WO1998035692A1 - Matieres et procedes relatifs a la protection de cellules immunitaires utiles - Google Patents

Matieres et procedes relatifs a la protection de cellules immunitaires utiles Download PDF

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Publication number
WO1998035692A1
WO1998035692A1 PCT/GB1998/000485 GB9800485W WO9835692A1 WO 1998035692 A1 WO1998035692 A1 WO 1998035692A1 GB 9800485 W GB9800485 W GB 9800485W WO 9835692 A1 WO9835692 A1 WO 9835692A1
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Prior art keywords
fas
cells
fasl
infected
expressing
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PCT/GB1998/000485
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English (en)
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Gavin Robert Screaton
Xiao-Ning Xu
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Isis Innovation Limited
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Publication of WO1998035692A1 publication Critical patent/WO1998035692A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally concerns materials and methods relating to the protection of useful immune cells .
  • materials and methods for blocking cellular interactions involved in the triggering of apoptosis (programmed cell death) .
  • It further relates to uses of such materials and methods in relation to the diagnostic, prognostic, or treatment (prophylactic and/or therapeutic) of diseases, particularly immune deficiency diseases (e.g. diseases caused by or associated with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) .
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • CTL cytotoxic T-cells
  • apoptosis has been well documented in infected individuals (16-18) .
  • those apoptotic cells include not only uninfected CD4 + cells but also CD8 +
  • Apoptosis of lymphocytes can be triggered by several cell surface receptors including TNF-R1, Fas and a newly cloned molecule WSL-1/DR3 (20,21). Each of these molecules will trigger apoptosis when it contacts its counter-receptor or ligand, TNF and Fas ligand (FasL) for TNF-R1 and Fas respectively (22) . Interaction between Fas and FasL plays an important role in the homeostatic regulation of normal immune responses (23) . The expression of Fas is quite diffuse being found on a variety of extra- lymphoid tissues such as liver, ovary and heart. The expression of FasL on the other hand is much more tightly controlled being restricted to activated lymphocytes and selected sites enjoying immune privilege (24,25) . Fas is upregulated on both CD4+ and CD8+ cells from HIV-infected individuals (26,27).
  • the present application is based on the determination of the mechanism by which virulent SIV (a simian model of HIV) infection leads to the depletion of T lymphocytes.
  • the present inventors have made the surprising discoveries that (1) FasL is expressed on CD4+ T lymphocytes which have been infected by the virulent pJ5 strain of SIV, whereas FasL is not expressed on CD4+ cells infected by the attenuated pC8 strain of SIV and that (2) an efficient CTL response may be regenerated against pJ5-infected CD4+ lymphocytes by incubating lymphocytes from an infected macaque with a soluble Fas-Fc fusion protein (known as "soluble Fas”) .
  • soluble Fas soluble Fas
  • FasL protein expressed on infected CD4+ cells is bound by the soluble Fas-Fc, preventing it from binding CD8+ membrane-bound Fas.
  • CD8+ cell apoptosis is avoided and CTL activity towards the infected CD4+ cells is regenerated.
  • virus-specific CTL responses were investigated in macaques infected with an attenuated SIV strain (pC8) , which has a defective nef gene, encoding a protein product having a 4 amino acid deletion, as compared with the virulent wild-type SIVmac32H clone (pJ5) . Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS- like symptoms in the 12 months following infection. The pC8- induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens.
  • pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after 3 months.
  • the latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4 + and CD8 + populations.
  • pJ5 but not pC8 leads to an increase in FasL expression on infected cells.
  • FasL protects infected cells from CTL attack, killing viral specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5.
  • pC8 does not induce FasL expression, allowing the development of a protective CTL response.
  • Fas-FasL interaction allows the regeneration of viral specific CTL responses in pJ5- infected animals.
  • the results indicate that the mechanisms of protection induced by the ⁇ ef-mutant SIV involves the induction of a viral specific CTL response.
  • the failure of pC8-infected CD4 + cells to upregulate FasL expression is fundamental to the generation of an efficient CTL response.
  • the pJ5-infected CD4" cells upregulate FasL expression, leading to the death of SIV-specific CTL expressing Fas. This results in a failure to check viral replication and a consequent progression to AIDS.
  • Fas-derived amino-acid sequence e.g. having the same or differing Fas-derived amino-acid sequence, optionally linked with the same Fc polypeptide or differing polypeptides may show the same or improved ability to bind FasL, thus preventing it from triggering apoptosis in Fas-expressing cells.
  • Non-activating, blocking, anti-Fas antibodies can prevent FasL from gaining access to the binding site of Fas; blocking anti-FasL antibodies, such as the anti -human FasL monoclonal antibodies 4A5 and 4H9 , described in (28) work in an analogous manner on the FasL binding site.
