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WO1998017305A1 - UTILISATION DE FasL OU DE LIGNEES CELLULAIRES CD4+FasL-/TH1 TRANSFECTEES AVEC DES FasL POUR LE TRAITEMENT DE MALADIES METTANT EN JEU LES TH1/TH2 - Google Patents

UTILISATION DE FasL OU DE LIGNEES CELLULAIRES CD4+FasL-/TH1 TRANSFECTEES AVEC DES FasL POUR LE TRAITEMENT DE MALADIES METTANT EN JEU LES TH1/TH2 Download PDF

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Publication number
WO1998017305A1
WO1998017305A1 PCT/EP1997/005589 EP9705589W WO9817305A1 WO 1998017305 A1 WO1998017305 A1 WO 1998017305A1 EP 9705589 W EP9705589 W EP 9705589W WO 9817305 A1 WO9817305 A1 WO 9817305A1
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Prior art keywords
fasl
treatment
cells
diseases
medicament
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PCT/EP1997/005589
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German (de)
English (en)
Inventor
Michael Hahne
Jürg TSCHOPP
Fatima Da Conceicao-Silva
Michael SCHRÖTER
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Apotech S.A.
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Priority to CA002269034A priority Critical patent/CA2269034A1/fr
Priority to EP97911208A priority patent/EP0930890A1/fr
Priority to AU48664/97A priority patent/AU4866497A/en
Publication of WO1998017305A1 publication Critical patent/WO1998017305A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • FasL or CD4 + / FasL7THl cell lines transfected with FasL for the treatment of TH1 / TH2 diseases
  • the invention relates to the use of FasL for the treatment of TH1 / TH2 diseases, in particular Leishmaniasen, AIDS or Listeriosen, a method for producing CD4 + / FasL + / THl cell lines from CD47FasL7THl cell lines, their use for the treatment of TH1 / TH2 Diseases, a composition of matter containing FasL and one or more cytokines, and their use as medicaments, in particular for the treatment of TH1 / TH2 diseases.
  • TH1 / TH2 diseases are a spectrum of immunologically-related disorders that are based on a shift in the immune response or an inadequate immune response after infection by pathogens, e.g. Viruses, bacteria or protozoa.
  • pathogens e.g. Viruses, bacteria or protozoa.
  • the mammalian or human immune system responds to pathogen infection through a cellular and / or humoral immune response A depending on the specific characteristics of the pathogen. While the cellular immune response is mostly used against intracellular parasites, the humoral immune response is characterized by the production of antibodies that extracellularly neutralize the pathogen.
  • T helper cells T helper cells
  • T helper cells are again divided into two subclasses, the so-called TH1 cells and the TH2 cells, respectively. Both cell classes occupy central positions in the fight against intra- or extracellular pathogens.
  • the inflammatory TH1 cells serve the immune response against intracellular pathogens, such as such as Leishmania, viruses or intracellular parasitic bacteria such as Listeria.
  • TH2 cells activate B lymphocytes, which - as mentioned above - control humoral immunity to extracellular pathogens
  • the TH1 or TH2-led immune response predominates.
  • the TH2-guided immune response predominates, ie there is no or insufficient TH1 - guided immune response, there is a TH1 / TH2 disease in the sense of this invention
  • TH1 cells develop a cytolytic effect via a path controlled by the Fas ligand (FasL) or Fas receptor (FasR)
  • FasL Fas ligand
  • FasR Fas receptor
  • KB Proc Natl Acad Sei USA 92, 11225 - 11229, 1995, Ju, ST, Cui, H., Panka, DJ, Ettinger, R., and Marshak, RA; Proc. Natl. Acad. Be. USA 91, 4185-4189, 1994; Richardson, BC, Buckmaster, T., Keren, DFjmd Johnson, KJ; Eur. J.
  • cloned TH1 cells have a high FasL expression rate in contrast to cloned TH2 cells (Hahn, S., Stalder, T., Wernli, M., Burgin, D., Tschopp, J., Nagata, S., and Erb, P., Eur.J. Immunol. 25, 2679 - 2685, 1995; Ramsdell, F., Seaman, MS, Miller, RE, Picha, KS, Kennedy, MK and Lynch, D. , Int. Immunol.
