EP1230357A2 - In tr1-zellen differentiell exprimierte gene und deren verwendung zur herstellung von immunoregulativen zusammensetzungen - Google Patents

In tr1-zellen differentiell exprimierte gene und deren verwendung zur herstellung von immunoregulativen zusammensetzungen

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Publication number
EP1230357A2
EP1230357A2 EP00969666A EP00969666A EP1230357A2 EP 1230357 A2 EP1230357 A2 EP 1230357A2 EP 00969666 A EP00969666 A EP 00969666A EP 00969666 A EP00969666 A EP 00969666A EP 1230357 A2 EP1230357 A2 EP 1230357A2
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EP
European Patent Office
Prior art keywords
gene
treg
cells
regulator
nucleic acid
Prior art date
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Application number
EP00969666A
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English (en)
French (fr)
Inventor
Elizabeth Adams
Herman Waldmann
Stephen Cobbold
Diana Zelenika
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication of EP1230357A2 publication Critical patent/EP1230357A2/de
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention is based, at least in part, on the elucidation of components of the Treg l mmunoregulatory pathway
  • the invention relates to genes which are differentially ex ⁇ ressed in certain T cell populations and to their expression products (e g cognate proteins), cognate receptors regulators, binding partners and inhibitors or mimetics thereof
  • the invention relates to pharmaceutical compositions and therapeutic and prophylactic methods based on the use, as targets for therapeutic intervention, of components of the Treg lmmunoregulatory pathway
  • Immunoregulation is of fundamental importance in the management and treatment of a wide variety of diseases and disorders
  • immunostimulation may be indicated m the treatment of infection and certain pro ferative disorders (such as cancer)
  • ThO cells naive CD4 + T helper cells
  • Thl lymphocytes contribute to cellular immunity
  • Th2 lymphocytes appear to contribute mainly to humoral immunity
  • Thl lymphocytes have characteristic cytokme profiles
  • Thl lymphocytes produce 1L- 2, IF ⁇ s- and TNF- ⁇
  • Th2 lymphocytes pro ⁇ uce IL-4, IL-5 and IL-10 Thl cells may be induced by actuation in the presence of IFN ⁇ and IL-12, whereas IL-4 can direct the differentiation of Th2 cells
  • T regulatory ceils T regulatory ceils
  • CD4 + T cell immunoregulatory network both in terms of its cellular elements (i.e. the nature and number of its constituent T cell subsets) and biochemical elements (i.e. the molecular composition of the regulatory network(s)) remains obscure.
  • biochemical elements i.e. the molecular composition of the regulatory network(s)
  • the present invention is based, at least in part, on the elucidation of elements of the Treg immunoregulatory network and on the development of a general approach for studying the network.
  • the pattern of gene expression in certain cell populations which are enriched in Treg cells is quite different from that in Th l- and Th2-enriched populations.
  • This finding was quite unexpected, since Treg cells have been suggested to suppress Thl and/or Th2 responses via a mechanism which does not require differential gene expression (viz. a passive competition for cytokines or antigen presenting cells - see Waldmann and Cobbold (1 98), Ann. Rev. Immunology 16, pages 619-644).
  • an isolated gene obtainable by a process comprising the steps of: (a) providing a Thl -enriched cell population, a Th2-enriched cell population and a Treg-enriched cell population; and (b) comparing the relative expression of one or more genes in said cell populations; thereby (c) identifying a gene which is differentially expressed in the cell populations; and then (d) isolating the gene identified in step (c).
  • the populations provided in step (a) need not be clonal populations, and they may therefore contain cells other than the T cells in which they are enriched.
  • the Treg-enriched population may contain ancillary cells which, though not themselves Treg cells, are supported and/or stimulated by the Treg cells and so neverthelss contribute to the Treg regulatory pathway.
  • the gene of the invention may be any gene which is differentially expressed in the populations when examined as a whole, so that the genes of the invention may include those which, at the cellular level, are differentially expressed not in Treg cells but in other ancillary cell subpopulations.
