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WO1989003221A1 - Agent antisyncytial - Google Patents

Agent antisyncytial Download PDF

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Publication number
WO1989003221A1
WO1989003221A1 PCT/US1988/003381 US8803381W WO8903221A1 WO 1989003221 A1 WO1989003221 A1 WO 1989003221A1 US 8803381 W US8803381 W US 8803381W WO 8903221 A1 WO8903221 A1 WO 8903221A1
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WO
WIPO (PCT)
Prior art keywords
sequence
cysteine
amino acids
hiv
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1988/003381
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English (en)
Inventor
Jeffrey D. Lifson
Kou Hwang
Lee E. Eiden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genelabs Inc
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Genelabs Inc
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Filing date
Publication date
Application filed by Genelabs Inc filed Critical Genelabs Inc
Publication of WO1989003221A1 publication Critical patent/WO1989003221A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to methods for modulating viral effects on a susceptible cell expressing cell surface antigen CD4 using polypeptides which interfere with interactions of viral proteins with CD4.
  • This invention is particularly directed to blocking virion infectivity and cytopathic effects of CD4-dependent retroviruses.
  • T cells regulate the immune response through "helper” and "suppressor” signals and also mediate part of the effector arm of cellular immunity.
  • Human T lymphocytes were initially subdivided into two functionally distinct sublineages on the basis of expression of mutually exclusive cell surface proteins designated CD4 (also referred to as T4 or Leu3) and CD8 (also referred to as T8 or Leu2).
  • CD4 also referred to as T4 or Leu3
  • CD8 also referred to as T8 or Leu2
  • T-cells expressing CD8 mediate Major Histocompatibility Complex (MHC) Class I restricted cytotoxic activity and non-cytotoxic antigen specific suppressor function.
  • T-cells expressing CD4 mediate helper function for B cell growth and differentiation, proliferative responses, and inducer function for differentiation of CD8 expressing cytotoxic cells.
  • MHC Major Histocompatibility Complex
  • CD4 and CD8 molecules identify T cells whose functional activity is restricted by MHC Class II and Class I determinants, respectively.
  • the significance of the CD4 and CD8 molecules in T cell-target cell interactions can be demonstrated by studies with monoclonal antibodies. Antibodies directed against specific epitopes of the CD4 molecule inhibit MHC Class II restricted functions, including antigen-induced T cell proliferation, lymphokine release, helper cell function and the cytotoxic activity of CD4 expressing cytotoxic T cells.
  • CD4 molecule acts as a receptor for the ret- rovirus HIV-1 (also referred to as HTLV-III, LAV-1 or ARV), the etiologic agent for Acquired Immunodeficiency Syndrome (AIDS) and other retroviruses such as HIV-2 and SIV (also referred to as STLV-III, which is similar to or identical to a virus designated HTLV-IV).
  • HIV-1 also referred to as HTLV-III, LAV-1 or ARV
  • AIDS Acquired Immunodeficiency Syndrome
  • STLV-III also referred to as STLV-III, which is similar to or identical to a virus designated HTLV-IV.
  • CD4 T lymphocytes and cells of the mononuclear phagocyte lineage appear to be the major targets of HIV infection, and constitute the major in vivo reservoirs of virus in infected individuals. Many of the clinical manifestations of HIV infection appear to be direct or indirect consequences of direct or indirect effects of the virus or viral components on cells expressing CD4. Agents which interfere with the obligate interactions of CD4-dependent viruses with cells expressing CD4 thus may find application in the treatment of diseases associated with retroviral infection, such as AIDS and AIDS-related complex.
  • CD4+ cells recognize MHC Class II determinants whereas CD8+ cells recognize MHC Class I determinants (Engleman et al., J. Immunol.
  • anti-CD4 monoclonal antibodies bind to the surface of human cells of the monocyte/macro- phage lineage and to cells of the Langerhans lineage. Wood et al., J. Immunol. (1983) 131:212-216. The CD4 antigen on human T lymphocytes and monocytes are similar molecules. Stewart et al., J. Immunol. (1986) 136: 3773-3778. Bank et. al. (J. Exp . Med. (1985) 162:1294- 1303) discloses that the CD4 molecule may function as an independent transducer of negative signals to the T cell surface.
  • CD4 may play a role acting in concert with the T cell antigen receptor to facilitate antigen receptor dependent cellular activation. Sleckman et al . , Nature (1987) 328:351-353; Rosoff et al ., Cell (1987) 49:845-853.
  • HIV-1 has a selective tropism for CD4+ T cell lymphocytes. Klatzmann et al. , Science (1984) 225:59-63. CD4 tropism of SIV and HIV-2 has also been reported. Kanki et al., Science (1985) 231 :951-954; Kornfeld et al., Nature (1987) 326:610-613; Hirsch et al., Cell (1987) 49:307-319; Guyader et al., Nature (1987) 326: 662-669. The CD4 molecule on T lymphocytes behaves as a receptor for the HIV-1 virus.
  • HIV-induced cell fusion a characteristic manifestation of HIV-induced cytopathology, is dependent upon interactions between the viral envelope glycoprotein and CD4. Dalgleish et al . (1984) supra; Lifson et al ., Science (1986) 232:1123-1127;