  • FasL binds to its receptor FasR, a cascade of signals is triggered which ultimately lead to cell death (apoptosis) (Nagata, S. and Golstein, P., Science 267, 1449 - 1456, 1995).
  • FasR FasR
  • FasR FasR
  • Leishmania is a characteristic example of the class of pathogens that cause intracellular infection.
  • Leishmania belongs to the Trypanosomatidae family and is caused by its diverse human pathogenic species (L. donovani, L. infantum, L. chagasi, L. tropica, L. ajor, L. braziliensis, L. mexicana) visceral, cutaneous or the mucosal leishmaniasis.
  • L. affects only the endolysosomal Compartments of macrophages The immune response is based solely on CD4 + cells
  • Heinzel et al Heinzel, FP, Sadick, MD, Holaday, BJ Coffman, RL and Locksley, RM, J Exp Medecine 169, 59-72, 1989
  • Kemp et al Kemp, M, Theander, TG and Kharazmi, A , Immunology today 17, 13-16, 1996) have infected two different resistant breeding lines (C57BL / 6 or C3H / HeN) with L. major based on the experimental mouse model for Leishmaniasis, which reflects the human immunological conditions.
  • the THI subclass of the CD4 + cells is responsible for the resistance of the two breeding lines From the writings of Heinzel, FP, Sadick, MD, Holaday, BJ, Coffman, RL and Locksley, RM, Journal of Experimental Medicine 169, 59-72, 1989, and Sadick, MD, Locksley, RM, Tubbs, C and Raff, HV, J Immunol 136, 655-661, 1986, are also known that the genetically non-resistant mouse strain, such as Balb / c, immunologically respond to the Leishmania infection by activating the TH2 cells However, as signalers for antibody production, they cannot adequately combat Leishmania infection.
  • TH1 cells produce especially interferon- ⁇ (IFN- ⁇ ), while TH2 cells produce interleukin-4 (IL-4)
  • intracellular parasitic pathogens which can trigger TH1 / TH2 diseases in the sense of the invention are to be understood in particular as follows:
  • Trypanosomoses (Trypanosoma cruzi, Trypanosoma equiperdium), Toxoplasmosen (Toxoplasma gondi ⁇ ), Mycosis (Mycosis fungoides), Candida infections (Candida), Tubercolosis (Mycobacterium titberculosis), Lepra (Mycobacterium leprae), Letteetussis (Bordetella) Bordelle , Chlamydiosen (Chlamydia) and BCG (bacille Calmette-Gu ⁇ rin).
  • the object of the present invention is to provide, by using suitable substances or by introducing new substance compositions, adequate medicaments which modulate the immune defense against TH1 / TH2 diseases in such a way that intracellular pathogens are attacked by an effective immune response carried by the inflammatory TH1 cells become.
  • compositions of matter and the use of these compositions or other substances for the treatment of TH1 / TH2 diseases are disclosed.
  • the present invention therefore relates to the use of FasL as an active pharmacological component for the treatment of TH1 / TH2 diseases and the corresponding use of FasL for the manufacture of a medicament for the treatment of Leishmaniasis, AIDS, Listeriosis or infections with other intracellular viral, bacterial or protozoological Excitement
  • TH1 / TH2 diseases have a reduced THl response.
  • An effective inflammatory TH1 cell response is crucial for immunological defense, particularly in the case of infections with intracellular pathogens.
  • the administration of FasL compensates for the immune response of the TH1 cells, which can effectively combat the intracellularly residing pathogens of viral, bacterial or protozoological origin in diseases such as leishmaniasis, AIDS, listeriosis, trypanosomosis, Bortedella, leprosy, etc.
  • the addition of FasL thus compensates for a missing or insufficient THI immune response.
  • the gene product of the human FasL gene sequence is used as a therapeutic agent for the treatment of TH1 / TH2 diseases.
  • physiologically active gene products of FasL gene segments that are potent for apoptosis for example N- or C-terminally shortened gene products, or gene products to which one or if several intracistronic gene segments are missing, they can be used.