  • Non-activating, molecules which block/interfere with the interaction between Fas and FasL may be obtainable by screening methods known in the art .
  • the present invention provides a pharmaceutical product which comprises a molecular entity which interferes with the interaction between Fas and FasL such that apoptosis of cells bearing Fas is reduced.
  • the present invention also provides treatment methods (prophylactic and therapeutic) which comprise administering a molecular entity which interferes with the interaction between Fas and FasL such that apoptosis of cells bearing Fas is reduced.
  • the present invention also provides for the use of a molecular entity which interferes with the interaction between Fas and FasL in the preparation of a medicament for the treatment (prophylactic and therapeutic) of a medical condition associated with an immunodeficiency disease .
  • An immunodeficiency disease may be caused by eg HIV-1 or HIV-2.
  • the molecular entity reduces the apoptosis of cells bearing Fas by virtue of the fact that it fails to mimic the full biological effects of binding of either wild-type membrane-bound Fas or FasL to their opposite partners.
  • the molecular entity may be considered as blocking and non-activating; either partially or fully blocking/non-activating.
  • the molecular entity may be selected from the group consisting of soluble Fas; soluble FasL; anti-Fas antibodies; or anti-FasL antibodies.
  • the molecular entity comprises a soluble Fas or soluble FasL
  • the entity differs from wild-type to the extent that when the molecular entity binds to its partner (ie soluble Fas to membrane bound FasL, or soluble FasL to membrane bound Fas) it is unable to mimic the full biological effects resulting from binding of wild-type, membrane bound Fas and FasL ie apoptosis of cells bearing Fas.
  • the molecular entity may also be provided by variants, mutants, derivatives, alleles and mimetics of any of the above-mentioned entities.
  • the immunodeficiency disease is preferably caused by human or simian immunodeficiency virus, i.e. HIV or SIV; the disease is preferably AIDS.
  • the present invention provides pharmaceutical products comprising a molecular entity as set out above for blocking the in vivo or in vi tro triggering of programmed cell death by the interaction of Fas with an activating ligand.
  • the programmed cell death is preferably associated with the expression of FasL by CD4+ cells on HIV or SIV infection and may affect CD8+ and/or uninfected CD4+ cells.
  • treatment methods which comprise administering a molecular entity as set out above, for blocking the in vivo or in vitro triggering of programmed cell death by the interaction of Fas with an activating ligand.
  • the hybridisation of antisense nucleic acid sequence to mRNA encoding FasL may prevent its expression.
  • a molecular entity for interfering with the interaction of Fas/FasL may thus also comprise a nucleic acid sequence.
  • the prevention of apoptosis preferably allows the maintenance or regeneration of cytotoxic T lymphocyte (CTL) activity towards cells infected with the infectious agent .
  • CTL cytotoxic T lymphocyte
  • cytotoxic T lymphocyte activity towards infected cells is prevented by the triggering of Fas- mediated apoptosis.
  • cells which would otherwise kill infected CD4+ cells are themselves killed due to the expression of FasL on infected CD4+ cells.
  • the treatment depends on the prevention of apoptosis by blocking the interaction of Fas with FasL, allowing maintenance or regeneration of the cytotoxic T lymphocyte activity towards the infected cells.
  • the teaching of the present invention is applicable to any disorder or disease caused by or involving a reduced immune response due to the Fas-mediated apoptosis of cells normally mediating the immune response, e.g. lymphocytes.
  • the invention provides a method for reducing depletion of activated Fas-expressing CD8 + TK cells in an immune cell population which also comprises Fas-ligand (FasL) -expressing activated CD4 + cells the method comprising contacting said immune cell population with an effective amount of an agent which is capable of interfering with any interaction between Fas and FasL.
  • FasL Fas-ligand
  • the invention also particularly provides use of an agent in the manufacture of a preparation for reducing depletion of activated Fas -expressing CD8 + TK cells in a population of immune cells which also comprises FasL- expressing activated CD4 + cells wherein said agent is capable of interfering with any interaction between Fas of said CD8 + cells and FasL of said CD4 + cells.
  • the agent may be capable of binding to FasL or Fas .
  • the agent may comprise a soluble version of native Fas .
  • the agent may comprise a soluble Fas-Fc fusion protein.
  • the FasL-expressing activated CD4 + cells may be infected with an immunodeficiency virus.
  • the nucleotide sequence for FasL comprises Fig. 9 herein.
  • FasL an amino acid sequence characteristic of FasL this may be done by employment of polypeptides with specificity for FasL eg soluble Fas and/or anti-FasL antibodies and/or their variants, mutants or derivatives and/or their mimetics.
  • kits and reagents for carrying out diagnostic methods as set out above.
