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WO2002034941A1 - Method for screening active molecules for treating and preventing atherosclerotic lesions and its uses - Google Patents

Method for screening active molecules for treating and preventing atherosclerotic lesions and its uses Download PDF

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Publication number
WO2002034941A1
WO2002034941A1 PCT/FR2001/003303 FR0103303W WO0234941A1 WO 2002034941 A1 WO2002034941 A1 WO 2002034941A1 FR 0103303 W FR0103303 W FR 0103303W WO 0234941 A1 WO0234941 A1 WO 0234941A1
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Prior art keywords
cells
molecules
lesions
screening method
molecule
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French (fr)
Inventor
Nathalie Thieblemont
Martin Mempel
Gabriel Gachelin
Philippe Kourilsky
Catherine Ronet
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Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
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Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
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Priority to AU2002214093A priority Critical patent/AU2002214093A1/en
Publication of WO2002034941A1 publication Critical patent/WO2002034941A1/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method of screening and selection of active molecules in the prevention and treatment of atherosclerotic or atheromatous lesions, to products modulating the activity and migration of cells carrying the invariant chain V l4-J ⁇ 281 of the receptor T cells
  • Atherosclerosis is a lesion of the inner layer (intima) of the arteries. More precisely, the elementary lesion of atherosclerosis or simple plaque consists of a localized thickening of the arterial intima, formed by a heap of hard fibrous tissue (sclerosis) encasing a soft center made up of modified or oxidized lipids (atheroma or lipid heart of the plaque) and lipoproteins (LDL or low density Hpoprotei). In humans, atherosclerosis is the dominant cause of obstructions that affect large and medium-sized arteries in large circulation. The diseases resulting from atherosclerosis consist of ischemia or insufficient blood supply to the organ irrigated by the obstructed artery.
  • Atherosclerosis Occurring primarily in people over the age of 40 and with an increased frequency as they age, atherosclerosis is with cancer, a leading cause of illness and death in Western countries.
  • Chronic inflammation that sets in is usually suspected of orchestrating the formation of atherosclerotic plaque.
  • the phenomena that come into play are multiple and complex, essentially involving the different categories of cells present in the diseased intima: muscle cells, endothelial cells, macrophages and lymphocytes.
  • lymphocytes present at the level of the plaque essentially consist of activated CD4 + T cells, due to the concomitant expression of HLA-DR molecules, of interleukin receptors, of antigen CD45R0 and VLA-1 antigen and various cytokines (International Application WO 00/20019).
  • mice TV cells participate, in a crucial manner, in the immune response at the level of the atherosclerotic lesion.
  • the subject of the present invention is a method for screening for active molecules capable of preventing and / or treating atherosclerosis in humans, characterized in that it essentially comprises the comparative detection of TV cells ⁇ 14-J ⁇ 281 present in atherosclerotic lesions or mimicking atherosclerotic lesions, in the absence and in the presence of the molecules to be tested (or screened).
  • active substance is understood to mean any substance capable of modulating the activity and migration of T cells carrying the invariant chain N ⁇ 14-J ⁇ 281.
  • the ⁇ 14-J ⁇ 281 TV cells are for example described in T. Kawano et al. (Science, 1997, 278, 1626-1629).
  • said detection is carried out by the comparative analysis of the AR ⁇ messengers produced by the V ⁇ 14-J ⁇ 281 cells: qualitative analysis [Immunoscope technique described in European Patent 0 613 503] or quantitative analysis: Taqman method (Perkin -Elmer, Courtaboeuf, France).
  • Such a model can thus be advantageously used to screen and select molecules capable of reducing the infiltration and therefore the migration of TN ⁇ 14-J ⁇ 281 cells in said lesions.
  • the Inventors unexpectedly found that the infiltration and migration of said TV ⁇ 14-J ⁇ 281 cells can reflect the infiltration of the lesion by other cells of the immune system (neutrophils, macrophages, other lymphocytes) and constitute a good marker for said screening.
  • the screening of molecules capable of preventing and / or treating atherosclerosis in humans can be implemented according to several variants:
  • the models of the prior art can also be used to quantify TN ⁇ 14-J ⁇ 281 cells present in atherosclerotic lesions, in the absence and / or in the presence of the molecules to be screened.
  • the induction of a subcutaneous lesion is carried out in mice, by administration of a substance present in atherosclerotic plaques, selected from the group consisting of lipoproteins LDL, cholesterol, modified molecules such as lysophospholipids, oxidized lipids or proteins, hsp65, glycolipids [phosphoinositol mannosides, lipopolysaccharides (LPS), lipo-techoic acid, glycolipids of pathogenic microorganisms, animals or humans chemically modified on the natural osidic part, synthetic glycolipids such as ceramides ( ⁇ or ⁇ ) ...].
  • a substance present in atherosclerotic plaques selected from the group consisting of lipoproteins LDL, cholesterol, modified molecules such as lysophospholipids, oxidized lipids or proteins, hsp65, glycolipids [phosphoinositol mannosides, lipopolysaccharides (LPS), lipo-techoic acid, glycolipids of path
  • said method comprises: the removal of TN tx 14-J ⁇ 281 cells from mice or from lesions suspected of containing said cells, - Stimulation of said cells in a suitable culture medium, in the presence of a substance present in atherosclerotic plaques, selected from the group consisting of substances present in atherosclerotic plaques such as:. LDL lipoproteins,
  • modified molecules such as lysophospholipids, oxidized lipids or proteins, hsp65,
  • glycolipids as defined above, preferably ⁇ GalCer [ct-galactoceramide or (2S, 3S, 4R) -lO- (oc-D-galactopyranosyl) -2- (N- hexacosanoylamino) - 1, 3, 4-octadecanetriol]
  • said detected substances are selected from the group consisting of interleukin 4 (IL-4), interferon ⁇ (IF ⁇ - ⁇ ) and the marker CD69.
  • Said substances are advantageously detected either by flow cytometry (CD69), or by ELISA (IL-4 and IF ⁇ - ⁇ ).
  • such screening methods have the following advantages: - rapid induction of lesions (approximately 7 days): the animal models of atherosclerosis currently proposed, such as mice deficient in ApoE or in LDL-R, develop detectable atherosclerotic lesions, when fed a high-fat diet, only after 4 to 5 weeks;
  • Such methods make it possible to detect different types of active substances on atherosclerosis, in particular substances inhibiting the migration of human TN ⁇ 24 cells and / or the stimulation of cytokines expressed by said T cells, such as MCP-1 inhibitors. , CCR2 or the CD ld molecule.
  • a subject of the present invention is also the use of the molecules obtained using the screening method, as defined above, for the preparation of a medicament intended for preventing and / or treating atherosclerosis.
  • said molecules are chemokine inhibitors involved in regulating the migration of TN ⁇ 14-J ⁇ 281 or TN ⁇ 24 cells, in particular inhibitors of MCP-1 or CCR2.
  • said molecules are inhibitors of the CDld molecule.
  • the present invention also relates to compositions capable of preventing and / or treating atherosclerosis (inhibition of the formation of atheroma plaques), characterized in that they comprise a molecule inhibiting the migration of human TV ⁇ 24 cells and / or the activity of said T cells and at least one pharmaceutically acceptable vehicle.
  • said molecule inhibiting the activity of human TN ⁇ 24 cells inhibits the production of cytokines expressed by said cells.
  • the present invention also relates to an active molecule screened by a process as defined above, characterized by its ability to modulate the migration and / or the activity of human N ⁇ 24 cells present in atherosclerotic lesions.
  • the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention as well as in the accompanying drawings, in which:
  • - Figure 1 shows the detection of N ⁇ l4-J ⁇ 281 T cells by Immunoscope in cholesterol-induced lesions.
  • - Figure 2 shows the differential immune responses of T cells to stimulation by LPS or ⁇ GalCer in the absence or in the presence of anti-CDl blocking antibodies.