  • FasL for the treatment of TH1 / TH2 diseases can be used in veterinary medicine in mammals, in particular in farm animals, especially in pigs, cattle and sheep, but also in pets, especially in dogs and cats, and in human pathology for support the THI immune response to intracellular pathogens. Farm animals are to be understood in particular as those mammals that humans use for transport tasks or in the agricultural sector for food production.
  • the use of FasL is particularly preferred for the treatment of human leishmaniasis, both cutaneous and visceral as well as mucosal leishmaniasis caused by L. donovani, L. infantum, L. chagasi, L. tropica, L. major, L. braziliensis, L mexicana triggered, addressed. Accordingly, the intracellularly parasitic Leishmania species can be controlled with FasL doses in farm animals.
  • FasL initiates apoptosis in infected cells.
  • One embodiment is the use of FasL in pharmacological form in vivo.
  • FasL can be used in vitro in the course of an external blood wash.
  • Infected blood cells i.e. differentiated cells of the hematopoietic system, especially macrophages, are killed ex vivo by administration of FasL.
  • the blood treated with FasL is again fed to the patient.
  • FasL is applied externally in order to avoid any undesirable side effects of administration of FasL.
  • FasL is administered topically to the patient.
  • Topical application means non-systemic application.
  • the substance according to the invention is then applied to the epidermis or instilled in the eyes, nose or ears, and essentially does not get into the bloodstream. ⁇
  • Liquid, semi-liquid or semi-solid formulations are suitable for topical application. They are applied to the cutaneous or mucosal lesions in a form capable of absorption. For example, cream-like formulations, ointments, pastes, drops or lotions are available.
  • the topical application of FasL is also carried out in solid form the provision of powder
  • Ointments, pastes or cream formulations contain the active substance as an aqueous or non-aqueous solution or suspension based on a fat-containing or non-fat-containing carrier substance
  • FasL is administered systemically, with oral, intravenous, intraperitoneal and intramuscular administration forms being particularly preferred.
  • oral, intravenous, intraperitoneal and intramuscular administration forms being particularly preferred.
  • the formulation for parenteral administration is particularly preferred
  • Carrier materials for systemic application are in particular the dosage forms cited below (Hartke & Mutschier, German Pharmacopoeia, 9th edition, 1986, volume 3 "Parenteralia”, S 2670-2677, Academicliche Verlagsgesellschaft Stuttgart, Govi- Verlag GmbH Frankrequisitet, Pharmacopoea Helvetica, Edition Septima 1 1 1991 and renewed version 1996, monograph "Parenteralia”, US Pharmacopeia, National Formulary 18, 1995, S 1650-1652)
  • FasL is given to the patient depending on the type of application (in particular depending on systemic or topical application, application in vivo or ex vivo), on the adjuvants, the respective galenical preparation, the type of infection, the stage of infection and the current course of infection as well as on the known clinical Parameters, for example of the patient's age and the specific patient's medical history, in a dose between 1 Picogram and 100 mg per kg body weight (of the patient) applied. Application in a concentration between 10 "8 and 10 " 3 g / kg body weight is preferred.
  • the active FasL substance is combined with further pharmacological carrier materials which, for example, improve the pharmacokinetic properties, but at the same time are also physiologically compatible.
  • FasL7CD4 + / THl cells or FasL only weakly expressing cells can be transfected with the FasL.
  • FasL gene material can be introduced by lipofection, microinjection, with the aid of viral vectors or by electroporation.
  • the FasL gene material can be provided with an upstream promoter and transfected in this form, or else can be inserted in a targeted manner at the position of the physiological promoter.
  • Clonogenic cells that are transfected by standard methods and stably express FasL are used therapeutically in TH1 / TH2 diseases in order to reinforce the TH1 immune response.
  • T lymphocytes are removed from the infected patient and THl cells are isolated whose T cell receptors recognize antigens of the respective infectious agent. These cells are cultivated and transfected with FasL gene material.
  • the FasL gene material can comprise the human FasL gene sequence as well as physiologically active and apoptosis-reactive FasL gene segments or gene segments that express the N- or C-terminally shortened FasL protein.