  • Kits and reagents may comprise primers for amplification of FasL nucleotide sequences.
  • Kits and reagents may comprise polypeptide binding partners which are specific for FasL.
  • Kits and reagents may comprise ancillary reagents and items for carrying out the diagnostic methods.
  • the present invention provides the use of any one of the above identified molecular entities in screening for compounds which are binding partners of either Fas or FasL, which may or may not mimic the full biological activity, e.g. antibodies or complementary peptides specific for Fas or FasL or a Fas or FasL mimetic.
  • the substances used for screening may be peptide fragments of Fas or FasL.
  • screening procedures for mimetics or binding partners include: (a) immobilising the Fas or FasL polypeptides or fragments on a solid support and exposing the support to a library of labelled peptides or other candidate compounds, and detecting the binding of the peptides or candidate compound to the Fas or FasL polypeptides or fragments;
  • yeast two hybrid screens to detect candidate peptides which bind to the Fas or FasL polypeptide or fragment (for a description of yeast two hybrid screens see W096/14334);
  • the present invention provides method of identifying compounds which compete with Fas or FasL, the method comprising:
  • the present invention provides a method of identifying mimetics of Fas or FasL, the method comprising :
  • the invention provides a method for screening for compounds capable of reducing depletion of activated Fas-expressing CD8 + TK cells in a population of immune cells which comprises contacting a population of immune cells comprising FasL-expressing activated CD4 + cells and Fas-expressing activated CD8 + TK cells with a test compound and selecting a candidate compound which alters the level of depletion of activated Fas-expressing CD8 + TK cells which occurs in a said population of immune cells in the absence of said test agent.
  • the FasL- expressing activated CD4 + cells may be infected with an immunodeficiency virus.
  • a method for manufacturing a preparation for reducing depletion of activated Fas -expressing CD8 + TK cells in an immune cell population which comprises the step of combining with a carrier or excipient an agent identified as above.
  • the candidate compounds can be selected from a synthetic combinatorial library.
  • the present invention provides reagents and kits for the above screening methods.
  • FIG. 1 Spontaneous DNA fragmentation of peripheral blood mononuclear cells (PBMCs) from pC8- induced protected (N113-N114) or pJ5-infected (N174-N177) macaques. Fresh isolated PBMCs (lxlO 6 ) were cultured in medium containing 10% human AB serum for 4 or 16 hours. Fragmented DNA was extracted as described in Materials and Methods.
  • PBMCs peripheral blood mononuclear cells
  • A DNA was analysed by electrophoresis on 1.5% agarose gels; B: Southern blot analysis of the DNA by hybridisation with 32 P-labelled genomic macaque DNA; C: The percentage of DNA fragmentation in B was calculated as dividing the total counts of each sample by the number of counts in the bottom 85 to 90% of the gel (below 23,000bp). The percentage of DNA fragmentation of PBMCs from naive macaques was always ⁇ 20% (data not shown) .
  • FIG. 3 Expression of Fas antigen on SIV- infected macaques.
  • A Representative histogram of Fas expression on PBMCs from naive, pC8-infected (C8+J5) , or pJ5- infected macaques.
  • SIV-infected cells kill Fas-sensitive target via FasL-Fas interaction.
  • Naive monkey PBMCs (A) or CEM cells (B) were superinfected with SIV pC8 or pJ5 as indicated in Materials and Methods.
  • SIV-infected cells were co-cultured with 51 Cr-labelled Fas-sensitive Jurkat cells in the presence or absence of Fas-Fc fusion proteins (lO ⁇ g/ml) for 12-16 hours. Chromium release was determined by a beta-counter. Specific lysis was calculated by subtracting the killing of Jurkat cells by mock-infected cells.
  • Infectivity of SIV pC8 or pJ5 in CEM cells was analysed (C) by flow cytometry using mAb to SIV nef antigen.
  • PBMCs from SIV pJ5- infected macaques kill Fas- sensitive target are CD4 -dependent .
  • ConA 5 ⁇ g/ml for 12 hours.
  • CD4 + or CD8 + T-cells were depleted by using anti-CD4 or CD8 magnetic beads, respectively.
  • Cells were then co-cultured with 51 Cr- labelled Jurkat cells in the presence or absence of fusion proteins (lO ⁇ g/ml) or anti -human FasL mAbs (5 ⁇ g/ml) for 12-16 hours. Specific lysis was assayed as described in Figure 4.
  • Soluble Fas-Fc fusion protein regenerates CTL response from SIV pJ5- infected macaques.
  • PBMCs were isolated from macaques (P93) 6 month post -infection and set up for bulk culture CTL in the presence or absence of either Fas-Fc fusion protein (lO ⁇ g/ml) or soluble CD4 (5 ⁇ g/ml) for 14 days. After washing the cells, the virus-specific CTL activities were determined as described in Materials and Methods. The data was representative of three experiments.