  • the expression of the CD69 marker on the cell surface is detected by Facscan and the production of cytokines (IL-4 and IF ⁇ - ⁇ ) by the T cells are detected by immunoassay, after stimulation of the cells.
  • APC alkaline phosphatase
  • biotin- anti-TCR ⁇ H57-597
  • PE phycoerythrin
  • PE-anti-CD19 PE-anti-CD69
  • PE-anti-CD14 PE-anti-CD14
  • biotin-anti-Macl Ml / 70
  • biotin-anti-Grl RB6-8C5
  • biotin-anti-CDl lb biotin-anti-CDld (1B1), Fc ⁇ III / II (Fc block, 2.4G2)
  • APC-streptavidin are provided by PharMingen (San Diego, CA).
  • FITC fluorescein isothiocyanate
  • RA3-6B2 biotin-anti-B220 conjugated antibodies, as well as streptavidin-tricolor, are available from Caltag (San Francisco, CA).
  • the anti-CD1 d monoclonal antibody (clone 20H2) is described in Roark et al., J. Immunol, 1998, 160, 3121-3127.
  • ⁇ GalCer (2S, 3S, 4R) -lO- ( ⁇ -D-galactopyranosyl) -2- (N-hexaco- sanoylamino) -l, 3,4-octadecanetriol) is marketed by the Pharmaceutical Research Laboratory Kirin Brewery Co. Ltd. (Gunma, Japan).
  • the stock solution is adjusted to 100 ⁇ g / ml in 10% DMSO and then diluted to a final concentration of 100 ng / ml in the culture medium.
  • a 10% DMSO vehicle solution, diluted to a final concentration of 0.1%, is used as a control.
  • LPS complexed with soluble CD14 is prepared by incubating LPS (10 ng / ml) with soluble CD 14 (200 ng / ml) overnight at 37 ° C, according to the protocol described in Hailman et al., J. Immunol ., 1996, 156, 4384-4390.
  • PBS phosphate buffer
  • mice are supplied by Iffa-Credo (L'Arbresle, France).
  • mice Homozygous CD1-deficient mice are crossed with C57BL / 6 mice as described by Mendiratta in Immunity, 1997, 6, 4, 469-477.
  • mice are anesthetized with 0.1% pentobarbital and then injected intrasplenically with 100 ⁇ l of a suspension of cells enriched in N ⁇ l4, in the presence or absence of 20H2 antibody (10 ⁇ g / 10 6 cells).
  • mice aged 3 to 7 weeks are injected with an antigen and then 7 days later the animals are sacrificed and extracts of their liver and their spleen are prepared and injected into animals as indicated below. Injection to animals and collection of samples
  • the extracts containing the inducing molecules present in atherosclerotic plaques (Ox-LDL, phospholipids ...), in the presence of 12.5% alu- gel-S (Serva, Heidelberg, Germany) in a volume of 100 ⁇ l are injected, subcutaneously at the base of the tail of the C57BL / 6 mice (Apostolou et al., P ⁇ AS, 1999, 96, 5141-5146).
  • the control animals receive 100 ⁇ l of PBS by the same injection route.
  • the animals are sacrificed 7 days after the injection and skin samples corresponding to the injection site, and possibly the livers are removed and frozen immediately. From these samples:
  • PCR primers are used as a specific marker, the practically invariable character of the N ⁇ l4-J ⁇ 281 chain of the T lymphocyte receptor (TCR) and a CDR3 long with 10 amino acids.
  • the primers are synthesized by Eurogentech (Orléans, France). For the technique based on RT-PCR, the specific primers having the following sequences were used: N ⁇ l4: CTAAGCACAGCACGCTGCACA (SEQ ID ⁇ O: l); C ⁇ : TGGCGTTGGTCTCTTTGAAG (SEQ ID NO: 2); C ⁇ labeled: FAM-ACACAGGAGGTTCTGGGTTC (SEQ ID NO: 3); J 281 marked: FAM- CAGCTGAGTCCCAGCTCC (SEQ ID NO: 4).
  • RNA is extracted using the Trizol reagent (Gibco BRL) then it is precipitated with ethanol and the pellets are resuspended in 20 ⁇ l of distilled water. 10 ⁇ l are reverse-transcribed in a final volume of 40 ⁇ l using reverse transcriptase (RT) of the avian myeloblastosis virus and an oligo-dT, according to standard protocols.
  • RT reverse transcriptase
  • Two microliters of the cDNA solution obtained are amplified by 40 cycles of chain polymerization (PCR) alternating a denaturation step at 94 ° C for 30 s, a step for fixing the primers at 60 ° C for 30 s and an extension step at 72 ° C for 30 s, using the specific primers N ⁇ l4- and C ⁇ previously described.
  • Two microliters of the products amplified by PCR are used in “run-off” experiments (5 cycles) using the fluorescent primers specific for the J ⁇ 281 or C ⁇ segments previously described.
  • the length of the CDR3 region and the fluorescence intensity of the “run-off” products are determined using an automatic sequencer (Perkin-Elmer) and analyzed using “Immunoscope” software (Pannetier, P ⁇ AS, 1993, 90, 4319-4323).
  • the size of the CDR3 region of a pair NC is represented by a family of peaks separated by 3 nucleotides (derived from AR ⁇ m in phase), the area of which is proportional to the initial quantity of AR ⁇ m.
  • the six to eight peaks observed exhibit a significant distribution- Gaussian.
  • a proliferation of T cells triggered by the antigen typically results in distortions of the Gaussian distribution.
  • N ⁇ l4- J ⁇ 281 T cells was measured using primers specific for the ⁇ -actin, C ⁇ , N ⁇ l4 and J ⁇ 281 genes.
  • Mononuclear cells isolated from the liver and spleen are cultured in 96-well round-bottom microplates (Nunc), in the presence of RPMI 1640 (Gibco BRL) containing 10% fetal calf serum (Gibco BRL), 100 IU / ml of penicillin and 100 ⁇ g / ml of streptomycin, supplemented with ⁇ GalCer (100 ng / ml) or various concentrations of antigen, and / or anti-CDl blocking antibodies. The supernatants are harvested 16 to 72 hours later and stored at -20 ° C until the assays of IL-4 and IFN- ⁇ are carried out.
  • the cells are labeled with fluorescent antibodies and the expression of the activation marker CD69 on the different populations is studied by flow cytometry (Facscan).
  • the sorted T cells are cultured for 16 to 72 hours in AIM V medium (Gibco-BRL) containing antibiotics, in the presence or in the absence of antigen presenting cells. Cell culture supernatants are harvested and frozen. Cytokine assays
  • the quantification of murine IFN- ⁇ , IL-4 and TNF- ⁇ present in sera and culture supernatants is determined using standard ELISA-sandwich tests, according to the manufacturer's recommendations.
  • the ELISA IFN- ⁇ , IL-4 and TNF- ⁇ kits are supplied by Pharmingen.
  • the samples are assayed according to a modified protocol using streptavidin-peroxidase (Amersham, Les Ulis, France) and the plates are read on a cytofluorimeter (Perspective Biosystems). Data are expressed as means ⁇ standard error (SEM). Flow cytometry & cell sorting
  • liver and spleen cell suspensions are stained using 4 colors using combinations of antibodies conjugated to FITC, PE, APC or biotin then to Tricolor ® or / and streptavidin conjugated to APC , and analyzed by flow cytometry (FACS; Becton Dickinson and Co., Mountain View, CA). Phenotypic analysis of liver T cells was performed at 4 ° C. The cells are first incubated for 10 min. with anti-Fc ⁇ III / II, then exposed for 30 min. to PE-NK1.1 and TCR ⁇ -biotinylated antibodies and, if necessary, FITC-CD69. After two washes, streptavidin-Tricolor ® (Caltag) or streptavidin-APC was added. After further washes, the cells are suspended in PBS containing fetal calf serum (FCS) and analyzed according to conventional procedures. The data are analyzed using Cell-Quest software (Becton-Dickinson).
  • the colored cells TCR ⁇ ⁇ Kl.l " are sorted using a" Facstar Plus “device (Becton-Dickinson) or an" Elite “(Coulter) equipped with double lasers (argon and dye) at a rate of 3500 events per second, and harvested in RPMI supplemented with 10% fetal calf serum (FCS) Under these conditions, the purity of each enriched cell fraction is greater than 94% after re-analysis.
  • FIG. 1 show that the subcutaneous injection of cholesterol or LPS in mice induces a major infiltration of T cells at the injection site, characterized by the detection of V ⁇ l4-J ⁇ 281 from the invariant ⁇ chain TCR by Immunoscope (Fig. 1). These observations indicate that under physiological conditions, antigens such as cholesterol or LPS are capable of promoting immune responses by recruiting T cells at the injection site.
  • FIG. 2 shows that the ⁇ GalCer and the LPS are capable of stimulating T cells, in a manner dependent on the CDl molecule.
  • the ⁇ GalCer glycolipid or LPS there is a marked increase in the expression of CD69 on the surface of T cells and in the production of dTFN- ⁇ and IL-4, which are completely inhibited in the presence anti-CDl antibodies.
  • FIG. 2 also shows that the stimulation of T cells can be measured also by the increase in the expression of CD69 in flow cytometry as by the assay in ELISA of IL-4 or of IFN- ⁇ .
  • the screening of molecules capable of inhibiting the stimulation of ⁇ 14-J ⁇ 281 TV cells by tests which measure the expression of CD69, IL-4 or dTFN- ⁇ by said stimulated f cells, such as those described above allows advantageously to screen for drugs capable of preventing and / or treating atherosclerosis in humans.

