EP1208115A2 - Interleukine-12 feline utilisee comme stimulant immunitaire - Google Patents

Interleukine-12 feline utilisee comme stimulant immunitaire

Info

Publication number
EP1208115A2
EP1208115A2 EP00954321A EP00954321A EP1208115A2 EP 1208115 A2 EP1208115 A2 EP 1208115A2 EP 00954321 A EP00954321 A EP 00954321A EP 00954321 A EP00954321 A EP 00954321A EP 1208115 A2 EP1208115 A2 EP 1208115A2
Authority
EP
European Patent Office
Prior art keywords
feline
sequence
carnivores
nucleic acid
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00954321A
Other languages
German (de)
English (en)
Inventor
Hans Lutz
Christian Leutenegger
Nils Pedersen
Matthias Schroff
Burghardt Wittig
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Zuerich
Mologen Forschungs- Entwicklungs- und Vertriebs GmbH
University of California
Original Assignee
Universitaet Zuerich
Mologen Forschungs- Entwicklungs- und Vertriebs GmbH
University of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaet Zuerich, Mologen Forschungs- Entwicklungs- und Vertriebs GmbH, University of California filed Critical Universitaet Zuerich
Publication of EP1208115A2 publication Critical patent/EP1208115A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to the long cytokine Interleuk ⁇ n-12 (IL-12) and its use as an immunostimulant in fids
  • Interleukin 12 belongs to the class of cytokines, a group of
  • IL-12 Proteins which mediate signals between different cells involved in the coordination and execution of the immune response.
  • IL-12 was published as a "Natural Killer Cell Stimulatory Factor" (Trinchien et al in EP 0 441 900, US 5,571, 515).
  • Interleukin 12 is a heterodimeric protein , consisting of the subunits p35 and p40 IL-12 belongs to a class of cytokines which are at the beginning of the formation of an immune response, close to the systems of innate immunity (macrophages, complement) and have a decisive influence on the development of the type of forming adaptive immune response have IL-12 in the series of so-called "type 1" cytokines, which favor the formation of a cytotoxic, T-cell-based response favor IL-12 stimulates, inter alia, the secretion of interferon gamma in CD4 -pos ⁇ t ⁇ ven T helper cell population Because of these properties, IL-12 was allowed to be used as an adjuvant or as an immunostimulant to cure pre-existing diseases are
  • the natural protection against infectious diseases is based on the recognition of structures of successfully controlled pathogens by the immune system. Two main activities can be distinguished. On the one hand, this is the activity of the humoral immune system, which is based on the synthesis of antibodies by the plasma cells from B-lymphocytes, but also humoral components of non-adaptive, innate immunity such as the complement system.
  • Antibodies are soluble protein molecules that are able to specifically attach to an antigen that is accessible to these antibodies either in soluble form or on the surface of cells, bacteria and viruses. By complexing with antibodies, the pathogens or toxins are either inactivated or identified for components of the non-adaptive immune system, which then eliminate them.
  • the cellular immune system is also known, which is based on the activity of T-lymphocytes in particular, but also "natural killer cells” (NK cells) and antigen-presenting cells of the innate immune system.
  • T-lymphocytes are able to recognize body cells infected with viruses as "foreign” if the infected cells present suitable structures recognizable for the T cells.
  • NK cells natural killer cells
  • T-lymphocytes are able to recognize body cells infected with viruses as "foreign” if the infected cells present suitable structures recognizable for the T cells.
  • T helper cells so-called CD4 + cells
  • CD8 + cells directly initiates the lysis of the cell recognized as foreign or infected
  • the cellular arm of the immune system is induced by activation of so-called type 1 helper cells and the humoral arm by activation of so-called type 2 helper cells (Mosmann et al., 1986).
  • the cellular arm is also referred to as the ' TH1 pathway ' and the humoral arm as the ' TH2 pathway ' of the immune system.
  • Bacteria are usually controlled by the TH2 arm by covering antigenic binding sites on the surface of the bacteria with antibodies. Bacteria covered in this way can then be eliminated by phagocytes.
  • the TH2 arm of the immune system is also important for neutralizing bacterial toxins and for fighting various parasites that are located in the extracellular space in the patient's body.
  • Infection pathogens which are primarily intracellular, as is known for individual types of bacteria and all viruses, are primarily combated by the TH1 arm of the immune system, i.e. by cytotoxic T cells.