  • Figure 7 The amino acid sequence of the known Fas protein.
  • Figure 8 A portion of the amino acid sequence shown in Fig 7, which was fused with an Fc polypeptide to form the soluble Fas-Fc fusion protein, and which corresponds to a portion of the extracellular domain of the wild-type Fas protein.
  • Figure 9 The nucleic acid (cDNA) sequence of the known FasL protein.
  • FIG. 10 The nucleic acid (cDNA) sequence of the known Fas protein.
  • the skilled person can use the techniques described herein and others well known in the art to produce large amounts of soluble Fas polypeptides, or soluble polypeptides having Fas function, for use as pharmaceuticals to block the Fas/FasL interaction, in diagnostic and/or prognostic applications for the presence of FasL in a sample, in the developments of drugs and for further study into its properties and role in vivo .
  • Soluble Fas is used to mean a soluble protein or polypeptide having at least a portion characteristic of the known Fas amino acid sequence, as shown in figure 7, which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated, such as other polypeptides or (for example if produced by expression in a prokaryotic cell) lacking in native glycosylation, e.g. unglycosylated.
  • a polypeptide comprising a portion characteristic of or corresponding to some or all of the extracellular domain of the known Fas protein is used to form the soluble Fas protein.
  • the soluble Fas protein may not comprise portions characteristic of or corresponding to the transmembrane or intracellular domains of the known Fas protein .
  • a polypeptide which is a variant, allele, derivative or mutant of Fas may have an amino acid sequence which differs from that given in figure 7 by one or more of addition, substitution, deletion and insertion of one or more amino acids.
  • Preferred such polypeptides have Fas function, that is to say have one or more of the following properties: immunological cross-reactivity with an antibody reactive with the known Fas protein, e.g.
  • a polypeptide which is an amino acid sequence variant, allele, derivative or mutant of the known Fas protein may comprise an amino acid sequence which shares greater than about 35% sequence identity with the sequence shown, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%.
  • the sequence may share greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity with the amino acid sequence of the known Fas protein.
  • Particular amino acid sequence variants may differ from the known Fas amino acid sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, 50-100, 100-150, or more than 150 amino acids.
  • Soluble proteins for use in accordance with the present invention may optionally be fusion proteins, wherein the polypeptide sequence having Fas function is fused with other polypeptide sequence or sequences, e.g. a polypeptide corresponding to the Fc fragment of an IgG molecule.
  • Other polypeptides which may be suitable for fusing with Fas polypeptides (or polypeptides having Fas function) are known in the art and their use in making soluble Fas proteins is included within the scope of the invention.
  • soluble Fas proteins are soluble active portions, fragments, derivatives and functional mimetics of the known Fas protein.
  • Fas protein means a peptide which is less than said full length Fas protein, but which retains its essential biological binding characteristics.
  • a “fragment" of the Fas protein means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids. Fragments of the Fas protein sequence preferably include domains which are capable of binding FasL.
  • a “derivative" of the Fas protein or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the Fas protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself .
  • Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the binding characteristics of the wild type Fas protein.
  • Fasmetic means a substance which may not contain an active portion of the Fas amino acid sequence, and probably is not a peptide at all, but which retains the essential FasL binding activity of natural Fas protein. The design and screening of candidate mimetics is described in detail below.
  • the soluble Fas polypeptides used in the present invention may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below) .
  • Polypeptides may also be generated wholly or partly by chemical synthesis.
  • the isolated and/or purified polypeptide may be used in formulation of a composition, which may include at least one additional component, for example a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.
  • a composition including a polypeptide according to the invention may be used in prophylactic and/or therapeutic treatment as discussed below.
  • a polypeptide, peptide fragment, allele, mutant or variant as described herein may be used as an immunogen or otherwise in obtaining specific antibodies.
  • Antibodies are useful in purification and other manipulation of polypeptides and peptides, diagnostic and /or prognostic screening and therapeutic contexts . This is discussed further below.
  • a polypeptide peptide fragment, allele, mutant or variant as described herein may be used in screening systems for molecules which affect or modulate its activity or function. Such molecules may be useful treatment agents and/or useful diagnostically .
  • a method of screening for a substance which modulates the Fas/FasL interaction may include contacting one or more test substances with the Fas or FasL polypeptide in a suitable reaction medium, testing the interaction of the treated polypeptide with its binding partner and comparing that interaction with the interaction of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in interaction with the binding partner between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances.
  • the screening system may be specifically for screening for compounds capable of reducing depletion of activated Fas-expressing CD8 + TK cells in a population of immune cells comprising in addition to such CD8 + TK cells, FasL- expressing activated CD4 + cells.