  • compositions for inhibiting HIV, the etiologic agent for AIDS, by use of a modified peptide having substantially the same sequence as at least a portion of the sequence of the human CD4 antigen. Inhibition is observed as a blockade of characteristic HIV-induced, CD4-dependent cell fusion.
  • compositions for inhibiting virally induced cytopathic effects due to infection by HIV.
  • the compositions are characterized by having a sequence comparable to a sequence of the CD4 molecule, in particular a sequence proximal to the N-terminus.
  • the sequence includes the cysteine at position 86 of CD4 at which the sulfur on the cysteine is blocked with a benzyl group.
  • the composition may be prepared by reacting underivatized peptide under mild conditions with reagents such as .a halogen benzyl, preferably benzyl bro mi e, which are known to react with mercaptans.
  • the derivatized peptide also can be synthesized directly, rather than by modification of the underivatized sequence, for example by substituting a cysteine modified as above in the synthesis protocol, when using an automatic synthesizer.
  • the peptide of interest will comprise substantially the same sequence as amino acids of the sequence 81 to 94, usually 83 to 94, or 79 to 94, preferably 76 to 94, where the sequence may be further extended by as many as 10 amino acids or more at either terminus, where the extension amino acids may be the same or different from the CD4 sequence.
  • the numbering of the amino acids is as set forth in Madden et al., Cell (1985) 4 :93-104.
  • the peptide sequences may be modified by terminal amino acylation, for example, acet- ylation; carboxy amidation, for example, with ammonia, methylamine, etc.
  • the amino acid sequence need not correspond exactly to the sequences given above, provided that the relevant peptide domain related to interference with interactions between CD4- dependent retroviruses and host cells is substantially retained. Therefore, the subject polypeptides may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non- conservative, where such changes might provide for certain advantages in their use.
  • conservative substitutions is intended combinations such as gly, ala; val , ile, leu; asp, glu; asn, gin; ser , thr; lys, arg; and phe , tyr, trp.
  • the sequence will not differ by more than 20?
  • the peptides of this invention may be conveniently linked for immobilization.
  • the arms will usually be at least about 5 amino acids and may be 50 or more amino acids.
  • the subject peptides may be employed linked to a soluble ma ⁇ romolecular carrier.
  • the carrier may be a polypeptide, either naturally occurring or synthetic, antibodies to which are unlikely to be encountered at high levels in human serum.
  • Illustrative polypeptides include poly-L-lysine, bovine serum albumin, keyhole limpet hemocyanin, bovine gamma globulin, etc.
  • conjugates there will be at least one molecule of at least one subject peptide per macro-molecule and generally not more than about 1 per 0.5 kDal, usually not more than about 1 per 2 kDal of the macromolecule.
  • One or more different peptides may be linked to the same macromolecule.
  • the manner of linking is conventional, employing such reagents as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acid anhydride, succinic acid anhydride, glutaraldehyde, etc.
  • the linkage may occur at the N-terminus, C-terminus, or at a site intermediate to the ends of the molecule.
  • the subject peptide may be derivatized for linking, may be linked while bound to a support, or the like.
  • the peptides can be prepared in a wide variety of ways.
  • the peptides because of their relatively short size, may be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, 1984; and Tam et al., J. Am. Chem. Soc. (1983) 105:6442.
  • hybrid DNA technology may be employed where a synthetic gene may be prepared by employing single strands which code for the polypeptide or substantially complementary strands thereof, where the single strands overlap and can be put together in an annealing medium so as to hybridize. The hybridized strands may then be ligated to form the complete gene and by choice of appropriate termini, the gene may be inserted into expression vectors, which are readily available today. See for example, Maniatis et al., Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982. Or, the region of the genome coding for the peptide may be cloned by conventional recombinant DNA techniques and expressed (see Maniatis, supra).
  • DNA coding sequences based upon the known sequence for CD4 may be used. Fragments from these sequences may be employed for expression of peptide fragments, conservative base changes can be made, where the modified codon(s) code for the same amino acid(s), or non-conservative changes in the coding sequence may be made, where the resulting amino acid may be a conservative or non-conservative change.
  • the coding sequence may be extended at either the 5'- or 3'-terminus or both termini to extend the peptide, while retaining the relevant sequence encoding the peptide domain related to interference with interaction between CD4-dependent retroviruses and host cells.
  • the extension may provide for an arm for linking, for providing antigenic activity, or the like.
  • the coding sequence will be provided with start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in a cellular host, for example prokaryotic or eukaryotic, bacteria, yeast, mammal, etc.
  • the thio group may be blocked with a benzyl or a p-methyl group which replaces the hydrogen of the mercaptan of the cysteine.