  • the gene identified in step (c) is one which is over- or under-expressed in the Treg-enriched population relative to the Thl- and Th2-enriched populations.
  • the differentially expressed genes of the invention comprise the constituent generic elements of the Treg immunoregulatory network. As such, they permit the identification of a wide range of novel targets for immunoregulatory agents. For example, those skilled in the art will appreciate that knowledge of (at least part of) the sequence of a given gene permits isolation or synthesis of the corresponding protein. Such proteins form a particularly important aspect of the invention, since they (or their agonises/antagonists) can be used as the basis for novel immunoregulatory drugs.
  • the Treg-enriched cell population is preferably that prepared as described herein (and referred to hereinafter as Trl/Treg cells).
  • Trl/Treg cells any other enriched population of Treg subsets (e.g. based on the known Tregl and Th3 cells mentioned earlier) from any suitable source can also be used.
  • the principal elements of the Treg immunoregulatory network may be elucidated by using any Treg subset as a reference population.
  • isolated is used herein to indicate that the material exists in a physical milieu distinct from that in which it occurs in nature.
  • the isolated gene or protein may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
  • the absolute level of purity is not critical, and those skilled in the art can readily determine appropriate levels of purity according to the use to which the material is to be put.
  • the isolated material will form part of a composition (for example a more or less crude extract containing many other substances), buffer system or pharmaceutical excipient, which may for example contain other components (including proteins, such as albumin).
  • a composition for example a more or less crude extract containing many other substances
  • buffer system or pharmaceutical excipient, which may for example contain other components (including proteins, such as albumin).
  • the isolated material may be purified to essential homogeneity, for example (and in the case of the proteins of the invention) as determined by PAGE or column chromatography (for example HPLC or mass spectrometry).
  • the isolated material of the invention may be essentially the sole active ingredient of the composition.
  • Particularly preferred are compositions in which the material of the invention is present as the sole active ingredient in a pharmaceutical composition.
  • isolated as applied to the genes and nucleic acids (both RNA and DNA) of the invention also indicates that the genes/nucleic acids may be present in any of a wide variety of vectors and in any of a wide variety of host cells (or other milieu, such as buffers, viruses or cellular extracts).
  • Table 1 Examples of genes according to the invention are listed in Table 1. In this table, the abundance of each tag is indicated on the left-hand side, while the columns on the right hand side indicate the degree to which a transcript is up/down-modulated with a statistical confidence of 95%. Table 1
  • TGAAACACTG MA-3 TIS (inducer of apoptosis) 1.2
  • homologue is used herein m two distinct senses It is used sensv stricto to define the corresponding gene from a different organism (l e a species variant), in which case there is a direct evolutionary relationship between the gene and its homologue This may be reflected in a structural and functional equivalence, the gene and its homologue performing the same role in each organism
  • Particularly preferred species variants according to the invention are the human homologues of each of the mouse genes shown in Table 1
  • homolog ⁇ is recognised on the basis of purely structural criteria by the presence of nucleic acid sequence
  • homologues may be recognised as those genes the corresponding DNAs of which-are capable of specifically or selectively cross-hybridizing, or which can cross-h b ⁇ d ⁇ ze under selective, appropriate and/or appropriately stringent hybridization conditions
  • a homologue may be used herein to define a protein which has an amino acid sequence which differs from the reference protein (e g the native mu ⁇ ne protein) because of one or more deletions, insertions or substitutions
  • the homologous amino acid sequence preferably is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical
  • the percent identity may be determined, for example by comparing sequence information using the GAP computer program, version 6 0 described by Devereux et al (1984), Nucl Acids Res 12.387 This program uses the alignment method of Needleman and Wunsch (1970), J Mol Biol 48 443, as revised by Smith and Waterman (1981), Adv Appl Math 2 482
  • the preferred default parameters for the GAP program include (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides.