  • the screen may be carried out by contacting a population of immune cells comprising such CD4 + and CD8 + cells with a test agent and selecting a candidate compound which alters the level of depletion of activated Fas-expressing CD8 + TK cells which occurs in such an immune cell population in the absence of test agent .
  • the teachings of the examples allows the skilled person to readily set up such a screening system.
  • the examples describe sources of CD4" cells and their infection with an immunodeficiency virus to stimulate expression of FasL and the detection of virus-specific CTL activities.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate the Fas/FasL interaction.
  • Such libraries and their use are known in the art.
  • the use of peptide libraries is preferred.
  • test substances Prior to or as well as being screened for modulation of the Fas/FasL interaction, test substances may be screened for ability to interact with either polypeptide, e.g. in a yeast two-hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid) .
  • This may be used as a coarse screen prior to testing a substance for actual ability to modulate the interaction.
  • the screen could be used to find mimetics of the Fas or FasL polypeptides, e.g. for testing as therapeutics.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • a substance identified using as a modulator of polypeptide function may be peptide or non-peptide in nature.
  • Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses.
  • a mimetic or mimic of the substance may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound s a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases m the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques, X- ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process . In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide based
  • further stability can be achieved by cyclising the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the Fas-FasL interaction may be blocked by non- activating, blocking anti-Fas antibodies.
  • blocking is described in Dhein, J. et al . Nature 373, 438-441 (1995), using a F(ab') 2 anti-Fas antibody as described in Dhein, J. et al . J. Immun. 149, 3166-3173 (1992) .
  • Monoclonal and polyclonal antibodies and antibody fragments may be produced and screened using methods known in the art to find other suitable anti -Fas antibodies .
  • blocking, non-activating anti- FasL antibodies similar to the anti-human FasL monoclonal antibodies 4A5 and 4H9 as described in (28) may be obtained using routine methods and used in accordance with the teaching of this invention.
  • Monoclonal antibodies can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs) , of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB-A- 2188638 or EP-A-239400.
  • a hybridoma producing a monoclonal antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • Antibodies may be specific in the sense of being able to distinguish between the polypeptide it is able to bind and other human polypeptides for which it has no or substantially no binding affinity (e.g. a binding affinity of about lOOOx worse) . Specific antibodies bind an epitope on the molecule which is either not present or is not accessible on other molecules. Antibodies may be employed in purifying the polypeptide or polypeptides to which they bind, e.g. following production by recombinant expression from encoding nucleic acid.
  • Antibodies may be isolated, in the sense of being free from contaminants such as antibodies able to bind other non-related polypeptides and/or free of serum components. Monoclonal antibodies are generally preferred in the art, though polyclonal antibodies may also be used in accordance with the present invention.
  • Antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, Nature, 357:80-82, 1992).
  • Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
  • an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • Antibodies may be modified in a number of ways . Indeed the term “antibody” should be construed as covering any binding substance having a binding domain with the required specificity. Thus the present application also concerns antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope.
  • Example antibody fragments capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the d_Ab fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • a hybridoma producing a monoclonal antibody for use in accordance with the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs) , of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB-A- 2188638 or EP-A- 0239400. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP- A-0125023.
  • Hybridomas capable of producing antibody with advantageous binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression.
  • the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
  • the reactivities of antibodies on a sample may be determined by any appropriate means . Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non- covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine .
  • Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge .
  • Antibodies to Fas or FasL may be used in screening for the presence of the relevant polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide for use according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid therefor.
  • An antibody may be provided in a kit, which may include instructions for use of the antibody, e.g. in determining the presence of a particular substance in a test sample .
  • One or more other reagents may be included, such as labelling molecules, buffer solutions, elutants and so on. Reagents may be provided within containers which protect them from the external environment, such as a sealed vial .
  • Forms of FasL may be produced (generally as described in relation to the production of soluble Fas) which can bind to and block Fas receptors without activating them, thus preventing wild-type FasL from gaining access to and activating the receptors.
  • FasL may be alleles, variants, mutants or derivatives of the known form of FasL whose nucleic acid sequence is shown in Fig 9, or a mimetic.
  • FasL in pharmaceuticals for blocking the interaction of Fas and FasL is also included within the scope of the present invention.
  • the soluble Fas polypeptides, appropriate blocking non- activating antibodies, binding non-activating FasL and mimetics can be formulated in pharmaceutical compositions.
  • These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient .
  • the precise nature of the carrier or ether material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutally effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands .
  • Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents could be produced in the target cells by expression from an encoding gene introduced into the cells, eg in a viral vector (a variant of the VDEPT technique - see below) .
  • the vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are switched on more or less selectively by the target cells .
  • the agent could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated.