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Abstract

The invention concerns a method for screening and selecting active molecules for preventing and treating atherosclerotic and atheromatous lesions, products modulating the activity and the migration of cells bearing the invariant chain V alpha 15-J alpha 281 of the T-cell receptor (TCR), and their uses. Said method comprises essentially comparative detection of T V alpha 15-J alpha 281 cells present in the atherosclerotic lesions or in miming atherosclerotic lesions, in the absence or in the presence of the molecules to be tested.

Description

PROCEDE DE CRIBLAGE DE MOLECULES ACTIVES DANS LA PREVENTION ET LE TRAITEMENT DES LESIONS ATHEROSCLEREUSES ET SES APPLICATIONS. METHOD FOR SCREENING ACTIVE MOLECULES IN THE PREVENTION AND TREATMENT OF ATHEROSCLERIC LESIONS AND ITS APPLICATIONS.

La présente invention est relative à un procédé de criblage et de sélection de molécules actives dans la prévention et le traitement des lésions athéroscléreuses ou athéromateuses, aux produits modulant l'activité et la migration des cellules portant la chaîne invariante V l4-Jα281 du récepteur des lymphocytes TThe present invention relates to a method of screening and selection of active molecules in the prevention and treatment of atherosclerotic or atheromatous lesions, to products modulating the activity and migration of cells carrying the invariant chain V l4-Jα281 of the receptor T cells

(TCR), ainsi qu'à leurs applications.(TCR), as well as their applications.

L'athérosclérose est une lésion de la couche interne (intima) des artères. De manière plus précise, la lésion élémentaire d'athérosclérose ou plaque simple consiste en un épaississement localisé de l'intima artérielle, formé d'un amoncellement de tissu fibreux dur (sclérose) enchâssant un centre mou constitué de lipides modifiés ou oxydés (athérome ou cœur lipidique de la plaque) et de lipoprotéines (LDL ou low density Hpoprotei ). Chez l'homme, l'athérosclérose est la cause dominante des obstructions qui touchent les artères de gros et de moyen calibre dans la grande circulation. Les maladies résultant de l'athérosclérose consistent en une ischémie ou insuffisance d'apport sanguin à l'organe irrigué par l'artère obstruée.Atherosclerosis is a lesion of the inner layer (intima) of the arteries. More precisely, the elementary lesion of atherosclerosis or simple plaque consists of a localized thickening of the arterial intima, formed by a heap of hard fibrous tissue (sclerosis) encasing a soft center made up of modified or oxidized lipids (atheroma or lipid heart of the plaque) and lipoproteins (LDL or low density Hpoprotei). In humans, atherosclerosis is the dominant cause of obstructions that affect large and medium-sized arteries in large circulation. The diseases resulting from atherosclerosis consist of ischemia or insufficient blood supply to the organ irrigated by the obstructed artery.

Se manifestant essentiellement chez les personnes âgées de plus de 40 ans et avec une fréquence accrue à mesure qu'elles vieillissent, l'athérosclérose est avec le cancer, une des causes premières de maladie et de mort dans les pays occidentaux.Occurring primarily in people over the age of 40 and with an increased frequency as they age, atherosclerosis is with cancer, a leading cause of illness and death in Western countries.

On suspecte généralement l'inflammation chronique qui s'installe d'orchestrer la formation de la plaque athéroscléreuse. Les phénomènes qui entrent en jeu sont multiples et complexes, impliquant essentiellement les différentes catégories de cellules présentes dans l'intima malade : cellules musculaires, cellules endothéliales, macrophages et lymphocytes.Chronic inflammation that sets in is usually suspected of orchestrating the formation of atherosclerotic plaque. The phenomena that come into play are multiple and complex, essentially involving the different categories of cells present in the diseased intima: muscle cells, endothelial cells, macrophages and lymphocytes.

On considère que les lymphocytes présents au niveau de la plaque sont essentiellement constitués de cellules T CD4+, activées, en raison de l'expression concomitante de molécules HLA-DR, de récepteurs aux interleukines, d'antigène CD45R0 et d'antigène VLA-1 et de diverses cytokines (Demande Internationale WO 00/20019).It is considered that the lymphocytes present at the level of the plaque essentially consist of activated CD4 + T cells, due to the concomitant expression of HLA-DR molecules, of interleukin receptors, of antigen CD45R0 and VLA-1 antigen and various cytokines (International Application WO 00/20019).

De manière inattendue, les Inventeurs ont trouvé que des cellules T Vα24 (chez l'homme) ou Nα14-Jα281 (chez la souris) participent, de manière cruciale, à la réponse immunitaire au niveau de la lésion athéroscléreuse.Unexpectedly, the inventors have found that α 24 or N α 14-J α 281 (in mice) TV cells participate, in a crucial manner, in the immune response at the level of the atherosclerotic lesion.

C'est la raison pour laquelle ils ont créé un modèle in vivo et in vitro adapté au criblage et à la sélection de molécules actives, aptes à prévenir et/ou à traiter l'athérosclérose chez l'homme, utilisables en complément des médicaments hypo- cholestérolémiants. La présente invention a pour objet un procédé de criblage de molécules actives, aptes à prévenir et/ou à traiter l'athérosclérose chez l'homme, caractérisé en ce qu'il comprend essentiellement la détection comparative des cellules T Vα14-Jα281 présentes dans des lésions athéroscléreuses ou mimant des lésions athéroscléreuses, en l'absence et en présence des molécules à tester (ou cribler). On entend, au sens de la présente invention, par substance active, toute substance apte à moduler l'activité et la migration des cellules T portant la chaîne invariante Nα14-Jα281.This is the reason why they have created an in vivo and in vitro model suitable for screening and selecting active molecules, capable of preventing and / or treating atherosclerosis in humans, usable in addition to hypo drugs. - cholesterol-lowering drugs. The subject of the present invention is a method for screening for active molecules capable of preventing and / or treating atherosclerosis in humans, characterized in that it essentially comprises the comparative detection of TV cells α 14-J α 281 present in atherosclerotic lesions or mimicking atherosclerotic lesions, in the absence and in the presence of the molecules to be tested (or screened). For the purposes of the present invention, the term active substance is understood to mean any substance capable of modulating the activity and migration of T cells carrying the invariant chain N α 14-J α 281.

Les cellules T Vα14-Jα281 sont par exemple décrites dans T. Kawano et al. (Science, 1997, 278, 1626-1629). De manière avantageuse, ladite détection est réalisée par l'analyse comparative des ARΝ messagers produits par les cellules Vα14-Jα281 : analyse qualitative [Technique Immunoscope décrite dans le Brevet européen 0 613 503] ou analyse quantitative : méthode Taqman (Perkin-Elmer, Courtaboeuf, France).The α 14-J α 281 TV cells are for example described in T. Kawano et al. (Science, 1997, 278, 1626-1629). Advantageously, said detection is carried out by the comparative analysis of the ARΝ messengers produced by the V α 14-J α 281 cells: qualitative analysis [Immunoscope technique described in European Patent 0 613 503] or quantitative analysis: Taqman method (Perkin -Elmer, Courtaboeuf, France).

Un tel modèle peut ainsi être avantageusement utilisé pour cribler et sélectionner des molécules aptes à réduire l'infiltration et donc la migration des cellules T Nα14-Jα281 dans lesdites lésions.Such a model can thus be advantageously used to screen and select molecules capable of reducing the infiltration and therefore the migration of TN α 14-J α 281 cells in said lesions.