  • Protem homologues may comprise conservatively substituted sequences, meaning that a given ammo acid residue is replaced by a residue having similar physiochemical characte ⁇ stics
  • Conservative substitutions are well known in the art and mclude substitution of one aliphatic residue for another (such as lie, Val, Leu or Ala for one another), or substitutions of one polar residue for another (such as between Lys and Arg, Glu and Asp, or Gin and Asn)
  • Conventional procedures and methods can be used for making and using such homologues
  • Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known and routineh performed
  • derivatives as applied herein to both genes and protems is used to define modified versions of the genes and proteins
  • Such derivatives may mclude fusion proteins, in which the protems of the invention have been fused to one or more different protems or peptides (for example an antibody or a protein domain conferring a biochemical activity, to act as a label, or to facilitate purification)
  • the derivatives ma% also be products of synthetic processes which use a gene or protem of the invention as a starting material or reactant
  • mutant form is used herein to define genes in which one or more nucleotides have been added deleted or substituted
  • the mutant forms of the invention therefore include fragments, truncates and chimeras
  • the mutant forms of the genes encode corresponding mutant proteins, termed "mutems' these are proteins in which one or more ammo acids have been added, deleted or substituted
  • the muteins of the invention therefore include fragments, truncates and fusion proteins
  • the muteins of the invention also include proteins in which mutations have been introduced which effectively promote or impair one or more activities of the protein, for example mutations which promote or impair the function of a receptor, a recognition sequence or an effector binding site
  • Muteins may be produced by any convenient method Conveniently, site-directed mutagenesis with mutagenic ohgonucleotides may be employed using a double stranded template (pBluesc ⁇ pt KS II TM construct containing the gene After verifying each mutant derivative by sequencing, the mutated gene is excised and inserted into a suitable vector so that the modified protein can be over-expressed and purified
  • a double stranded template pBluesc ⁇ pt KS II TM construct containing the gene
  • the mutated gene is excised and inserted into a suitable vector so that the modified protein can be over-expressed and purified
  • the term "equivalent” as used herein and applied to the materials of the invention defines materials (e.g. protems, DNA etc.) which exhibit substantially the same functions as those of the materials of the invention while differing in structure (e.g. nucleotide or amino acid sequence).
  • Such equivalents may be generated for example by identifying sequences of functional importance (e.g. by identifying conserved or canonical sequences or by mutagenesis followed by functional assay), selecting an amino acid sequence on that basis and then synthesising a peptide based on the selected amino acid sequence.
  • Such synthesis can be achieved by any of many different methods known in the art, including solid phase peptide synthesis (to generate synthetic peptides) and the assembly (and subsequent cloning) of oligonucleotides.
  • homologues, fragments, muteins, equivalents or derivatives of the proteins of the invention may also be defined inter alia as those proteins which cross-react with antibodies to the proteins of the invention.
  • the relative expression of the genes of the invention may be compared by any one of a number of standard techniques. Such techniques may measure expression at various levels, for example at the level of gene transcription or at the level of translation. A particularly convenient technique is described by Velculescu et al. (1995), Science, Vol. 270, pages 484-487. The technique, referred to herein as serial analysis of gene expression (SAGE), allows the quantitative and simultaneous analysis of a large number of transcripts. Other techniques based on an analysis of the nature and quantities of the proteins produced by the cells (proteomic analysis), for example using 2-dimensional chromatography and microsequencing, may also be used.
  • SAGE serial analysis of gene expression
  • the invention also contemplates an isolated regulator of the gene of the invention.
  • the gene regulator may be an activator or repressor of transcription of the gene, or antisense nucleic acid corresponding to the gene.
  • the invention also contemplates an isolated regulator of the protein of the invention.
  • the protein regulator may be an agonist or antagonist of the protein, and may be selected from a cognate receptor of the protein, an antibody or antibody fragment which blocks the activity of the protein or its cognate receptor, a mimetic, an activator or repressor of transcription of a gene encoding a cognate receptor of the protein, a nucleic acid encoding any of (a) to (d) or antisense DNA corresponding to the nucleic acid of(e).
  • the invention also contemplates an isolated binding partner for the gene of the invention.