  • Different pathogens stimulate only one arm of the immune system, and certain infectious diseases, as a result of which only the TH2 arm is activated, cannot be controlled by the immune system, or cannot be controlled efficiently. In such cases, stimulation of a TH2 response by vaccination is also ineffective. Induction of the TH1 immune response after vaccination is only possible if it is the vaccine antigen is a reproductive agent. Vaccines that are unable to reproduce in the animal can only induce a humoral, but not a cellular, immune response.
  • Invention to provide a functional feline IL-12 and / or a necessary nucleic acid-based sequence, and thereby in the fleins to induce a TH1 immune response in the target cells via synthesis of interferon gamma or other biologically active molecules.
  • this object is achieved by expressing the two polypeptide chains of the subunits p35 and p40 of feline interleukin 12 in eukaryotic or prokaryotic cells by means of recombinant gene expression and to provide the proteins formed in such a way that they are present in equimolar concentrations in the presence of an immunization agent suitable antigen can be used. It is irrelevant whether the antigen is administered together with the IL-12 by co-injection or other forms of external delivery, or whether the antigen is already in the course of an existing infection or allergy to be treated (not -human) animal, e.g. Cat, is present and contributes locally to the development of the desired answer.
  • the immunostimulant of the present invention can also be achieved by the control of cat-operable promoter and terminator or polyadenylation sequences of one or more DNA constructs consisting of genes encoding the p35 and p40 subunits of feline IL-12 , are placed directly in the cells of the cat, where they bring about the synthesis of functioning IL-12 and thus induce the desired cascade of immunostimulatory signals, primarily primarily the induction of the synthesis of interferon gamma.
  • Another aspect of the invention is an immunostimulant for the therapy of certain diseases or. Adjuvant for co-injection with antigen.
  • Immunostimulant can also be used for
  • TH1 response is helpful. This has already been postulated for various animal species and for humans (Gately and Mulqueen, 1996), but has so far not been demonstrated.
  • diseases in which the feline IL-12 adjuvant of the present invention can be used are infection with the feline coronavirus, which leads to the widespread and feared feline infectious peritonitis (FIP).
  • feline infectious peritonitis FIP
  • this disease results in a massive excess weight of a TH2 response, which leads to vasculitis, peritonitis and death. This early assumption was confirmed by measuring the cytokine activity in cats with FIP.
  • cytokines specific for TH2 could be detected in abundance, but IL-12 and interferon gamma could hardly be detected or not at all. Further examples result from infection with the FIV or the feline leukemia virus (FeLV). These two retrovirus infections are characterized by the intracellular presence of the virus, which eludes a humoral immune response. By stimulating the TH1 response by administering IL-12 it is possible to make the amount of virus in infected cats disappear or at least to reduce it significantly. These are just a few application examples; the list is not exhaustive.
  • the present invention generally also encompasses polypeptides with at least 95% sequence similarity to that of the polypeptide encoded by the nucleotide sequence flL12p40 (p40 subunit of feline IL-12: SEQ ID NO 1) and flL12p35 (IL-12SEQ ID NO 2) as Immune stimulant, especially for prevention and therapy in carnivores, especially in the domestic cat.
  • flL12p40 p40 subunit of feline IL-12: SEQ ID NO 1
  • IL-12SEQ ID NO 2 flL12p35
  • nucleic acid constructs that contain sequences with at least 95% sequence similarity to the sequences of flL12p40 (p40 subunit of the feline IL-12: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:
  • Contain NO 1) and flL12p35 (p35 subunit of the feline: SEQ ID NO 2), and in which the sequences are under the control of an operable promoter and terminator sequence in higher animals, such as carnivores, in particular furds, especially domestic cats, operable promoter and terminator sequence, are suitable immunostimulants , for immunization against infectious diseases and for the therapy of infectious and
  • Corresponding nucleic acid constructs are preferably those in which the construct consists of a linear, double-stranded DNA double strand which contains only one promoter and the coding sequence per strand.
  • a polypeptide according to the invention is suitable as a therapeutic agent for tumor diseases and autoimmune diseases, or for diseases in which a TH1- There is a deficit, especially in the case of pre-existing FIV, FeLV and coronavirus infections.
  • a nucleic acid construct according to the invention is suitable as an adjuvant for the prophylactic immunization against viral diseases of carnivores, in particular baits, especially domestic cats, especially for immunization against the FIV
  • nucleic acid construct according to the invention is also suitable as a therapeutic agent for diseases in which there is a TH1 deficit, in particular in the case of pre-existing FlV infection, FeLV infection or FCoV infection.