  • Active halogen benzyl reagents may find use.
  • the temperature will generally be mild, 0-50°C, usually 10-30°C, with time ranging from about 0.5 to 24 hours.
  • Polar solvents particularly aqueous solvents, may be employed, where organic solvents may be present up to about 60 volume % .
  • Organic solvents include acetonitrile, acetone, diethyl ether, dimethylformamide, etc.
  • a mild basic acid acceptor will be present, conveniently, carbonate, bicarbonate, etc. Usually an excess of the blocking agent will be employed.
  • the product may be isolated and purified according to conventional techniques.
  • the subject compositions may be used in vitro and in vivo.
  • the subject compositions may be employed for detecting the role of CD4 in viral infection t preventing infection of CD4-bearing cells including T cells and macrophages susceptible to infection by HIV, and the like.
  • the subject compositions may be used prophylactically or therapeutically for preventing infection or inhibiting dissemination of the virus and infection of or cytopathic effects on additional T cells or other CD4-bearing cells by inhibiting HIV-induced CD4-dependent cytopathic effects.
  • the subject compositions will generally be administered so as to enter the blood stream, being administered intramuscularly, intraperitoneally, intravenously, parenterally, intranasally, topically, or the like.
  • any physiologically acceptable medium may be employed, such as deionized water, saline, phosphate buffered saline, aqueous ethanol, and the like.
  • concentration of the subject composition will vary, depending upon the use, frequency of administration, and the like. The amount used will depend upon the relative antisyncytial activity of the composition employed. Generally, the dosage will be in the range of 0.2 mg to 500 mg, more usually 10 mg to 100 mg. The following examples are offered by way of illustration and not by way of limitation.
  • Synthetic Peptides Corresponding to Discrete Regions of the CD4 Molecule Synthetic peptides corresponding to regions of the CD4 molecule (peptides E-K) were generated from the predicted amino acid sequence of CD4 (Maddon et al., Cell (1985) 42:93-104), using automated solid phase peptide synthesis methodology (Applied Biosystems, Foster City, CA). Additional peptides not encoded by the CD4 gene (peptides A-D) were likewise prepared. All preparations were heterogeneous as assessed by HPLC. The following synthetic peptides were prepared.
  • HIV Envelope Induced Cell Fusion An HIV envelope glycoprotein-induced cell fusion assay was performed essentially as described in Lif son et al . , Nature ( 1 986 ) 323 : 725 -728. Bri efly 5 ⁇ 10 4 H9 HIV HXB -2 cells were preincubated with varying concentrations of the peptide to be tested in the wells of flat-bottomed 96-well microtiter plates for 30 minutes at 37°C. 5 ⁇ 10 4 CD4+ VB cells were then added. The total volume was 100 ⁇ l.
  • syncytia (defined as at least four nuclei within a common cell membrane) were scored by inverted phase microscope ( ⁇ 300) as described (Lifson et al., supra). Titer indicates nominal peptide concentration required for complete blockade of HIV envelope induced CD4 dependent cell fusion after an overnight incubation in a standard assay. Molarities are calculated based on formula weight for the expected dominant synthetic product. None of the non-derivatized peptides A-H exhibited any ability to block HIV-induced cell fusion at the concentrations tested (up to 500 ⁇ M). However, peptide H modified with a benzyl group in the cysteine residue did block HIV-induced cell fusion, as did various fragments of benzyl-substituted peptide H. The results are shown in Table 1, below.
  • the benzyl derivatized parent 19-mer also completely inhibited fusion induced by SIV and two additional HIV isolates at nominal peptide concentrations between 60 and 250 ⁇ M, but did not show any inhibition of cell fusion induced by HTLV-I, a non-CD4-dependent human retrovirus. Concentrations of the derivatized 19-mer which completely blocked HIV induced cell fusion did not affect the mixed leukocyte reaction, a CD4-dependent immune response.
  • compositions which inhibit retrovirus-induced CD4-dependent cytopathic effects. These compositions can be used prophylactically and therapeutically for inhibiting HIV infection and syncytial formation.
  • the subject compositions provide for protection against infection and progression of symptoms associated with AIDS-related complex and from AIDS.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
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  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention décrit des compositions peptides qui comprennent une séquence de CD4 incluant la cystéine en position 86 et dans lesquelles le soufre de la cystéine est substitué par un substituant contenant un aryle ou par un thioéther résultant de la réaction entre le groupe thio de la cystéine et une maléimide ou par un disulfure résultant de la réaction entre le groupe thio de la cystéine et d'autres composés contenant un groupe thio. Les compositions qui en résultent peuvent être utilisées prophylactiquement ou thérapeutiquement pour inhiber les interactions entre le CD4 et des protéines rétrovirales.
PCT/US1988/003381 1987-10-13 1988-09-30 Agent antisyncytial Ceased WO1989003221A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10815987A 1987-10-13 1987-10-13
US108,159 1987-10-13