En effet, les Inventeurs ont trouvé, de manière inattendue, que l'infiltration et la migration desdites cellules T Vα14-Jα281 peuvent refléter l'infiltration de la lésion par d'autres cellules du système immunitaire (neutrophiles, macrophages, autres lymphocytes) et constituent un bon marqueur pour ledit criblage. Conformément à ce principe général, le criblage des molécules aptes à prévenir et/ou à traiter l'athérosclérose chez l'homme, peut être mis en œuvre selon plusieurs variantes :In fact, the Inventors unexpectedly found that the infiltration and migration of said TV α 14-J α 281 cells can reflect the infiltration of the lesion by other cells of the immune system (neutrophils, macrophages, other lymphocytes) and constitute a good marker for said screening. In accordance with this general principle, the screening of molecules capable of preventing and / or treating atherosclerosis in humans can be implemented according to several variants:

- comparaison des lésions induites par un antigène, en présence et en l'absence de la molécule à tester ;- comparison of lesions induced by an antigen, in the presence and in the absence of the molecule to be tested;

- induction de lésions chez des souris nourries pendant au moins une semaine avec la molécule à tester.- induction of lesions in mice fed for at least one week with the molecule to be tested.

Bien que moins avantageux, les modèles de l'art antérieur (souris déficientes en ApoE ou en LDL-R) peuvent également être utilisés pour quantifier les cellules T Nα14-Jα281 présentes dans les lésions athéroscléreuses, en l'absence et/ou en présence des molécules à cribler.Although less advantageous, the models of the prior art (mice deficient in ApoE or in LDL-R) can also be used to quantify TN α 14-J α 281 cells present in atherosclerotic lesions, in the absence and / or in the presence of the molecules to be screened.

Selon un mode de mise en œuvre avantageux dudit procédé, il comprend essentiellement :According to an advantageous embodiment of said method, it essentially comprises:

- l'induction d'une lésion sous-cutanée chez des souris, par admi- nistration d'une substance présente dans les lésions athéroscléreuses, et- induction of a subcutaneous lesion in mice, by administration of a substance present in atherosclerotic lesions, and

- la détection qualitative et/ou quantitative comparée des cellules T N l4-Jα281, à partir d'un échantillon de ladite lésion, avant et après la mise en contact (administration in vivo ou in vitro) avec une molécule à tester.- the comparative qualitative and / or quantitative detection of TN l4-J α 281 cells, from a sample of said lesion, before and after contact (administration in vivo or in vitro) with a molecule to be tested.

Selon une disposition avantageuse de ce mode de mise en œuvre, l'induction d'une lésion sous-cutanée est effectuée chez des souris, par administration d'une substance présente dans les plaques d'athérosclérose, sélectionnée dans le groupe constitué par les lipoprotéines LDL, le cholestérol, des molécules modifiées telles que les lysophospholipides, des lipides ou des protéines oxydés, des hsp65, des glycolipides [phosphoinositol mannosides, lipopolysaccharides (LPS), acide lipo- techoïque, glycolipides de microorganismes pathogènes, animaux ou humains modifiés chimiquement sur la partie osidique naturelle, glycolipides de synthèse tels que céramides (α ou β)...].According to an advantageous arrangement of this embodiment, the induction of a subcutaneous lesion is carried out in mice, by administration of a substance present in atherosclerotic plaques, selected from the group consisting of lipoproteins LDL, cholesterol, modified molecules such as lysophospholipids, oxidized lipids or proteins, hsp65, glycolipids [phosphoinositol mannosides, lipopolysaccharides (LPS), lipo-techoic acid, glycolipids of pathogenic microorganisms, animals or humans chemically modified on the natural osidic part, synthetic glycolipids such as ceramides (α or β) ...].

Selon un autre mode de mise en œuvre avantageux dudit procédé, il comprend : - le prélèvement des cellules T Ntx14-Jα281 de souris ou des lésions suspectées de contenir lesdites cellules, - la stimulation desdites cellules dans un milieu de culture convenable, en présence d'une substance présente dans les plaques d'athérosclérose, sélectionnée dans le groupe constitué par des substances présentes dans les plaques d'athérosclérose telles que : . les lipoprotéines LDL,According to another advantageous embodiment of said method, it comprises: the removal of TN tx 14-J α 281 cells from mice or from lesions suspected of containing said cells, - Stimulation of said cells in a suitable culture medium, in the presence of a substance present in atherosclerotic plaques, selected from the group consisting of substances present in atherosclerotic plaques such as:. LDL lipoproteins,

. le cholestérol,. cholesterol,

. des molécules modifiées telles que les lysophospholipides, des lipides ou des protéines oxydés, des hsp65,. modified molecules such as lysophospholipids, oxidized lipids or proteins, hsp65,

. des glycolipides, tels que définis ci-dessus, de préférence l'αGalCer [ct-galactocéramide ou (2S, 3S, 4R)-l-O-(oc-D-galactopyranosyl)-2-(N- hexacosanoylamino)- 1 ,3,4-octadecanetriol]. glycolipids, as defined above, preferably αGalCer [ct-galactoceramide or (2S, 3S, 4R) -lO- (oc-D-galactopyranosyl) -2- (N- hexacosanoylamino) - 1, 3, 4-octadecanetriol]

- la mise en contact desdites cellules stimulées avec une molécule à tester et- bringing said stimulated cells into contact with a molecule to be tested and

- la détection qualitative des cellules T Nα14-Jα281 et/ou quantita- tive comparée d'une substance exprimée par lesdites cellules en l'absence et en présence de ladite molécule à tester.- the qualitative detection of TN α 14-J α 281 and / or quantitative compared cells of a substance expressed by said cells in the absence and in the presence of said molecule to be tested.

L'ensemble de ces étapes est réalisé in vitro. Selon une disposition avantageuse de ce mode de mise en œuvre, lesdites substances détectées sont sélectionnées dans le groupe constitué par l'interleukine 4 (IL-4), l'interféron γ (IFΝ-γ) et le marqueur CD69.All of these steps are carried out in vitro. According to an advantageous arrangement of this embodiment, said detected substances are selected from the group consisting of interleukin 4 (IL-4), interferon γ (IFΝ-γ) and the marker CD69.

Lesdites substances sont avantageusement détectées soit par cyto- métrie de flux (CD69), soit par ELISA (IL-4 et IFΝ-γ).Said substances are advantageously detected either by flow cytometry (CD69), or by ELISA (IL-4 and IFΝ-γ).

De manière avantageuse, de tels procédés de criblage présentent les avantages suivants : - une induction rapide des lésions (environ 7 jours) : les modèles animaux d'athérosclérose actuellement proposés, tels que des souris déficientes en ApoE ou en LDL-R, développent des lésions athéroscléreuses détectables, lorsqu'ils sont nourris avec un régime riche en graisses, seulement au bout de 4 à 5 semaines ;Advantageously, such screening methods have the following advantages: - rapid induction of lesions (approximately 7 days): the animal models of atherosclerosis currently proposed, such as mice deficient in ApoE or in LDL-R, develop detectable atherosclerotic lesions, when fed a high-fat diet, only after 4 to 5 weeks;

- une dissection rapide des lésions : lésions sous-cutanées contre lésions du sinus de l'aorte dans les modèles de l'art antérieur ;- rapid dissection of lesions: subcutaneous lesions against lesions of the sinus of the aorta in the models of the prior art;

- une détection qualitative et quantitative du nombre de cellules T Vα14-Jα281 ayant migré au niveau de la lésion, par une méthode simple ; - une détection et une quantification de la réponse immunitaire à des antigènes présents dans les lésions athéroscléreuses à l'aide de tests in vitro simples (analyse de type Facscan, production de cytokines).- a qualitative and quantitative detection of the number of α 14-J α 281 TV cells which have migrated to the level of the lesion, by a simple method; - detection and quantification of the immune response to antigens present in atherosclerotic lesions using simple in vitro tests (Facscan type analysis, production of cytokines).

De tels procédés permettent de détecter différents types de substances actives sur l'athérosclérose, en particulier des substances inhibant la migration des cellules T Nα24 humaines et/ou la stimulation des cytokines exprimées par lesdites cellules T, telles que des inhibiteurs de MCP-1, de CCR2 ou de la molécule CD ld.Such methods make it possible to detect different types of active substances on atherosclerosis, in particular substances inhibiting the migration of human TN α 24 cells and / or the stimulation of cytokines expressed by said T cells, such as MCP-1 inhibitors. , CCR2 or the CD ld molecule.