  • the binding pa ⁇ ner may be nucleic acid (optionally, single stranded nucleic acid).
  • an isolated binding partner for the protem of the invention as well as an isolated binding partner for the regulator of the invention
  • the binding partner for the protem or regulator preferably comprises an antibody (or antibody derivative) specific for (or selectively reactive with) the protein or regulator
  • the antibody may be a monoclonal antibody, and may be labelled
  • the invention relates to an isolated single stranded nucleic acid comprising the complement of the coding or non-codmg strand of the gene of the invention
  • nucleic acid hyb ⁇ dizable with the gene of the invention Such nucleic acids may function as probes
  • anti-sense DNA corresponding to the gene of the invention
  • Such DNA may be used to regulate the expression of the cognate gene in VTVO, and may therefore form the basis of therapeutic compositions
  • the un entio ⁇ also contemplates a vector (e g an expression vector) comprising the gene of the invention
  • a vector e g an expression vector
  • Any suitable vector may be used, including plasmid, virus, bacte ⁇ ophage, transposon, minichromosome, posome or mechanical carrier
  • the expression vectors of the invention are DNA constructs suitable fo> expressing which may mclude (a) a regulatory element (e g a promoter, operator, activator, repressor and/or enhancer), (b) a structural or coding sequence which is transcribed into mRNA and (c) appropriate transcription, translation, initiation and termination sequences They may also contain sequence encoding any of various tags (e g to facilitate subsequent purification of the expressed protem, such as affinity (e g His) tags)
  • vectors which comprise an expression element or elements operably linked to the DNA of the invention to provide for expression thereof at suitable levels
  • expression element or elements may for example be selected from promoters, enhancers, ⁇ bosome binding sites, operators and activating sequences
  • expression elements may comprise an enhancer, and for example may be regulatable, for example being inducible the addition of an inducer
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence
  • DNA for a signal peptide secretory leader
  • a promoter is operabK linked to a coding sequence if it controls the transcription of the sequence
  • a ⁇ bosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation
  • the vector may further compnse a positive selectable marker and/or a negative selectable marker The use of a positive selectable marker facilitates the selection and/or identification of cells containing the vector
  • the invention relates to a host cell comprismg tne vector of the invention
  • a host cell comprismg tne vector of the invention
  • Any suitable host cell may be used, including prokaryotic host cells (such as Escher chia coli, Streptomyces spp and Bacillus s btilis) and eukaryotic host cells
  • composition comprismg the gene, protem, regulator binding partner, nucleic acid, vector or host cell of the mvention as an active ingredient, the gene, protein, regulator, binding partner, nucleic acid, vector or host cell optionally being present at a concentration sufficient to confer biological activity on the pharmaceutical composition
  • a pharmaceutical composition is a solid or liquid composition m a form, concentration and level of purity suitable for administration to a patient (e g a human or animal patient) upon which administration it can elicit the desired physiological changes
  • the composition may be an immunoregulatory composition, for example an lmmuno-suppressive composition, immuno-stimulatory composition or anti-inflammatory composition
  • the gene protem, regulator, bindmg partner, nucleic acid, vector or host cell of the invention may be for use in medicine (for example in therapy, prophylaxis or diagnosis) and/or provided in a pharmaceutical excipient, a unit dosage form or in a form suitable for local or systemic administration
  • the indention also contemplates a diagnostic kit or reagent comprismg the gene, protem, regulator, binding partner, nucleic acid, vector or host cell of the invention
  • the invention also contemplates a cell typing reagent comprising the gene, protein, regulator, binding partner nucleic acid, vector or host cell of the invention
  • the mvention provides an ex vivo method for analysing the status of the Treg immunoregulatory pathway in a biological sample comprising the step of determining the presence or activity of (a) a gene of the invention, or (b) a cognate expression product(s) of said gene, or (c) a biochemical marker of the activity of said gene, or (d) Thl and/or Th2 and/or Treg cells
  • the invention provides an ex vivo method for detectmg Thl and/or Th2 and/or Treg cells in a biological sample comprising the step of contacting the sample with a reagent which selecrivel) binds to the gene, protem, regulator or bmdmg partner of the mvention
  • the reagent comprises the cell typing reagent of the mvention
  • the detecting method of the mvention may be for detecting the presence of the cells or for determining the number of Thl and/or Th2 and/or Treg cells.