  • Stable transfected, clonogenic, autologous cells are again administered to the patient with FasL deficiency or a suppressed THI immune response.
  • TH1 cells transfected with FasL are used to treat TH1 / TH2 diseases.
  • the same spectrum of infections is covered that has already been mentioned above for the treatment with FasL.
  • the transfected, autologous cells mobilize that FasL / FasR apoptosis system and kill infected cells, especially macrophages.
  • THI lymphocytes can kill macrophages infected with Leishmania in vitro.
  • THl lymphocytes were cocultivated with Leishmania-induced THl cells in the presence of interferon- ⁇ .
  • the THl cells were removed from lymph nodes from C57BL / 6 mice that had previously been infected with Leishmania major for 5 to 8 weeks by removing the lymph nodes that disposed of the lesions and from them the THl cells by the MACS method (Miltenyi Biotech GmbH, Bergisch-Gladbach). This material was grown in vitro by L.
  • the present invention relates to a composition of matter containing FasL and one or more cytokines, in particular interferon- ⁇ , in particular such a composition of matter as a medicament, in particular for in vivo treatment.
  • a composition of matter containing FasL and one or more cytokines, in particular interferon- ⁇ in particular such a composition of matter as a medicament, in particular for in vivo treatment.
  • the use of a corresponding composition of the invention for the treatment of TH1 / TH2 diseases is claimed, for example for the use for the treatment of leishmaniasis, AIDS, listeriosis or infections with other intracellular viral, bacterial or protozoological pathogens or in a preferred form for the treatment of cutaneous , visceral or mucosal leishmaniasis.
  • the invention also relates to the use of autologous CD4 + / FasL7TH1 cell lines of the patient transfected with the FasL gene sequence or physiologically active sections of the FasL gene sequence for the treatment of TH1 / TH2 diseases, for example the use of CD4 + transfected with FasL / FasL7THl cell lines for the treatment of Leishmaniasis, AIDS, Listeriosen or infections with other intracellular viral, bacterial or protozoological pathogens, in particular the use of CD4 + / FasL7THl cell lines transfected with FasL for the treatment of cutaneous, visceral or mucosal leishmaniasis is claimed.
  • FIG. 1 shows that the resistant control strain C57BL / 6, as expected, showed almost no signs of the lesions 12 weeks after the infection event.
  • FIG. 1 shows that a progressive course of the size of the lesions can be observed in the two other mouse strains Ipr and gld.
  • This experiment underlines the importance of functional FasL and FasR for an effective inflammatory TH1 effector function.
  • the axes represent the number of weeks after the infection event on the abscissa and the lesion size in mm on the ordinate.
  • FIG. 2 shows by series of dilutions that the number of Leishmania parasites in the lesions of the Ipr or / mice is significantly increased compared to samples from the lesions of the mouse control group which have a functional FasL / FasR signal transfer system (C57BL / 6) .
  • the number of parasites per mg tissue is plotted on the abscissa.
  • the number of parasites was determined after 30 and 90 days for each of the three strains of mice (exception: Ipr mice). The number of parasites was not determined for the Ipr-Mmise after 90 days (ND).
  • FIG. 3 shows that after an in vitro stimulation of the THl cells in all three mouse strains (control group, Ipr and gld) a comparable production of interferon- ⁇ can be demonstrated.
  • the JL-4 characteristic of TH2 cells (not shown in the figures) is almost undetectable. This proves experimentally that both mutant strains have wild-type properties with regard to cell proliferation and cytokine production.
  • this experiment shows that there is adequate cytokine production for the initiation of a THl immune response in the mutant strains.
  • FIG. 4 shows that potential influences of the immunologically active CD8 + T cells with their characteristic cytotoxic effector function by perforin secretion in a Leishmania infection can be excluded.
  • Perforin-deficient mouse strain shows the same kinetic profile when eliminating the infectious agents compared to wild-type strains.
  • the axes represent the number of weeks after the infection event on the abscissa and the lesion size in mm on the ordinate.
  • the size of the lesions as a function of the time after the original Leishmania infection with L. major in gW mice is known to be FasL-deficient (1) after administration of FasL in the form of a supernatant from FasL-expressing cell cultures, (2) without FasL and (3) in control C57BL / 6 mice who also did not receive any FasL injections.