  • an activating agent produced in, or targeted to, the cells to be treated.
  • This type of approach is sometimes known as ADEPT or VDEPT; the former involving targeting the activating agent to the cells by conjugation to a cell- specific antibody, while the latter involves producing the activating agent, eg an enzyme, in a vector by expression from encoding DNA in a viral vector (see for example, EP-A-415731 and WO 90/07936) .
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • a further aspect of the present invention relates to the detection of FasL on lymphocytes.
  • FasL is upregulated on the surface of HIV/SIV- infected CD4+ lymphocytes
  • the presence (either quantitatively or qualitatively) of FasL in a population of lymphocytes, T cells or CD4+ T cells may provide an indication of the HIV/SIV status of the individual and/or the progress of infection and/or the onset of AIDS.
  • Various diagnostic and/or prognostic methods are available for detecting the presence of a particular polypeptide in a biological sample. The methods make use of biological samples from individuals that are suspected of contain the polypeptide. Examples of biological samples include blood, plasma, serum, tissue samples.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies) , specific for the FasL polypeptide or variants thereof.
  • a specific binding member such as an antibody (or mixture of antibodies)
  • the anti- human FasL monoclonal antibodies 4A5 and 4H9 described in (28) .
  • the specific binding member may be a Fas protein having the amino acid sequence shown in figure 7 or a polypeptide having Fas function.
  • the sample may be tested by being contacted with a specific binding member such as an antibody under appropriate conditions for specific binding, before binding is determined, for instance using a reporter system as discussed.
  • a specific binding member such as an antibody under appropriate conditions for specific binding
  • different reporting labels may be employed for each antibody so that binding of each can be determined.
  • PCR comprises steps of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerisation.
  • the nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA.
  • PCR can be used to amplify specific sequences from genomic DNA, specific RNA sequences and cDNA transcribed from mRNA, bacteriophage or plasmid sequences.
  • the FasL nucleic acid sequence provided herein readily allows the skilled person to design PCR primers, see for example figure 9. References for the general use of PCR techniques include Mullis et al, Cold Spring Harbor Symp . Quant.
  • Antisense oligonucleotide sequences based on the FasL nucleic acid sequence described herein may also be used for blocking the Fas/FasL interaction according to the present invention.
  • Antisense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of polypeptide encoded by a given DNA sequence (e.g. native FasL polypeptide), so that its expression is reduced or prevented altogether.
  • antisense techniques can be used to target the control sequences of the FasL gene, e.g.
  • antisense oligonucleotides can interfere with FasL control sequences .
  • the construction of antisense sequences and their use is described in Pey an and Ulman, Chemical Reviews, 90:543- 584, (1990), Crooke, Ann. Rev. Pharmacol. Toxicol . , 32:329-376, (1992), and Zamecnik and Stephenson, P.N.A.S, 75:280-284, (1974) .
  • Oligonucleotide probes or primers, as well as the full- length sequence (and mutants, alleles, variants and derivatives) are also useful in screening a test sample for the presence of mRNA encoding FasL and/or its alleles, mutants and variants, to determine the presence or absence of FasL in a biological sample, for diagnosis and/or prognosis of immunodeficiency disease, the probes hybridising with a target sequence from a sample obtained from the individual being tested.
  • the conditions of the hybridisation can be controlled to minimise non-specific binding, and preferably stringent to moderately stringent hybridisation conditions are preferred.
  • the skilled person is readily able to design such probes, label them and devise suitable conditions for the hybridisation reactions, assisted by textbooks such as Sambrook et al (1989) and Ausubel et al (1992) .
  • binding agents such as antibodies or nucleic acid sequences
  • the present invention provides a kit comprising a support or diagnostic/prognostic chip having i immobilised thereon one or more binding agents capable of specifically binding FasL polypeptide and/or its functional variants, optionally in combination with other reagents (such as labelled developing reagents) needed to carrying out an assay.
  • Antibodies anti-human Fas monoclonal IgM was obtained from UBI, New York; anti-human Fas ligand monoclonal antibodies, 4A5 and 4H9 , were described previously (28) .
  • MAb to SIV-nef protein were made by NIBSC, London.
  • PE- conjugated anti-CD4 and CD8 mAbs were purchased from Becton Dickinson and Dako diagnostics, respectively.
  • Fas-Fc fusion protein PCR primers F Fas Kpn AAT GCG GTA CCT AGA TTA TCG TCC AAA AGT GTT AAT GCC C and R Fas Bel GCA CTT TGA TCA GAT CTG GAT CCT TCC TCT TTG CAC TT were 5 used to amplify sequences encoding the extracellular region of the Fas protein from PHA blasted PBMC cDNA. Following digestion with the appropriate restriction enzymes the fragment was cloned into a CMV driven expression vector forming a fusion with the Fc region of 0 human IgGl . The plasmid DNA was then used for transient transfection of COS cells by DEAE dextran (29) .