  • the number of Thl cells relative to Th2 and/or Treg cells, or Th2 cells relative to Thl and/or Treg cells, or Treg cells relative to Thl and'or Th2 cells may be determined
  • the mvention relates to an ex vivo method for monitoring the progress of an anti-mflammatory or immunoregulatory treatment in a sub j ect or the progress of an inflammatory or immune disorder in a subject, the method comprismg the step of analysing the status of the Treg immunoregulatory pathway in a sample from the subject
  • the method monitors the efficacy or endpoint of a treatment
  • Also contemplated by the mvention is an ex ⁇ ivo method for determining the posology of an anti- lnflammatory or immuno-regulatory drug administration regime in a subject undergoing treatment comprising the step of analysing the status of the Treg immunoregulatory pathway m a biological sample comprising the step of determining the presence or activity of (a) a gene of the mvention, or (b) a cognate expression product(s) of said gene, or (c) a biochemical marker of the activity of said gene, or (d) Thl and/or Th2 and/or Treg cells
  • the samples for use in the foregoing methods may be from an accessible body site, for example a mucous membrane of the vagma, anus, nose, urethra, cervix, skin, conjunctiva, mouth or throat
  • the sample mav comprise a fluid or semi-solid (for example a bodily fluid or semi-solid, e g discharge, vomit, secretion, excreta, urine, sputum, plasma, serum or blood)
  • the sample may compnse a solid (e g stool, tissue or biopsy sample) or a cell culture (e g a lymphocyte culture)
  • a cell culture e g a lymphocyte culture
  • Also contemplated by the invention is the use of the gene, protem, regulator, bmdmg partner, nucleic acid, vector or host cell of the invention for the manufacture of a medicament for use in the treatment of an inflammatory or immune disorder
  • Also contemplated by the invention is the use of the gene, protein, regulator, binding partner, nucleic acid, vector or host cell of the invention for the manufacture of an agent for use in a bipartite anti- mflammatory or immunoregulatory treatment, the first part comprising administration of an anti- lnflammatory or immunoregulatory agent and the second part comprising monitormg the progress of the treatment by analysing the status of the Treg immunoregulatory pathway m a biological sample comprising the step of determmmg the presence or activity of (a) a gene of the mvention, or (b) a cognate expression product(s) of said gene, or (c) a biochemical marker of the activity of said gene, or (d) Thl and/or Th2 and/or Treg cells
  • the efficacy and or endpoint of treatment is monitored by the method of the invention
  • an anti-mflammatory or immunoregulatory agent for the manufacture of a medicament for the treatment of an inflammatory or immune disorder m a sub j ect, characterized in that the treatment comprises the step of analysing the status of the Treg immunoregulatory pathway the subject determmmg the presence or activity of (a) a gene of the mvention, or (b) a cognate expression product(s) of said gene, or (c) a biochemical marker of the activity of said gene, or (d) Thl and or Th2 and/or Treg cells
  • the biochemical marker may for example comprise a molecule which is produced, directly or indirecth by the gene and which therefore serves as an index of the expression of said gene
  • the step of analysing the status of the Treg immunoregulatory pathway in the subject may be for monitormg the efficacy of an anti-inflammatory or unmunoregulatory treatment, or for determmmg the posolog of an anti-mflammatory or immunoregulatory drug administration regime
  • the mvention provides a process for producing a pharmaceutical composition comprismg the steps of
  • step (d) synthesising or purifying a drug having pharmaceutical activity on the basis of the identity of the candidate drug screened in step (c)
  • composition produced by (or obtainable by) the aforementioned process or a derivative thereof
  • the invention provides a process for producing the gene, protein, regulator, binding partner, nucleic acid or vector of the invention comprising the steps of (a) cultu ⁇ ng the host cell of the invention, and (b) purifying the gene, protem, regulator, binding partner, nucleic acid or vector from the cultured host cell (e g from a culture supernatant or cell fraction)