  • the number of weeks after infection is plotted on the abscissa, and the lesion size in mm is plotted on the ordinate.
  • gW mice were infected with L.
  • gW mice that were not injected with FasL showed an average lesion size of 1.3 mm nine weeks after infection.
  • wild-type mice with functional FasL and FasR production even without administration of FasL only have an average lesion size of 0.3 mm after the same time.
  • Mice of the gW strain that received injections with FasL samples have a significantly reduced lesion size of 0.75 mm on average compared to the untreated g / ⁇ i mouse.
  • FIG. 6 the number of parasites per mg of tissue for the mouse strains gld, treated with FasL or with a control sample, and for the control strain C57BL / 6 is plotted on the abscissa.
  • the result of this experiment is comparable to the result from FIG. 5.
  • the control group of resistant C57BL / 6 mice had almost no parasites in their lesions 63 days after the infection event.
  • interferon- ⁇ interferon- ⁇
  • mice with a genetically resistant disposition to leishmaniases show an unfavorable course of infection as soon as they do not have a functional EFN- ⁇ receptor.
  • IFN- ⁇ promotes the expression of FasR on macrophages which are infected with Leishmania.
  • FIG. 7 shows that the expression of FasR is increased both in the wild type and in gW mice after administration of LFN- ⁇ . In contrast, this effect is not detectable in the FasR-deficient Ipr mice.
  • Bone marrow macrophages were six week old mice of the strains Bone marrow macrophages were taken from six week old mice of the strains C57BL / 6, Ipr and gld and according to the information in Feng et al. (Feng, ZY, Louis, J., Kindler, V., Pedrazzini, T., Eliason JF, Behin, R. and Vasalli, P .; Eur. J. Immunol. 18, 1245-1251, 1988).
  • the macrophages were then infected with promastigotes from Leishmania major in a ratio of 5: 1, washed after 12 hours and grown in the culture in the presence of IFN- ⁇ (50 U / ml) for a further 48 hours. After labeling with anti-Mac 1 monoclonal antibodies Ml / 70, 98% of the macrophages had a positive signal.
  • the macrophages were also labeled with a control antibody, a biotinylated anti-Fas antibody (JO2, Pharmingen, USA) or a biotinylated monoclonal anti-MHC-II antibody (monoclonal Ak BP 107) and then with fluorescent streptavidin (Caltag, USA). The analysis was carried out on a FACScanr flow cytometer (Becton Dickinson, Mountain View, CA, USA).
  • FasR the activation of the expression of FasR on the macrophages initiated by IFN- ⁇ leads to an increased sensitivity of the cells to apoptosis controlled by FasL.
  • IFN- ⁇ -activated and Leishmania-infected macrophages from the three above-mentioned mouse strains were used. As described in the last paragraph, these cells were grown for 48 hours in the culture and then with the supernatant from recombinant FasL-producing neuro-2a cells or from sham-transfected neuro-2a cells (Rensing-Ehl, A., Frei, K.
  • TH1 cells can therefore initiate apoptosis of macrophages infected with Leishmania or other intracellular infectious agents via the FasL / FasR system if and insofar as the macrophages have been previously activated with IFN- ⁇ .
  • IFN- ⁇ production for stimulation NO synthesis according to the present invention is not sufficient to explain the experiments on the immune responses of the mouse strains C57BL / 6, gld and Ipr against the Leishmania pathogen
  • FIG. 8 shows that the macrophages of the three strains have an approximately comparable NO synthesis rate after the addition of IFN- ⁇ .
  • mice C57BL / 6 and C3H / HeN were purchased from the company LFFA-CREDO (Saint Germain-sur-Abresle, France).
  • the two deficient mouse strains gld (FasL “ ) and Ipr (FasR " ) are derived from the strains C57BL / 6 or C3H / HeN and were bred in Jackson's laboratory (Bar Harbor, Maine, USA). All mice were six to eight weeks old at the time of the experiments
  • the Leishmania parasites of the L. major LV 39 strain were preserved in vivo and in vitro according to the method of Louis, JA, Moedder, E, Benin, R, Engers, HD (Eur J Immunol, 9, 841-847, 1979)
  • the mice were infected with the parasites subcutaneously in a paw with 2 ⁇ 10 6 promastigotes in the stationary phase with a final volume of 50 ⁇ l.