  • Fas-Fc fusion protein was passed over a protein A sepharose column and eluded with 0.1M citric acid (pH3.0) .
  • the Fas-Fc fusion contained 5 the Fas-derived amino acid sequence shown in figure 8.
  • Cells CEM or Jurkat CD4 + T-lymphoblastoid cell lines were obtained from the American Type Culture Collection. Macaque PBMCs were isolated on Ficoll-Hypaque and cultured in a R10H medium (RPMI containing 10% human AB serum) as indicated.
  • SIVmac251 virus pool (1) The pC8 clone differs from the pJ5 clone by a 4 amino acid deletion in the nef open reading frame and expresses an attenuated phenotype in vivo (4) .
  • Four cynomolgus macaques were infected intravenously with pC8 (10 4 half maximal tissue culture infectious dose, TCID 50 ) for 12 months and then challenged with a pathogenic SIVmac32H clone, pJ5 (50 macaque infectious dose, MID 50 ) for further 3 months.
  • Another group of four naive macaques was infected with pJ5 (50 MID 50 ) only.
  • SIV-specific CTL activity was measured in bulk cultures as previously described (30) . Briefly, PBMC were isolated on Ficoll-Hypaque and one-tenth of the autologous PBMC were stimulated with ConA (5 ⁇ g/ml) for 24 hours. Cells were infected with lOO ⁇ l SIV pC8 supernatant for 2 hours, washed and then added back to the remaining cells. Infected cells were then cultured in R10H medium for 3 days and maintained for another 7-14 days in medium supplemented with 10 U/ml IL-2. H.
  • Papio- transformed autologous B cell lines infected with recombinant vaccinia viruses carrying the SIV mac nef, gag/pol , env, RT, rev ta t or control (influenza NP) gene were used as target.
  • soluble Fas-Fc fusion protein (5 ⁇ g/ml) was added initially in bulk cultures and the cells were then washed before using as effector cells in CTL assay. Cytotoxicity was determined by culturing sl Cr- labelled target cells with effector cells at various effector : target (E:T) ratios for 4 hours in 96-well U-bottomed plates.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • lOO ⁇ l lysis buffer 1% NP-40, 20mM EDTA, 50mM Tris-HCL, pH7.5
  • the supernatant was collected and treated with 1% SDS and RNAseA (5 ⁇ g/ml) at 56 °C for 2 hours.
  • DNA extracted from naive controls had ⁇ 20% of the counts as determined by this method.
  • PBMCs (5xl0 5 ) were incubated first with anti-Fas IgM monoclonal antibody and then with a secondary FITC-conjugated rabbit anti -mouse IgM (Sigma) . Fas stained cells were then counterstained with a PE-conjugated anti-CD4 or CD8 mAbs .
  • SIV nef antigen the infected cells were incubated with the anti-SIV nef mAb together with 0.3% saponin (Sigma) and then stained with a secondary FITC-conjugated rabbit anti-mouse Ig (Sigma) . Labelled cells were analysed on a Becton Dickinson
  • FasL Fas ligand
  • FasL expression was assessed using a bioassay for FasL (33) .
  • SIV-infected cells were co-cultured with sl Cr- labelled Fas-sensitive Jurkat cells at various E:T ratios in the presence or absence of the human Fas-Fc fusion protein (lO ⁇ g/ml) or blocking anti-FasL mAbs (5 ⁇ g/ml) for 12-16 hours.
  • the level of Chromium release into the supernatant was determined using a -plate counter.
  • T-cells in particular CD8 + T-cells, from pJ5- infected animals are more vulnerable to apoptosis that those from pC8- infected animals.
  • Fas expression was analysed on lymphocyte subsets obtained from pJ5 and pC8-infected animals ( Figure 3) . Fas expression was higher on both CD4 + and CD8 + T-cells in pJ5 group .
  • FasL Engagement of FasL is required for Fas-induced apoptosis, so to search for the source of FasL a sensitive bioassay was used.
  • the assay exploits the sensitivity of Jurkat cells to Fas-mediated killing which can be blocked by the addition of an excess of soluble Fas-Fc fusion protein or anti-FasL mAbs.
  • PBMC or CEM cells were infected in vi tro with either pC8 or pJ5 and then co-cultured with 51 Cr-labelled Jurkat cells ( Figure 4) .
  • Loss of functional immune cells is a hallmark of AIDS. This was initially thought to result from direct viral cytotoxicity on CD4 + T-cells with a consequent loss of T- cell help (35) . However it is now clear that a considerable loss of uninfected bystander lymphocytes occurs in HIV-infected individuals. Much of this loss is due to apoptosis occurring in both CD4 + and CD8 + T-cells (18, 26, 32, 36). Why this happens is not understood.