  • the invention provides an isolated Treg cell (which may be capable of suppressing the proliferation and cytokme production of Thl and Th2 cells), said ce'l having a gene expression profile substannally as shown in Table 1
  • the gene expression profile is such that the expression of at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the genes listed in Table 1 are up- or down- regulated relative to Thl and Th2 cells at least to the extent mdicated in the Table
  • the cell of the mvention may be used as a test system in the preparation and screenmg of candidate drugs, or directly (in adoptive immunotherapy protocols) in therapy or prophylaxis In the latter case, the cell may be used in immunoregulation (e g immunosuppression or lmmunostimulation) or the treatment of inflammation
  • the cells of the invention may be administered by any convenient method, for example by site-specific instillation (e g into a capillary bed or into a vessel via a catneter)
  • the mvention permits the isolation, synthesis and rational design of a wide l ange of novel medicaments and pharmaceuticals for use m therapy, prophylaxis and diagnosis
  • the various forms of therapy, prophylaxis and diagnosis in which the materials of the invention find application involve targeting the Treg immunoregulatory pathway
  • the mvention finds application in therapies based on immunoregulation
  • the mvention is used to effect immunosuppression, while m other embodiments lmmunostimulation is achieved
  • the invention provides anti-mflammatory agents for use in a wide range of applications, for example in the treatment or prophylaxis of chronic inflammatory disorders
  • the mvention may also be used to treat damage to cells resulting from the effects of inflammatory response
  • the mvention finds application in the treatment of rheumatoid and osteo-arth ⁇ tis and of glomerular nephritis, diabetes, inflammatory bowel disease, vascular diseases such as atheroclerosis and vascuhtis and skin diseases such as psoriasis and dermatitis
  • the mvention may also be used to prevent or treat graft rejection
  • diseases which may be treated according to the invention include Crohn's disease, ulcerative colitis, Type I diabetes, lupus erythematosus, auto-immune disorders, hypersensitivity disorders and multiple sclerosis
  • a variety of lung diseases are associated with airway inflammation, including chronic bronchitis, emphysema, ldiopathic pulmonary fibrosis and asthma, and these also may be treated accordmg to the invention
  • the lmmunostimulants of the invention find particular application in the treatment of infections and prohferative disorders (such as cancer)
  • the genes and their cognate protein products described herein form part of the Treg immunoregulatory network As such their activity can be used as an index of the status of the network they can be used as "markers' which are either present or absent in accessible body fluids and tissues (usually blood, serum urine or biopsy material)
  • the mvention finds application in monitoring during and after therapy for multiple sclerosis
  • the blood may initially show evidence of Thl behaviour directed to brain antigens-there are numerous documented examples of increases in the frequency of antigen-specific cells m acute episodes of the disease. These cells activate particular genes of the Treg immunoregulatory network and can be identified according to the invention. Successful treatment would damp this activation, lowering the expression of these genes and allowing the efficacy and/or endpoint of the treatment to be determined.
  • 5xl 0 3 spleen cells were cultured with 5x l 0 6 mitomycin C treated male CBA spleen cells (male stimulators) in 2 ml volumes in RPMI 1640 medium containing 10% foetal calf serum (FCS) and 50ng/ml recombinant murine IL-10 (Genzyme) for 7 days at which time spent medium was removed and fresh stimulator cells and medium containing IL-10 added.
  • FCS foetal calf serum
  • Genzyme recombinant murine IL-10
  • the viable cells were harvested by separation over Ficoll/Hypaque and cloned by limit dilution ( 1 cell/200 microlitre well) on anti-CD3 coated plates (1452C1 1, 50 microgram/ml) in the presence of mitomycin treated female CBA spleen feeder cells (10 7 /ml) in RPMI 1640 containing 10% FCS and 20U/ml recombinant murine IL-2 (rmIL-2).