  • the recombinant FasL was in neuro-2a cells according to the method of Rensing-Ehl, A., Frei, K, Flury, R., Matiba, B., Mariani, SM, Weller, M., Aebischer, P., Krammer , PH and Fontana, A. (Eur. J. Immunol. 25, 2253-2258, 1995).
  • the apoptotic cells were analyzed using the method of Renno, T., Hahne, M., Tschopp, J. and MacDonald, H.R. (J. Exp. Med. 183, 431-437, 1996) by the content of subdiploid DNA.
  • the bone marrow macrophages are derived from bone marrow progenitor cells (after in v / ' tr ⁇ differentiation), which are obtained using the method of Feng et al. (Feng, ZY, Louis, J., Kindler, V., Pedrazzini, T., Eliason JF, Behin, R. and Vasalli, P .; Eur. J. Immunol. 18, 1245-1251, 1988).
  • Cultivation of the lymphocytes The cells were removed from those lymph nodes associated with the L. / w / or-related lesions (3xl0 6 / ml) and in vitro with UV-irradiated L.
  • the supernatants from appropriate cultures were pooled and stored at -20 ° C until they were used for cytokine determination.
  • the CD4 + T cells were removed for certain experiments in the cell suspensions by incubating Ig-M monoclonal antibodies against the CD4 marker (RL 172.4) and low-tox rabbit complement (Cedarlane, Horny, Ontario , (RL 172.4) and low-tox rabbit complement (Cedarlane, Homy, Ontario, Canada) according to the method of Swihart, K., Fruth, U., Messmer, N., Hug, K., Behin, R., Huang, S., Del Giudice, G., Aguet, M. and Louis, JA (J. Exp. Med. 181, 1995).
  • the level of proliferation was also checked by measuring the incorporation of 3 H-methylthymidine ( 3 htdr, Amersham, GB).
  • the CD47TH1 cells were taken from mice of the breeding strain C57BL / 6 (IFFA-CREDO, Saint Germain-sur-PAbresle, France), which had been infected with L. major 5 to 8 weeks previously.
  • the lymph nodes in the area of the Leishmania lesions were removed and cleaned up using the MACS method according to the method specified by the manufacturer (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany).
  • the purified CD4 + cells (2 ⁇ 10 5 per “well”) were stimulated in vitro by L. major promastigotes (previously treated with UV radiation) in the presence of irradiated (3000 rad) lymph node cell populations without CD4 + cells to use them as material for antigen-presenting cells (APC) After 72 h, the CD4 + "blast” cells (see above) were isolated and washed.
  • bone marrow macrophages which had previously been labeled with L major infected and activated with interferon-, co-cultivated in a ratio of 1: 1 (in a final volume of 2 ml in Petri dishes). After 6 h of co-cultivation, the bone marrow macrophages were harvested with a plastic scraper, washed and the portion of the apoptotic cells was determined by FACS analysis, for which the macrophage population was labeled with monoclonal anti-Mac 1 antibodies Ml / 70. After labeling, 98% of the Macrophages a positive signal.
  • the macrophages were also labeled with a control antibody, a biotinylated anti-Fas antibody (JO2, Pharmingen, USA) or a biotinylated monoclonal anti-MHC-II antibody (BP 107) and then with fluorescent streptavidin (Caltag, USA) transferred.
  • the analysis was carried out on a FACScanr flow cytometer (Becton Dickinson, Mountain View, CA, USA).
  • mice which had also been infected, were treated with a supernatant of neuro-2a sham transfectants that do not express FasL (Rensing-Ehl, A., Frei, K., Flury, R., Matiba, B., Mariani, SM , Weller, M., Aebischer, P., Krammer, PH and Fontana, A .; Eur. J. Immunol. 25, 2253-2258, 1995). Seven days after the end of treatment (or nine weeks after infection) the lesion size and the number of parasites were in the lesions in the g / t / mice treated in this way.