  • FasL expression is tightly regulated being confined to activated lymphocytes, Sertoli cells, stromal cells of the anterior chamber of the eye, and neurons (22) .
  • the expression at these non lymphoid sites of immune privilege suggests that FasL may play a role in protection from immunological attack (37) .
  • allogeneic transplanted testes from gld mice which lack FasL expression are rapidly rejected (38) .
  • Tumour cells may also express FasL, gaining immune privilege and escaping an anti-tumour immune response (25, 39, 40) .
  • HIV has been shown to upregulate FasL expression on macrophages (41) and HIV tat/gpl20 can enhance anti-CD3 induced apoptosis by increasing the expression of FasL on CD4+ cells (42) .
  • This study has assessed the effects of FasL upregulation upon apoptosis and the course of infection in vivo . It is demonstrated that freshly isolated PBMC show increased FasL expression and kill Fas-sensitive target which is blocked by soluble Fas-Fc fusion protein or anti-FasL mAb.
  • FasL The activity of FasL is contained within the CD4 + population, possibly SIV-infected cells.
  • nef-mutant pC8 SIV-infected cells do not upregulate FasL expression.
  • the nef gene codes for a protein that is not essential for viral growth in vi tro, but which is essential to the development of AIDS (43) .
  • Nef leads to the downregulation of CD4 expression and is believed to increase the state of T-cell activation through interactions with proteins involved in cellular activation and signalling such as Src family tyrosine kinases (44) .
  • T-cell activation vai several modalities leads to an increase in FasL expression (33) , so nef through enhancing T-cell activation may similarly lead to the expression of FasL.
  • the mechanisms underlying the failure of the ⁇ ef-mutant SIV pC8 to induce FasL expression require clarification.
  • the present inventors have shown that full length nef expression by vaccinia does not upregulate FasL. This may be due to either counter-activity induced by vaccinia gene products or other HIV/SIV genes may be involved.
  • FasL is the cause for the pathogenicity of wild type SIV pJ5.
  • the FasL expression by infected CD4 + cells can trigger apoptosis of virus-specific CTL which themselves express Fas. This situation thus mimicks the expression of FasL at sites of immune privilege, or the upregulation of FasL by certain tumours. In this way the virus can evade the immune response by preventing the development of an effective CTL response.
  • the effective CTL response developed by macaques infected with pC8 (which does not cause FasL expression in CD4 + cells) suggests that inhibition of the FasL activity on infected cells may restore CTL responses. This is indeed the case, the results show that incubation of cells from the infected macaque with soluble Fas leads to the generation of an efficient anti-nef CTL response.
  • Monkey N113-116 were infected intravenously with 10 4 TCID 50 of SIVpC ⁇ for 35 months and then together with naive N174-177 monkeys challenged intravenously with 50 MID 50 SIV pJ5 clone.
  • Antibody to SIV gpl40 was determined by ELISA using anti-SIV gpl40 mAbs and data is end point titres expressed as log 10 .
  • Herpes papio-transformed autologous B cell lines were infected with recombinant vaccinia viruses (10 pfu/cell) expressing SIV proteins for 2 hours at 37°C. After washing the cells were culture in RPMI 10% FCS for 12 hours and then used as target cells.
  • ⁇ Specific lysis was calculated by subtracting the background killing of rW fluNP-infected target.
  • HIV-1 human immunodeficiency virus type 1
  • CTL cytotoxic T lymphocyte
  • CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells. Proc . Na tl . Acad . Sci . USA . 92:7312-7316.
  • Fas ligand mediates activation-induced cell death in human T lymphocytes. J " . Exp. Med. 181:71-77.

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Abstract

L'invention concerne, entre autres choses, un procédé permettant de réduire la déplétion de cellules de TK CD8+, exprimant le récepteur Fas, d'une population de cellules immunitaires qui comporte également des cellules CD4+ activées exprimant un ligand Fas. Le procédé consiste à mettre en contact la population de cellules immunologiques avec une quantité suffisante d'un agent qui est susceptible d'interférer dans toute interaction entre les récepteurs Fas et FasL.
PCT/GB1998/000485 1997-02-17 1998-02-17 Matieres et procedes relatifs a la protection de cellules immunitaires utiles WO1998035692A1 (fr)

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CN102939536A (zh) * 2009-11-14 2013-02-20 匡-宇·邱 用于治疗和/或预防动脉粥样硬化的免疫调节方法和系统

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102939536A (zh) * 2009-11-14 2013-02-20 匡-宇·邱 用于治疗和/或预防动脉粥样硬化的免疫调节方法和系统

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