  • Wells containing growing cells were progressively expanded into 48 x 1 l and then 24 x 2ml well plates with male stimulators in RPMI 1640 containing 10% FCS, rmIL-2 (20U/ml) and recombinant murine IL-4 (rmIL-4 20U/mI).
  • RPMI 1640 containing 10% FCS, rmIL-2 (20U/ml) and recombinant murine IL-4 (rmIL-4 20U/mI).
  • Thl and Th2 cells for use in the analysis were prepared as described in Zelenika et al. (supra).
  • Example 2 Testing for mRNA expression of differentially expressed Treg gene sequences by quantitative real-time RT-PCR.
  • RNA was obtained from each of the Thl, Th2 and Treg clones, as well as normal spleen cells from normal CBA/Ca mice that were in the process of rejecting a B 10.BR skin graft, using standard methodology.
  • Each sample was then treated with DNA-ase to ensure that no PCR product would be generated from contaminating genomic DNA.
  • First strand cDNA was then generated using Reverse Transcriptase (RT) together with either random hexamers or polyA primers.
  • RT Reverse Transcriptase
  • This material was then subjected to real time quantitiative PCR using the standard protocols (User Bulletin #5: Multiplex PCR with TaqMan VIC Probes, and User Bulletin -2: Relative Quantitation of Gene Expression) for the Perkin Elmer Applied Biosystems ABI Prism 7700 Sequence Detection System.
  • this uses standard PCR 5' and 3 ' primers to generate a specific PCR product for the gene of interest, and utilises an internal oligonucleotide specific for the product that is doubly labelled with a fluorescent dye (eg. FAM or VIC) and a quencher dye (eg.
  • a fluorescent dye eg. FAM or VIC
  • quencher dye eg.
  • This fluorescence is detected in the ABI Prism 7700 machine by scanning the PCR reactions in an optical 96 well plate multiple times for each cycle, generating a real time semi-logarithmic plot of the PCR reaction that can be used to accurately calculate a threshold cycle (Ct) for each reaction.
  • Ct threshold cycle
  • the primers and probes of the gene of interest FAM probe
  • a housekeeping gene such as HPRT or ribosomal RNA (VIC probe)
  • HPRT ribosomal RNA
  • This approach would allow rapid, thigh throughput, simple and automatic, measurement of a number of Thl , Th2 and Treg differential genes in, for example, blood samples from normal individuals compared to patients undergoing treatment, in order to monitor the status of the patient during their course of therapy.
  • PAFAH tgatcagtgggaccctctgg gagagtgttgctgagccgg FAM-ctgggtcaccctttgacgcagtca-TAMRA
  • Example 3 Testing for expression of differentially expressed Treg gene by immunofluorescence specific for a biochemical product
  • Treg differentially expressed gene tryptophan hydroxylase is tested for indirectly by accumulation in Treg cells or tolerant T cells of the product of the biochemical pathway for which tryptophan hydroxylase is the rate limiting enzyme (i.e. serotonin).
  • Both polyclonal antisera and at least one monoclonal antibody are widely available for identification of cells containing serotonm after fixation oy formalin to generate the specific serotonin-protein con j ugate that the antibodies recognise
  • a sample of blood cells, tissue cells or other source of cells it is necessary to first fix the sample in, for example 1% formaldehyde or paraformaldehyde, and then permeabilise the cells, for example using phosphate buffered saline contaming 0 5% saponm, and then stammg by standard methods of immunofluorescence with the anti- serotomn antibody, most conveniently conjugated directly with an appropriate fluorescent dye such as fluorescein lsothiocyanate (FITC) or indirectly using anti-species specific immunoglobulm (IgG) antibodies coupled to FITC or other appropriate dye
  • FITC fluorescein lsothiocyanate
  • IgG anti-species specific immunoglobulm
  • Virtual clon z uses the data produced by random sequencing of short cDNA fragments, called ESTs If one or more matching EST is found, they are analysed and compared usmg different softwares made available to all on the web A "virtual" cDNA sequence can be produced by usmg this procedure
  • the initial tag sequence is as follows GCAGTGGTTC. All the tags were generated using an Nlalll restriction enzyme (CATG), therefore they all start with the corresponding restriction site Thus, the complete tag sequence, of 14 nucleotides, is CATGGCAGTGGTTC
  • the tag is underlined
  • the next step was to create a contig (organised overlapping ESTs fragments) and to derive a consensus sequence from it This was done usmg freely available software (available on the website http //gcg tigem lt/BLASTEXTRACT/estextract html)
  • the tag is underlined and it is indeed located next to the most 3' Nlalll site
  • the real cloning is technically completely independent from the virtual cloning. However, t he obtention of a virtual cDNA, whenever it is possible, can be useful to cross-check the real clonin°.