  • the number of parasites in the lesions on the infected paws was determined by a dilution test at certain intervals according to the method of Titus, RG, Marchand, M., Boon, T. and Louis, JA (Parasite Immunology 7, 545-555, 1985) certainly.
  • FIGS. 5 and 6 show the lesion size as a function of time (in weeks after infection). The three curves symbolize 1. ( ⁇ ) gW mice that were treated with FasL from FasL-expressing cells five weeks after infection, 2. (D) g / mice that were treated with the supernatant from FasL mock transfectants and 3.) (o) Control mice from strain C57BL / 6.
  • the g / mice Five weeks after infection and before treatment with the different supernatants, the g / mice showed a lesion size of approximately 0.9 mm. While the lesion size increased in the gW mice to which no functional FasL was injected (to approximately 1.2 mm nine weeks after infection), the lesion size decreased in the mice treated with functional FasL. Nine weeks after infection, these mice have an average lesion size of only 0.6 mm. This experiment documents that the treatment of FasL-deficient g / mice by FasL has a clear therapeutic effect on the course of the infection.
  • FIG. 6 shows the number of parasites per mg of tissue determined by a dilution assay in the lesions of the C57BL / 6 mice, the gld mice treated with FasL and the gld mice treated with the supernatant from transfectants. The number of parasites was determined nine weeks after infection and seven days after the end of treatment. £ 1
  • the number of parasites is significantly reduced by the injection of FasL (less than 10 parasites per mg tissue).
  • the mice not treated with FasL have more than 40 parasites per mg of tissue.
  • This experimental arrangement also shows the therapeutic effect of FasL in the treatment of TH1 / TH2 diseases, here using the example of Leishmania infection.
  • bone marrow cells from C57BL / 6, gld and Ipr mice, the gld and Ipr males having a C57BL / 6 origin were placed in 10 ml plastic petri dishes (Sterilin, Hounslow, GB) in DMEM (see above) ) with the addition of 20% horse serum and 30% senim-free supernatant from L929 cells, as donor of M-CSF. After seven days of cultivation, the macrophages were detached and suspended in DMEM with 10% FCS (see above), L-glutamine (216 mg / ml), 10 mM Hepes, 100 U / ml penicillin and 100 mg / ml streptomycin.
  • the macrophages were then infected with L. m ⁇ jor promastigotes in a ratio of 5: 1 and taken up in a final volume of 2 ml in plastic petri dishes.
  • the bone marrow macrophages were washed twelve hours after infection and incubated in the medium described above for 48 h with recombinant IFN- (50 U / ml).
  • the supernatant from either FasL-producing neuro-2a cells or neuro-2a sham transfectants (20% by volume) was added to the macrophage cultures.
  • the FACS method described above see under a)
  • Cell surface expression of MHC-II major class II isocompatibility complex
  • FasR molecules FasR molecules.
  • FIG. 7 The result of this experiment is shown in FIG. 7 as a plot of the cell number against the number of Co (control), MHC-II and FasR molecules for the wild-type, the gld and the g / mice.
  • the comparable activation of the three mouse strains by IFN- ⁇ was demonstrated by the increased expression of MHC-II molecules.
  • the integral of the three strains is comparable to the number of cells and the number of expressed MHC-II molecules.
  • the number of FasR molecules expressed as a result of treatment with IFN- ⁇ affects the individual strains with characteristic differences.
  • Both the wild type strain and the gW strain show an increased expression rate, but not the FasR-deficient gW strain. It is thus clear that the treatment with IFN- ⁇ has a direct influence on the FasL / FasR-controlled apoptosis signal in that the expression rate of the FasR molecules is up-regulated by EFN- ⁇ .

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Abstract

L'invention concerne l'utilisation de ligands Fas(FasL) comme principe pharmacologique actif pour le traitement de maladies mettant en jeu les cellules TH1/TH2, une composition contenant des ligands Fas et au moins une cytokine, notamment l'interféron η, l'utilisation d'une composition selon la revendication 11 ou 12 pour le traitement de maladies mettant en jeu les cellules TH1/TH2, l'utilisation de lignées cellulaires CD4+/FasL-/TH1 autologues du patient transfectées avec la séquence génique FasL ou avec des fragments physiologiquement actifs de la séquence génique FasL pour lutter contre les maladies mettant en jeu les TH1/TH2.