La présente invention a également pour objet l'utilisation des molé- cules obtenues à l'aide du procédé de criblage, tel que défini ci-dessus, pour la préparation d'un médicament destiné à prévenir et/ou à traiter l'athérosclérose.A subject of the present invention is also the use of the molecules obtained using the screening method, as defined above, for the preparation of a medicament intended for preventing and / or treating atherosclerosis.

Selon un mode de réalisation avantageux de ladite utilisation, lesdites molécules sont des inhibiteurs de chemokines intervenant dans la régulation de la migration des cellules T Nα14-Jα281 ou T Nα24, notamment des inhibiteurs de MCP-1 ou de CCR2.According to an advantageous embodiment of said use, said molecules are chemokine inhibitors involved in regulating the migration of TN α 14-J α 281 or TN α 24 cells, in particular inhibitors of MCP-1 or CCR2.

Selon un autre mode de réalisation avantageux de ladite utilisation, lesdites molécules sont des inhibiteurs de la molécule CDld.According to another advantageous embodiment of said use, said molecules are inhibitors of the CDld molecule.

La présente invention a également pour objet des compositions aptes à prévenir et/ou à traiter l'athérosclérose (inhibition de la formation des plaques d'athérome), caractérisées en ce qu'elles comprennent une molécule inhibant la migration des cellules T Vα24 humaines et/ou l'activité desdites cellules T et au moins un véhicule pharmaceutiquement acceptable.The present invention also relates to compositions capable of preventing and / or treating atherosclerosis (inhibition of the formation of atheroma plaques), characterized in that they comprise a molecule inhibiting the migration of human TV α 24 cells and / or the activity of said T cells and at least one pharmaceutically acceptable vehicle.

Selon un mode de réalisation avantageux desdites compositions, ladite molécule inhibant l'activité des cellules T Nα24 humaines inhibe la production des cytokines exprimées par lesdites cellules.According to an advantageous embodiment of said compositions, said molecule inhibiting the activity of human TN α 24 cells inhibits the production of cytokines expressed by said cells.

La présente invention a également pour objet une molécule active criblée par un procédé tel que défini ci-dessus, caractérisée par sa capacité à moduler la migration et/ou l'activité des cellules Nα24 humaines présentes dans les lésions athéroscléreuses. Outre les dispositions qui précèdent, l'invention comprend encore d'autres dispositions, qui ressortiront de la description qui va suivre, qui se réfère à des exemples de mise en œuvre du procédé objet de la présente invention ainsi qu'aux dessins annexés, dans lesquels :The present invention also relates to an active molecule screened by a process as defined above, characterized by its ability to modulate the migration and / or the activity of human N α 24 cells present in atherosclerotic lesions. In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention as well as in the accompanying drawings, in which:

- la figure 1 représente la détection des cellules T Nαl4-Jα281 par Immunoscope dans des lésions induites par le cholestérol. - la figure 2 représente les réponses immunitaires différentielles des cellules T à la stimulation par le LPS ou l'αGalCer en l'absence ou en présence d'anticorps bloquants anti-CDl. L'expression du marqueur CD69 à la surface cellulaire est détectée par Facscan et la production de cytokines (IL-4 et IFΝ-γ) par les cellules T sont détectées par immunoessai, après stimulation des cellules. II doit être bien entendu, toutefois, que ces exemples sont donnés uniquement à titre d'illustration de l'objet de l'invention, dont ils ne constituent en aucune manière une limitation. Exemple : Matériel et méthodes Anticorps et réactifs- Figure 1 shows the detection of Nαl4-Jα281 T cells by Immunoscope in cholesterol-induced lesions. - Figure 2 shows the differential immune responses of T cells to stimulation by LPS or αGalCer in the absence or in the presence of anti-CDl blocking antibodies. The expression of the CD69 marker on the cell surface is detected by Facscan and the production of cytokines (IL-4 and IFΝ-γ) by the T cells are detected by immunoassay, after stimulation of the cells. It should be understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation. Example: Materials and methods Antibodies and reagents

Les anticorps conjugués suivants : APC (phosphatase alcaline)- ou biotine- anti-TCR β (H57-597), phycoérythrine (PE)-anti-ΝKl.l (PK136), PE-anti- CD19, biotine-anti-CD69, PE-anti-CD14 (rmC5-3), biotine-anti-Macl (Ml/70), biotine-anti-Grl (RB6-8C5), biotine-anti-CDl lb, biotine-anti-CDld (1B1), Fcγ III/II (Fc bloc, 2.4G2) et APC-streptavidine sont fournis par PharMingen (San Diego, CA). Les anticorps conjugués FITC (Isothiocyanate de fluorescéine)-anti-TCRαβ (H57- 597) et biotine-anti-B220 (RA3-6B2), ainsi que la streptavidine-tricolore sont fournis par Caltag (San Francisco, CA). L'anticorps monoclonal anti-CDl d (clone 20H2) est décrit dans Roark et al., J. Immunol, 1998, 160, 3121-3127. Le αGalCer (2S, 3S, 4R)-l-O-(α-D-galactopyranosyl)-2-(N-hexaco- sanoylamino)-l,3,4-octadecanetriol) est commercialisé par le Laboratoire de Recherche Pharmaceutique Kirin Brewery Co. Ltd. (Gunma, Japon). La solution stock est ajustée à 100 μg/ml dans du DMSO 10% puis diluée à une concentration finale de 100 ng/ml dans le milieu de culture. Une solution véhicule de DMSO à 10%, diluée à une concentration finale de 0, 1%, est utilisée comme contrôle. Le LPS complexé au CD14 soluble est préparé en incubant du LPS (10 ng/ml) avec du CD 14 soluble (200 ng/ml), une nuit à 37°C, selon le protocole décrit dans Hailman et al., J. Immunol., 1996, 156, 4384-4390.The following conjugate antibodies: APC (alkaline phosphatase) - or biotin- anti-TCR β (H57-597), phycoerythrin (PE) -anti-ΝKl.l (PK136), PE-anti-CD19, biotin-anti-CD69, PE-anti-CD14 (rmC5-3), biotin-anti-Macl (Ml / 70), biotin-anti-Grl (RB6-8C5), biotin-anti-CDl lb, biotin-anti-CDld (1B1), Fcγ III / II (Fc block, 2.4G2) and APC-streptavidin are provided by PharMingen (San Diego, CA). FITC (fluorescein isothiocyanate) -anti-TCRαβ (H57-597) and biotin-anti-B220 (RA3-6B2) conjugated antibodies, as well as streptavidin-tricolor, are available from Caltag (San Francisco, CA). The anti-CD1 d monoclonal antibody (clone 20H2) is described in Roark et al., J. Immunol, 1998, 160, 3121-3127. ΑGalCer (2S, 3S, 4R) -lO- (α-D-galactopyranosyl) -2- (N-hexaco- sanoylamino) -l, 3,4-octadecanetriol) is marketed by the Pharmaceutical Research Laboratory Kirin Brewery Co. Ltd. (Gunma, Japan). The stock solution is adjusted to 100 μg / ml in 10% DMSO and then diluted to a final concentration of 100 ng / ml in the culture medium. A 10% DMSO vehicle solution, diluted to a final concentration of 0.1%, is used as a control. LPS complexed with soluble CD14 is prepared by incubating LPS (10 ng / ml) with soluble CD 14 (200 ng / ml) overnight at 37 ° C, according to the protocol described in Hailman et al., J. Immunol ., 1996, 156, 4384-4390.

L'aprotinine, le tampon phosphate (PBS) de Dubelcco et la BSA dégraissée sont fournis par Sigma Chemical Co. (St Louis, MO).Aprotinin, phosphate buffer (PBS) from Dubelcco and defatted BSA are supplied by Sigma Chemical Co. (St Louis, MO).

Animaux et traitement in vivoAnimals and in vivo treatment

Les souris C57BL/6 sont fournies par Iffa-Credo (L'Arbresle, France).C57BL / 6 mice are supplied by Iffa-Credo (L'Arbresle, France).

Les souris homozygotes déficientes en CD1 sont croisées avec les souris C57BL/6 comme décrit par Mendiratta dans Immunity, 1997, 6, 4, 469-477.Homozygous CD1-deficient mice are crossed with C57BL / 6 mice as described by Mendiratta in Immunity, 1997, 6, 4, 469-477.

Les souris transgéniques pour N l4 sont décrites dans Bendelac et al. (J. Exp. Med., 1996, 184, 4, 1285-1293).Transgenic mice for N l4 are described in Bendelac et al. (J. Exp. Med., 1996, 184, 4, 1285-1293).

