptides and antibodies) that are potentially neutralizing 5by virtue of the ability to prevent binding of virus to target cells (See Figure 13) .
  • the binding assay comprises exposing cells to virus (e.g. HIV) in medium for a period of time sufficient to allow binding of virus to cells to occur, centrifuging the cells and virus through a high specific ⁇ gravity liquid such as, preferably, silicon oil, freezing the media and cell pellet, separating the cell pellet from the frozen medium, and assaying the cell pellet for viral core protein.
  • This assay may be used to test the ability of a compound (e.g. a peptide) to block virus biding.
  • CS3-HSA may be incubated with MT4 cells prior to the addition of HIV (5-6 infectious particles per cell) .
  • Cells may then be centrifuged (preferably microfuged) through silicon oil, the tube may then be frozen (for example in liquid N_) then the pellet snipped off to separate it from the frozen medium.
  • the cell pellet may then be assayed for p24 core antigen content using, for example, the Abbott assay or any suitable HIV assay system known in the art.
  • freezing the tube is essential to prevent any HIV from the medium from contaminating the cell pellet.
  • Analysis of core protein is also an important feature, since gpl20 from particles that do not infect would result in high backgrounds if antibody to gpl20 was used. This assay may be used to identify compounds which block HIV/cell binding such as compounds which block HIV binding to the CS3 receptor.
  • Soluble CD4 and irrelevant peptide conjugates were used as positive and negative controls, respectively.
  • HIV to kill and infect CD4 + cells can be inhibited by interfering with HIV binding to the receptor for gp41 (TM) .
  • the CS3 peptide is the CS3 receptor (also known as the TM receptor) domain which appears to be a major determinant for
  • CS3-HSA can block the cell killing and infection by all HIV Isolates examined. This is consistent with the conserved nature of the CS3 sequence in HIV isolates (Los Alamos data base) .
  • PBMC peripheral blood mononuclear cells
  • Dual fluorescence flow cytometry was used to determine the percentage of each subset positive for expression of CS3,using CS3-HSA-Rhodamine or FITC.
  • Subset markers were monoclonal antibodies from Coulter as follows: T cells, T4 for helper, T8 for suppressor cytotoxic, Til for pan T Cbells, 12 for HLA-Dr, MOl and M02 for monocyte/macrophag .
  • RH9 cells have been stimulated with antibody to CD3 or with TPA or incubated in medium alone.
  • CD4 and CS3-HSA binding Fig. 12
  • CD4 was ⁇ Lost upon treatment with TPA; however, CS3 binding was increased.
  • Stimulation with antibody to CD3 resulted in a time dependent increase in receptor density.
  • control cultures demonstrated variation. in CD4 expression which is probably related to cell cycle and division (MOLT 4 %nd MT do not express CD3, so anti-CD3 stimulation was not attempted) .
  • Expression of the CS3 binding domain can be regulated in a fashion that may be dependent on the activating agent and cell. Thus, there may be specificity to regulation of the CS3 domain via distinct signal transduction pathways.

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Abstract

Procédés d'inhibition de la destruction de cellules due au VIH consistant à inhiber l'interaction entre la région CS3 de la glycoprotéine gp41 virale et son récepteur à la surface des lymphocytes. Les procédés proposés sont l'emploi de peptides, de dérivés peptidiques ou d'anticorps visant à inhiber l'interaction CS3/récepteur CS3. L'invention porte également sur le récepteur CS3. L'invention se fonde en partie sur la découverte qu'un récepteur cellulaire spécifique CS3 est largement réparti sur les lymphocytes humains et forme un complexe de 108 kd avec le conjugué peptidique CS3-HSA. On a aussi découvert que le peptide CS3 bloque effectivement l'infection et la destruction de cellules dues au VIH. En conséquence, on propose des procédés de traitement et de prophylaxie de l'infection par le VIH ainsi qu'un moyen de mieux comprendre la physiologie du syndrome d'immunodéficience acquise (SIDA).
PCT/US1991/001549 1990-03-09 1991-03-07 Effet de blocage de l'inhibiteur peptidique de l'infection causee par le virus de l'immunodeficience humaine sur les interactions entre le virus et un nouveau recepteur cellulaire WO1991013911A1 (fr)

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US49113790A 1990-03-09 1990-03-09
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US62665290A 1990-12-12 1990-12-12
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0323157A2 (fr) * 1987-12-24 1989-07-05 The University Of Melbourne Composés antiviraux et méthodes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0323157A2 (fr) * 1987-12-24 1989-07-05 The University Of Melbourne Composés antiviraux et méthodes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
IMMUNOLOGY LETTERS, Volume 19, Number 1, issued 1988, G.J. CIANCIOLO et al., "Human Retrovirus-Related Synthetic Peptides Inhibit T Lymphocyte Proliferation", pages 7-13. *

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