PCT/EP1997/005589 1996-10-18 1997-10-10 UTILISATION DE FasL OU DE LIGNEES CELLULAIRES CD4+FasL-/TH1 TRANSFECTEES AVEC DES FasL POUR LE TRAITEMENT DE MALADIES METTANT EN JEU LES TH1/TH2 WO1998017305A1 (fr)

Priority Applications (3)

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CA002269034A CA2269034A1 (fr) 1996-10-18 1997-10-10 Utilisation de fasl ou de lignees cellulaires cd4+fasl-/th1 transfectees avec des fasl pour le traitement de maladies mettant en jeu les th1/th2
EP97911208A EP0930890A1 (fr) 1996-10-18 1997-10-10 UTILISATION DE FasL OU DE LIGNEES CELLULAIRES CD4?+ FasL?- /TH1 TRANSFECTEES AVEC DES FasL POUR LE TRAITEMENT DE MALADIES METTANT EN JEU LES TH1/TH2
AU48664/97A AU4866497A (en) 1996-10-18 1997-10-10 Use of FASL or FASL transfected CD4+/FASL-/TH1-cell lines for the treat ment of TH1/TH2 diseases

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DE19643093A DE19643093C2 (de) 1996-10-18 1996-10-18 Verwendung von FasL oder mit FasL transfizierten CD4·+·/FasL·-·/TH1-Zellinien oder von FasL in Kombination mit einem oder mehreren Cytokin(en) zur Bekämpfung von TH1/TH2-Krankheiten

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US20030044352A1 (en) * 2001-04-24 2003-03-06 Phillips Nigel C. Method for evaluating the efficacy of treatment with bacterial DNA and bacterial cell walls

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WO1995018819A1 (fr) * 1994-01-07 1995-07-13 Immunex Corporation Ligand fixant l'antigene fas
WO1997012632A1 (fr) * 1995-10-05 1997-04-10 Tkb Associates Limited Partnership Methodes de traitement de maladies associees a une deficience de l'activite du ligand fas

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EP0723556A4 (fr) * 1993-10-14 2003-03-19 Immunex Corp Antagonistes de fas et leurs applications
JPH08127594A (ja) * 1993-11-10 1996-05-21 Mochida Pharmaceut Co Ltd Ｆａｓ抗原に結合する新規蛋白質およびそれをコードするＤＮＡ
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WO1995018819A1 (fr) * 1994-01-07 1995-07-13 Immunex Corporation Ligand fixant l'antigene fas
WO1997012632A1 (fr) * 1995-10-05 1997-04-10 Tkb Associates Limited Partnership Methodes de traitement de maladies associees a une deficience de l'activite du ligand fas

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ASHANY D. ET AL.: "Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 92, no. 24, 21 November 1995 (1995-11-21), WASHINGTON US, pages 11225 - 11229, XP002055702 *
ESTAQUIER J ET AL: "T helper type 1- T helper type 2 cytokines and T cell death: Preventive effect of interleukin 12 on activation-induced and CD95 ( FAS -APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons.", JOURNAL OF EXPERIMENTAL MEDICINE 182 (6). 1995. 1759-1767, XP002055700 *
FUSE Y ET AL: "CD95 ( Fas ) may control the expansion of activated T cells after elimination of bacteria in murine listeriosis.", INFECTION AND IMMUNITY 65 (5). 1997. 1883-1891, XP002055705 *
HAHN S. ET AL.: "Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells vis Fas/Fas-ligand interaction", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 25, September 1995 (1995-09-01), pages 2679 - 2685, XP002055703 *
KEMP M. ET AL.: "The contrasting roles of CD4+ T cells in intracellular infections in humans: leishmaniasis as an example", IMMUNOLOGY TODAY, vol. 17, no. 1, January 1996 (1996-01-01), pages 13 - 16, XP002055704 *
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DE19643093A1 (de) 1998-04-23
AU4866497A (en) 1998-05-15

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