Publications (1)

Publication Number Publication Date
WO1989003221A1 true WO1989003221A1 (fr) 1989-04-20

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PCT/US1988/003381 Ceased WO1989003221A1 (fr) 1987-10-13 1988-09-30 Agent antisyncytial

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WO (1) WO1989003221A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0394297A4 (en) * 1987-10-13 1990-12-27 Genelabs Incorporated Anti-retroviral agent
EP0410178A1 (fr) * 1989-07-05 1991-01-30 The Calpis Food Industry Co., Ltd. Fragment de CD4 avec de l'activité anti-HIV
EP0432693A1 (fr) * 1989-12-11 1991-06-19 Sumitomo Chemical Company, Limited Liposomes cytotoxiques à des cellules infectées par des virus
US5171838A (en) * 1990-03-23 1992-12-15 Kureha Kagaku Kogyo Kabushiki Kaisha Leu3a binding peptides
WO1995000538A1 (fr) * 1993-06-22 1995-01-05 Peptech(Uk) Limited Peptides derives de l'interleukine-2 humaine destines a etre utilises en medecine
EP0832971A1 (fr) * 1987-10-02 1998-04-01 Genentech, Inc. Variantes d'adhésions
US6117655A (en) * 1987-10-02 2000-09-12 Genentech, Inc. Nucleic acid encoding adhesion variants
US6710169B2 (en) 1987-10-02 2004-03-23 Genentech, Inc. Adheson variants

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU631357B2 (en) * 1987-10-14 1992-11-26 United States of America, as represented by the Secretary, U.S. Department of Commerce, The New anti-receptor peptides and therapeutic agents

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 104, issued 09 June 1986, (Columbus, OH.), "Structure of the CD4 (W3/25) T-helper lymphocyte glycoprotein", (JEFFERIES), See abstract no. 205113b. *
NATURE, Vol. 312, issued 20/27 December 1984, pages 763-768, (DALGLEISH), "The CD4 (T4) Antigen is an essential component of the receptor for the AIDS retrovirus". (See summary). *
NATURE, Vol. 322, issued 31 July 1986, pages 470-474, (SODROSKI), "Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity". (See summary). *
NATURE, Vol. 323, issued 23 October 1986, pages 725-728, (LIFSON), "Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein". (See summary). *
SCIENCE, Vol. 232, issued 30 May 1986, pages 1123-1127, (LIFSON), "AIDS Retrovirus Induced Cytopathology: Giant Cell Formation and Involvement of CD4 Antigen". (See summary). *
SCIENCE, Vol. 234, issued 28 November 1986, pages 1120-1123, (SATTENTAU), "Epitopes of the CD4 Antigen and HIV Infection". (See summary). *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0832971A1 (fr) * 1987-10-02 1998-04-01 Genentech, Inc. Variantes d'adhésions
US6117655A (en) * 1987-10-02 2000-09-12 Genentech, Inc. Nucleic acid encoding adhesion variants
US6710169B2 (en) 1987-10-02 2004-03-23 Genentech, Inc. Adheson variants
EP0394297A4 (en) * 1987-10-13 1990-12-27 Genelabs Incorporated Anti-retroviral agent
EP0410178A1 (fr) * 1989-07-05 1991-01-30 The Calpis Food Industry Co., Ltd. Fragment de CD4 avec de l'activité anti-HIV
EP0432693A1 (fr) * 1989-12-11 1991-06-19 Sumitomo Chemical Company, Limited Liposomes cytotoxiques à des cellules infectées par des virus
US5171838A (en) * 1990-03-23 1992-12-15 Kureha Kagaku Kogyo Kabushiki Kaisha Leu3a binding peptides
WO1995000538A1 (fr) * 1993-06-22 1995-01-05 Peptech(Uk) Limited Peptides derives de l'interleukine-2 humaine destines a etre utilises en medecine

Also Published As

Publication number Publication date
AU2784789A (en) 1989-05-02

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