Pour le transfert de cellules enrichies en Nαl4, des souris sont anesthésiées avec du pentobarbital à 0,1% puis injectées par voie intrasplénique avec 100 μl d'une suspension de cellules enrichies en Nαl4, en présence ou en l'absence d'anticorps 20H2 (10 μg/106 cellules).For the transfer of cells enriched in Nαl4, mice are anesthetized with 0.1% pentobarbital and then injected intrasplenically with 100 μl of a suspension of cells enriched in Nαl4, in the presence or absence of 20H2 antibody (10 μg / 10 6 cells).

Des souris âgées de 3 à 7 semaines sont injectées avec un antigène puis 7 jours plus tard les animaux sont sacrifiés et des extraits de leur foie et de leur rate sont préparés et injectés à des animaux comme indiqué ci-après. Injection aux animaux et prélèvement des échantillonsMice aged 3 to 7 weeks are injected with an antigen and then 7 days later the animals are sacrificed and extracts of their liver and their spleen are prepared and injected into animals as indicated below. Injection to animals and collection of samples

Les extraits contenant les molécules inductrices présentes dans les plaques d'athérosclérose (Ox-LDL, phospholipides...), en présence de 12,5% d'alu- gel-S (Serva, Heidelberg, Allemagne) dans un volume de 100 μl sont injectés, par voie sous-cutanée à la base de la queue des souris C57BL/6 (Apostolou et al., PΝAS, 1999, 96, 5141-5146). Les animaux témoin reçoivent 100 μl de PBS par la même voie d'injection. Les animaux sont sacrifiés 7 jours après l'injection et des échantillons de peau correspondant au site d'injection, et éventuellement les foies sont prélevés et congelés immédiatement. A partir de ces échantillons :The extracts containing the inducing molecules present in atherosclerotic plaques (Ox-LDL, phospholipids ...), in the presence of 12.5% alu- gel-S (Serva, Heidelberg, Germany) in a volume of 100 μl are injected, subcutaneously at the base of the tail of the C57BL / 6 mice (Apostolou et al., PΝAS, 1999, 96, 5141-5146). The control animals receive 100 μl of PBS by the same injection route. The animals are sacrificed 7 days after the injection and skin samples corresponding to the injection site, and possibly the livers are removed and frozen immediately. From these samples:

- des coupes des lésions sont analysées par histologie, - les cellules T Vαl4-Jα281 sont détectées par une technique basée sur la RT-PCR (Im unoscope) qui utilise comme marqueur spécifique, le caractère pratiquement invariable de la chaîne Nαl4-Jα281 du récepteur des lymphocytes T (TCR) et un CDR3 long de 10 acides aminés. Amorces PCR- sections of the lesions are analyzed by histology, - the Vαl4-Jα281 T cells are detected by a technique based on RT-PCR (Im unoscope) which uses as a specific marker, the practically invariable character of the Nαl4-Jα281 chain of the T lymphocyte receptor (TCR) and a CDR3 long with 10 amino acids. PCR primers

Les amorces sont synthétisées par Eurogentech (Orléans, France). Pour la technique basée sur la RT-PCR, les amorces spécifiques présentant les séquences suivantes ont été utilisées : Nαl4 : CTAAGCACAGCACGCTGCACA (SEQ ID ΝO:l) ; Cα : TGGCGTTGGTCTCTTTGAAG (SEQ ID NO:2) ; Cα marqué : FAM-ACACAGGAGGTTCTGGGTTC (SEQ ID NO:3) ; J 281 marqué : FAM- CAGCTGAGTCCCAGCTCC (SEQ ID NO:4).The primers are synthesized by Eurogentech (Orléans, France). For the technique based on RT-PCR, the specific primers having the following sequences were used: Nαl4: CTAAGCACAGCACGCTGCACA (SEQ ID ΝO: l); Cα: TGGCGTTGGTCTCTTTGAAG (SEQ ID NO: 2); Cα labeled: FAM-ACACAGGAGGTTCTGGGTTC (SEQ ID NO: 3); J 281 marked: FAM- CAGCTGAGTCCCAGCTCC (SEQ ID NO: 4).

Analyse automatisée (immunoscope) ou semi-automatisée de la diversité des cellules TAutomated (immunoscope) or semi-automated analysis of T cell diversity

L'ARN total est extrait en utilisant le réactif Trizol (Gibco BRL) puis il est précipité à l'éthanol et les culots sont remis en suspension dans 20 μl d'eau distillée. 10 μl sont réverse-transcrits dans un volume final de 40 μl en utilisant la réverse transcriptase (RT) du virus de la myéloblastose aviaire et un oligo-dT, selon des protocoles standards. Deux microlitres de la solution d'ADNc obtenue sont amplifiés par 40 cycles de polymérisation en chaîne (PCR) alternant une étape de dénatura- tion à 94°C pendant 30 s, une étape de fixation des amorces à 60°C pendant 30 s et une étape d'extension à 72°C pendant 30 s, en utilisant les amorces spécifiques Nαl4- et Cα précédemment décrites. Deux microlitres des produits amplifiés par PCR sont utilisés dans des expériences de « run-off » (5 cycles) en utilisant les amorces fluorescentes spécifiques des segments Jα281 ou Cα précédemment décrites. La longueur de la région CDR3 et l'intensité de fluorescence des produits du « run-off» sont déterminées à l'aide d'un séquenceur automatique (Perkin-Elmer) et analysées en utilisant un logiciel "Immunoscope" (Pannetier, PΝAS, 1993, 90, 4319-4323). La taille de la région CDR3 d'un couple N-C est représentée par une famille de pics séparés par 3 nucléotides (dérivé de l'ARΝm en phase), dont la surface est proportionnelle à la quantité initiale d'ARΝm. En l'absence d'une prolifération des cellules T, déclen- chée par l'antigène, les six à huit pics observés présentent une distribution sensible- ment gaussienne. Une prolifération des cellules T déclenchée par l'antigène résulte typiquement en des distorsions de la distribution gaussienne. TaqmanThe total RNA is extracted using the Trizol reagent (Gibco BRL) then it is precipitated with ethanol and the pellets are resuspended in 20 μl of distilled water. 10 μl are reverse-transcribed in a final volume of 40 μl using reverse transcriptase (RT) of the avian myeloblastosis virus and an oligo-dT, according to standard protocols. Two microliters of the cDNA solution obtained are amplified by 40 cycles of chain polymerization (PCR) alternating a denaturation step at 94 ° C for 30 s, a step for fixing the primers at 60 ° C for 30 s and an extension step at 72 ° C for 30 s, using the specific primers Nαl4- and Cα previously described. Two microliters of the products amplified by PCR are used in “run-off” experiments (5 cycles) using the fluorescent primers specific for the Jα281 or Cα segments previously described. The length of the CDR3 region and the fluorescence intensity of the “run-off” products are determined using an automatic sequencer (Perkin-Elmer) and analyzed using “Immunoscope” software (Pannetier, PΝAS, 1993, 90, 4319-4323). The size of the CDR3 region of a pair NC is represented by a family of peaks separated by 3 nucleotides (derived from ARΝm in phase), the area of which is proportional to the initial quantity of ARΝm. In the absence of a proliferation of T cells, triggered by the antigen, the six to eight peaks observed exhibit a significant distribution- Gaussian. A proliferation of T cells triggered by the antigen typically results in distortions of the Gaussian distribution. Taqman

Dans une approche quantitative, l'infiltration des cellules T Nαl4- Jα281 a été mesurée en utilisant des amorces spécifiques des gènes β-actine, Cα, Nαl4 et Jα281.In a quantitative approach, the infiltration of Nαl4- Jα281 T cells was measured using primers specific for the β-actin, Cα, Nαl4 and Jα281 genes.