  • Cloning by RACE-PCR is a standard technique, although usually used from longer starting sequences.
  • cDNAs produced from our T-cell clones have been ligated at their both ends by specific adaptors. Starting from the tag sequence of 14 nt, two fragments can be generated by PCR extension towards the 3' or the 5' end of the cDNA.
  • the primers used to generate the 5' fragment are underlined :
  • the sequence of the 5' primer corresponds to the 5' SMART adaptor designed by Clontech.
  • the 3' primer is the tag itself (CATGGCAGTGGTTC)
  • the primers used to generate the 3' fragment are underlined :
  • the sequence of the 5' primer is the tag itself.
  • the 3' primer sequence is derived from the polyA- anchored-3'adaptor.
  • a 3 rd round of PCR (and cloning) has been done using an internal primer in order to show that the 5' and the 3' fragments have been generated from a unique cDNA.
  • Tne complete cDNA sequence is: TTCGGCACGATGGGTGAACACACAATCAAAACTTCTCCTGGAATAAATCATCTGACATCAG ATCCTTCCCATTGTCTGTACTGTTTCTGCTGCCCTGGAAACCATGCAAGGACAGGAACAGA CCACCCATGGCAGTGGTTCCTGGAGGTGCTCCACCTTCAGAGAATTCTGTTATGACATCA CAAATGTGGAACGAGAAGAAGGAGAAATTCTTGAAGGGGGAACCAAAGTCCTTGGGGTTT TACAAGGTATGATTGCTATCATAAACCTCAGCTTAGGAATAATAATTTCGACAACTTTGGT TTTCTGAACTACCCACTTCAGTGATGTTAATGGTCCCAATTGGGGGATCAGTAGTGTCCAT TGTCTCCGGATCCCTGGCCATTGCAGCAGGAGTGACACCTACAAAATGCCTGGTAAT TTGGTATGTTTTGGTAAT TTGGTATGTTTTGGTAAT TTGGTATGTTTTTTGGTAAT TTGGTATGTTTTGGTA
  • This new cDNA sequence contains an ORF:
  • the predicted protein deduced from that ORE shows 32% homology with 2 tetraspan proteins: Mouse CD20 receptor and the Human FcRIa.
  • This example shows that two independent methodologies can be used to determine the complete sequence of each of the gene transcripts corresponding to the genes listed in Table 1 and so permit their synthesis or isolation.
  • the particular example described above encodes a previously unidentified protem that has 4 transmembrane domains (as shown by the analysis of hydrophobicity) and probably belongs to the 4TM family of proteins together with the CD20 antigen and the FcRIa.
  • this protein may be potentially used as a biomarker of the Thl subset.
  • An antibody could be raised against the predicted protein using standard methodology. Such an antibody would be a very useful tool for monitoring Thl cells in normal and pathological situations quite independently of the biological function of the predicted protein (for which further studies can be carried out).

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EP00969666A 1999-10-08 2000-10-06 In tr1-zellen differentiell exprimierte gene und deren verwendung zur herstellung von immunoregulativen zusammensetzungen Withdrawn EP1230357A2 (de)

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