Préparation des cellulesCell preparation

Les foies et les rates sont prélevés des souris avec précaution. Les suspensions cellulaires des foies et des rates sont préparées à l'aide d'un homogénéi- sateur. La totalité des cellules du foie est remise en suspension dans une solution isotonique de Percoll à 80% (Pharmacia, Uppsala, Suède) et recouverte d'une solution isotonique de Percoll à 40%. Après 30 min de centrifugation à 3000 rpm, les cellules mononucleées qui se rassemblent à l'interface entre les solutions à 40 et à 80% sont recueillies et lavées avec du PBS additionné de 2% de sérum de veau fœtal (FCS). Quand cela est nécessaire, les cellules mononucleées du foie sont marquées avec des anticorps biotine-anti-Macl et les cellules présentatrices d'antigène sont isolées en utilisant des billes de streptavidine magnétiques (Dynal, Oslo, Norvège). Cultures primaires de cellules répondeusesLivers and rats are carefully removed from mice. Cell suspensions of livers and spleens are prepared using a homogenizer. All the liver cells are resuspended in an 80% isotonic Percoll solution (Pharmacia, Uppsala, Sweden) and covered with a 40% Percoll isotonic solution. After 30 min of centrifugation at 3000 rpm, the mononuclear cells which gather at the interface between the 40 and 80% solutions are collected and washed with PBS added with 2% of fetal calf serum (FCS). When necessary, the mononuclear cells of the liver are labeled with biotin-anti-Mac1 antibodies and the antigen presenting cells are isolated using magnetic streptavidin beads (Dynal, Oslo, Norway). Primary cultures of responder cells

Des cellules mononucleées isolées du foie et de la rate sont cultivées dans des microplaques de 96 puits à fond rond (Nunc), en présence de RPMI 1640 (Gibco BRL) contenant 10% de sérum de veau fœtal (Gibco BRL), 100 Ul/ml de pénicilline et lOO μg/ml de streptomycine, additionné d'αGalCer (100 ng/ml) ou de concentrations variées d'antigène, et/ou d'anticorps bloquants anti-CDl. Les surnageants sont récoltés 16 à 72 heures plus tard et stockés à -20°C jusqu'à ce que les dosages d'IL-4 et d'IFN-γ soient réalisés. Les cellules sont marquées à l'aide d'anticorps fluorescents et l'expression du marqueur d'activation CD69 sur les différentes populations est étudiée par cytométrie de flux (Facscan). Les cellules T triées sont cultivées pendant 16 à 72 heures dans un milieu AIM V (Gibco-BRL) contenant des antibiotiques, en présence ou en l'absence de cellules présentatrices d'antigène. Les surnageants de culture cellulaire sont récoltés et congelés. Dosages de cytokineMononuclear cells isolated from the liver and spleen are cultured in 96-well round-bottom microplates (Nunc), in the presence of RPMI 1640 (Gibco BRL) containing 10% fetal calf serum (Gibco BRL), 100 IU / ml of penicillin and 100 μg / ml of streptomycin, supplemented with αGalCer (100 ng / ml) or various concentrations of antigen, and / or anti-CDl blocking antibodies. The supernatants are harvested 16 to 72 hours later and stored at -20 ° C until the assays of IL-4 and IFN-γ are carried out. The cells are labeled with fluorescent antibodies and the expression of the activation marker CD69 on the different populations is studied by flow cytometry (Facscan). The sorted T cells are cultured for 16 to 72 hours in AIM V medium (Gibco-BRL) containing antibiotics, in the presence or in the absence of antigen presenting cells. Cell culture supernatants are harvested and frozen. Cytokine assays

La quantification des IFN-γ, IL-4 et TNF-α murins présents dans les sera et les surnageants de culture est déterminée en utilisant des tests ELISA- sandwich classiques, selon les recommandations du fabricant. Les kits ELISA IFN-γ, IL-4 et TNF-α sont fournis par Pharmingen. Dans certains essais, les échantillons sont dosés d'après un protocole modifié utilisant la streptavidine-peroxydase (Amersham, Les Ulis, France) et la lecture des plaques est effectuée sur un cytofluorimètre (Perspective Biosystems). Les données sont exprimées en moyennes ± erreur standard (SEM). Cytométrie de flux & tri des cellulesThe quantification of murine IFN-γ, IL-4 and TNF-α present in sera and culture supernatants is determined using standard ELISA-sandwich tests, according to the manufacturer's recommendations. The ELISA IFN-γ, IL-4 and TNF-α kits are supplied by Pharmingen. In some tests, the samples are assayed according to a modified protocol using streptavidin-peroxidase (Amersham, Les Ulis, France) and the plates are read on a cytofluorimeter (Perspective Biosystems). Data are expressed as means ± standard error (SEM). Flow cytometry & cell sorting

Les suspensions cellulaires de foie et de rate sont colorées à l'aide de 4 couleurs en utilisant des combinaisons d'anticorps conjugués au FITC, PE, APC ou à la biotine puis au Tricolor® ou/et à la streptavidine conjuguée à l'APC, et analysées par cytométrie de flux (FACS ; Becton Dickinson and Co., Mountain View, CA). L'analyse phénotypique des lymphocytes T de foie a été réalisée à 4°C. Les cellules sont d'abord incubées 10 min. avec de l'anti-Fc γ III/II, puis exposées 30 min. à des anticorps PE-NK1.1 et TCRβ-biotinylés et, si nécessaire, FITC-CD69. Après deux lavages, de la streptavidine-Tricolor® (Caltag) ou de la streptavidine-APC a été ajoutée. Après d'autres lavages, les cellules sont mises en suspension dans du PBS conte- nant 2% de sérum de veau fœtal (FCS) et analysées selon des procédures conventionnelles. Les données sont analysées en utilisant un logiciel Cell-Quest (Becton- Dickinson).The liver and spleen cell suspensions are stained using 4 colors using combinations of antibodies conjugated to FITC, PE, APC or biotin then to Tricolor ® or / and streptavidin conjugated to APC , and analyzed by flow cytometry (FACS; Becton Dickinson and Co., Mountain View, CA). Phenotypic analysis of liver T cells was performed at 4 ° C. The cells are first incubated for 10 min. with anti-Fc γ III / II, then exposed for 30 min. to PE-NK1.1 and TCRβ-biotinylated antibodies and, if necessary, FITC-CD69. After two washes, streptavidin-Tricolor ® (Caltag) or streptavidin-APC was added. After further washes, the cells are suspended in PBS containing 2% fetal calf serum (FCS) and analyzed according to conventional procedures. The data are analyzed using Cell-Quest software (Becton-Dickinson).

Les cellules colorées TCRαβ^Kl.l " sont triées en utilisant un appareil "Facstar Plus" (Becton-Dickinson) ou un "Elite" (Coulter) équipé de lasers doubles (argon et colorant) à un débit de 3500 événements par seconde, et récoltées dans du RPMI additionné de 10%. de sérum de veau fœtal (FCS). Dans ces conditions, la pureté de chaque fraction cellulaire enrichie est supérieure à 94% après nouvelle analyse. RésultatsThe colored cells TCRαβ ^ Kl.l " are sorted using a" Facstar Plus "device (Becton-Dickinson) or an" Elite "(Coulter) equipped with double lasers (argon and dye) at a rate of 3500 events per second, and harvested in RPMI supplemented with 10% fetal calf serum (FCS) Under these conditions, the purity of each enriched cell fraction is greater than 94% after re-analysis. Results

Infiltration in vivo de cellules V l4-Ja281 dans des lésions induites par le cholestérol ou le LPSIn vivo infiltration of l4-Ja281 V cells into lesions induced by cholesterol or LPS

Les résultats illustrés à la figure 1 montrent que l'injection sous- cutanée de cholestérol ou de LPS chez des souris induit une infiltration majeure des cellules T au site de l'injection, caractérisée par la détection du Vαl4-Jα281 de la chaîne α invariante du TCR par Immunoscope (Fig. 1). Ces observations indiquent que dans des conditions physiologiques, des antigènes tels que le cholestérol ou le LPS sont susceptibles de promouvoir des réponses immunitaires en recrutant des cellules T au site de l'injection.The results illustrated in FIG. 1 show that the subcutaneous injection of cholesterol or LPS in mice induces a major infiltration of T cells at the injection site, characterized by the detection of Vαl4-Jα281 from the invariant α chain TCR by Immunoscope (Fig. 1). These observations indicate that under physiological conditions, antigens such as cholesterol or LPS are capable of promoting immune responses by recruiting T cells at the injection site.

Réponses in vitro des cellules T aux antigènes Les foies et rates de souris transgéniques pour V 14 sont isolés et les cellules mononucleées sont stimulées in vitro.In vitro T cell responses to antigens The livers and spleens of V 14 transgenic mice are isolated and the mononuclear cells are stimulated in vitro.

La figure 2 montre que l'αGalCer et le LPS sont capables de stimu- 1er des cellules T, d'une façon dépendante de la molécule CDl. En effet, après stimulation avec le glycolipide αGalCer ou le LPS, on observe une nette augmentation de l'expression du CD69 à la surface des cellules T et de la production dTFN-γ et d'IL-4, qui sont complètement inhibées en présence d'anticorps anti-CDl.FIG. 2 shows that the αGalCer and the LPS are capable of stimulating T cells, in a manner dependent on the CDl molecule. In fact, after stimulation with the αGalCer glycolipid or LPS, there is a marked increase in the expression of CD69 on the surface of T cells and in the production of dTFN-γ and IL-4, which are completely inhibited in the presence anti-CDl antibodies.

La figure 2 montre également que la stimulation des cellules T peut être mesurée aussi par l'augmentation de l'expression du CD69 en cytométrie de flux que par le dosage en ELISA de l'IL-4 ou de l'IFN-γ.FIG. 2 also shows that the stimulation of T cells can be measured also by the increase in the expression of CD69 in flow cytometry as by the assay in ELISA of IL-4 or of IFN-γ.

Des résultats similaires sont observés à partir de cellules T triées, stimulées en présence ou en l'absence de cellules présentatrices d'antigène et d'un antigène d'intérêt présent dans les lésions d'athérosclérose. Ces résultats montrent que les cellules T Vα14-Jα281 sont stimulées par des antigènes d'intérêt présents dans les lésions d'athérosclérose (cholestérol, glycolipide).Similar results are observed from sorted T cells, stimulated in the presence or absence of antigen presenting cells and an antigen of interest present in atherosclerotic lesions. These results show that the α 14-J α 281 TV cells are stimulated by antigens of interest present in atherosclerotic lesions (cholesterol, glycolipid).

Par conséquent, le criblage de molécules aptes à inhiber la stimulation des cellules T Vα14-Jα281 par des tests qui mesurent l'expression du CD69, d'IL- 4 ou dTFN-γ par lesdites cellules f stimulées, tels que ceux décrits ci-dessus permet avantageusement de cribler des médicaments aptes à prévenir et/ou à traiter l'athérosclérose chez l'Homme.Consequently, the screening of molecules capable of inhibiting the stimulation of α 14-J α 281 TV cells by tests which measure the expression of CD69, IL-4 or dTFN-γ by said stimulated f cells, such as those described above allows advantageously to screen for drugs capable of preventing and / or treating atherosclerosis in humans.

Ainsi que cela ressort de ce qui précède, l'invention ne se limite nullement à ceux de ses modes de mise en oeuvre, de réalisation et d'application qui viennent d'être décrits de façon plus explicite ; elle en embrasse au contraire toutes les variantes qui peuvent venir à l'esprit du technicien en la matière, sans s'écarter du cadre, ni de la portée, de la présente invention. As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.

Claims

REVENDICATIONS 1°) Procédé de criblage de molécules actives, aptes à prévenir et/ou à traiter l'athérosclérose chez l'homme, caractérisé en ce qu'il comprend essentiellement la détection comparative des cellules T Vα14-Jα281 présentes dans des lésions athéroscléreuses ou mimant des lésions athéroscléreuses, en l'absence et en présence des molécules à tester.1) CLAIMS Method for screening active molecules capable of preventing and / or treating atherosclerosis in humans, characterized in that it essentially comprises the comparative detection of TV α 14-J α 281 cells present in atherosclerotic lesions or mimicking atherosclerotic lesions, in the absence and in the presence of the molecules to be tested. 2°) Procédé de criblage selon la revendication 1, caractérisé en ce que ladite détection est réalisée par l'analyse comparative des ARN messagers produits par les cellules Vα14-Jα281. 3°) Procédé de criblage selon la revendication 1 ou la revendication2 °) screening method according to claim 1, characterized in that said detection is carried out by the comparative analysis of the messenger RNA produced by the cells V α 14-J α 281. 3 °) screening method according to claim 1 or claim 2, caractérisé en ce qu'il comprend essentiellement :2, characterized in that it essentially comprises: - l'induction d'une lésion sous-cutanée chez des souris, par administration d'une substance présente dans les lésions athéroscléreuses, et- induction of a subcutaneous lesion in mice, by administration of a substance present in atherosclerotic lesions, and - la détection qualitative et/ou quantitative comparée des cellules T Vα14-Jα281, à partir d'un échantillon de ladite lésion, avant et après la mise en contact avec une molécule à tester.- Qualitative and / or quantitative detection of TV α 14-J α 281 cells, from a sample of said lesion, before and after contact with a molecule to be tested. 4°) Procédé de criblage selon la revendication 3, caractérisé en ce que l'induction d'une lésion sous-cutanée est effectuée chez des souris, par administration d'une substance présente dans les plaques d'athérosclérose, sélectionnée dans le groupe constitué par les lipoprotéines LDL, le cholestérol, des molécules modifiées telles que les lysophospholipides, les lipides ou protéines oxydés, les hsp65 ou des glycolipides.4 °) screening method according to claim 3, characterized in that the induction of a subcutaneous lesion is carried out in mice, by administration of a substance present in the atherosclerotic plaques, selected from the group consisting by LDL lipoproteins, cholesterol, modified molecules such as lysophospholipids, oxidized lipids or proteins, hsp65 or glycolipids. 5°) Procédé de criblage selon la revendication 1 ou la revendication 2, caractérisé en ce qu'il comprend : - le prélèvement de cellules T Vα14-Jα281 de souris ou des lésions suspectées de contenir lesdites cellules,5) screening method according to claim 1 or claim 2, characterized in that it comprises: the removal of TV α 14-J α 281 cells from mice or from lesions suspected of containing said cells, - la stimulation desdites cellules dans un milieu de culture convenable, en présence d'une substance présente dans les plaques d'athérosclérose, sélectionnée dans le groupe constitué par les lipoprotéines LDL, le cholestérol ou des molécules modifiées telles que les lysophospholipides, les lipides ou protéines oxydés, les hsp65 ou des glycolipides, - la mise en contact desdites cellules T Vα14-Jα281 stimulées avec une molécule à tester et- stimulation of said cells in a suitable culture medium, in the presence of a substance present in atherosclerotic plaques, selected from the group consisting of LDL lipoproteins, cholesterol or modified molecules such as lysophospholipids, lipids or oxidized proteins, hsp65 or glycolipids, - bringing said stimulated α 14-J α 281 TV cells into contact with a molecule to be tested and - la détection qualitative desdites cellules T Vα14-Jα281 et/ou quantitative comparée d'une substance exprimée par lesdites cellules en l'absence et en présence de ladite molécule à tester.- qualitative detection of said TV α 14-J α 281 cells and / or quantitative comparison of a substance expressed by said cells in the absence and in the presence of said molecule to be tested. 6°) Procédé de criblage selon la revendication 5, caractérisé en ce. que lesdites substances détectées sont sélectionnées dans le groupe constitué par l'interleukine 4 (IL-4), l'interféron γ (IFN-γ) et le marqueur CD69.6 °) screening method according to claim 5, characterized in. that said detected substances are selected from the group consisting of interleukin 4 (IL-4), interferon γ (IFN-γ) and the marker CD69. 7°) Utilisation des molécules obtenues à l'aide du procédé de criblage selon l'une quelconque des revendications 1 à 6 pour la préparation d'un médicament destiné à prévenir et/ou traiter l'athérosclérose.7 °) Use of the molecules obtained using the screening method according to any one of claims 1 to 6 for the preparation of a medicament intended to prevent and / or treat atherosclerosis. 8°) Utilisation selon la revendication 7, caractérisée en ce que lesdites molécules sont des inhibiteurs de chemokines intervenant dans la régulation de la migration des cellules T Vα14-Jα281, notamment des inhibiteurs de MCP-1 ou de CCR2.8 °) Use according to claim 7, characterized in that said molecules are chemokine inhibitors involved in the regulation of the migration of TV cells α 14-J α 281, in particular inhibitors of MCP-1 or CCR2. 9°) Utilisation selon la revendication 7, caractérisée en ce que lesdites molécules sont des inhibiteurs de la molécule CDld. 9 °) Use according to claim 7, characterized in that said molecules are inhibitors of the CDld molecule.
PCT/FR2001/003303 2000-10-24 2001-10-24 Method for screening active molecules for treating and preventing atherosclerotic lesions and its uses Ceased WO2002034941A1 (en)

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