  • FIG. 1 shows the cloning strategy of the p35 construct, showing how the feline p35 gene was stably supplemented by sequences from the human 11-12 p35.
  • Figure 2 is a sequence comparison of human and feline p35.
  • 1st line human sequence pG-hLL12p;
  • 2nd line feline sequence according to Fehr et al .; fil12p35a;
  • 3rd line recombinant sequence pMOL-flL12 p35;
  • FIG. 3 shows an IRES construct, the construct being preceded by a CMV and a T7 promoter and important restriction sites being shown.
  • FIG. 4 shows the expression of interferon (IFN) -
  • FIG. 5 shows the RNA virus load in animals of the third
  • the core of the invention is the provision of both subunits of the IL-12, in a form which enables their expression, preferably in feline cells or tissues.
  • the basis for this is the successful cloning of the coding sequences of both subunits in recombinant expression constructs
  • Periodic blood tests can then be used to determine how the immunization behaves Four cats each showed that the animals in group 1 (vaccinated with FIV-DNA and IL-12) performed better in all relevant parameters with which the infection can be characterized than animals in group 2, which only with the FIV- DNA had been vaccinated (see Example 4) embodiments
  • Example 1a Recombinant feline IL-12, p40
  • the confirmed sequence was again PCR-amplified using the primers 5'-GTAGCGGATA AGGTACCATG CATCCTCAGC AGTTGGT (SEQ ID NO 4) and 5'-GAGAGTTCTC AGAGCTCATC CTGGGGGTGG AACCTAA (SEQ ID NO 5) according to standard conditions and the isolated amplification and restriction enzyme was then cloned and cloned using the restriction enzyme digested and used between the Kpnl and Sstl interfaces of the vector pMol using known methods. The result was the plasmid pMol-flL12p40.
  • Example 1b Recombinant feline IL-12, p35
  • a human p35-encoding plasmid pMOL-hlL12p35 was used as a template for the amplification with the primers f12p35-l-long (71 mer) 5 ' - TGCTGACAGC TATTGATGAG CTGTTACAGG CCCTGAATGT CAACAGTGTG ACTGTGCCAC AGAACTCCTC p (SEQL ID12TC) C (SEQ -) - r (76mer)
  • the amplificate was digested using the residual functional endonucleases SstI and Kpnl and used between the Kpnl and Sstl interfaces of the vector pMol using known methods.
  • the result was the plasmid pMol-flL12p35.
  • the resulting reaction mixture was concentrated and after buffering with 100 U restriction endonuclease HindIII and 100 U T7 DNA polymerase in the absence of deoxyribonucleotides above sea level. digested.
  • the resulting product was purified by anion exchange chromatography and, after control by gel electrophoresis and PCR control, was free of residues of the undesired fragment.
  • Example 2a In vitro transcription / translation of the two IL-12 p35 and p40 chains
  • Construct 1 was based on the p40 sequence produced by PCR, which was incorporated into the pCIneo Vector (Promega).
  • the plasmid contained the CMV and the T7 promoter and was named pCI-p40.
  • Construct 2 was based on the p35 sequence produced by PCR, which was incorporated into the vector pCITE4a (+) (Novagen)
  • the plasmid contained the IRES (infernal nbosomal entry site), which preceded the p35 sequence. This plasmid was called pCITE-p35.
  • Construct 3 corresponded to the construct shown in FIG. 3, it was called pCI-flL-12. In vitro translation was carried out with the three constructs mentioned above to check the correct translation of the two subunits p35 and p40
  • the plasmids pCI-p40, pCITE-p35 and pCI-flL-12 were heaned and transcribed in vitro using the T7 Cab Scribe Kit (Boehnnger Mannheim).
  • the RNA was purified and translated in vitro using the Flexi TM Rabbit Reticulocyte Lysate Systems (Promega) was used.
  • the translation products were labeled with 35 S methionones.
  • the newly synthesized proteins were separated on an SDS gel according to their molecular weight. The gel was dried under vacuum and exposed to a film which was then attached The bands found on the film corresponded in molecular weight to that expected for p35 and p40 (Table 1)
  • Example 2b In vitro induction of IFN gamma in cat cells by incubation with cell culture supernatant containing IL-12
  • the experiment mentioned below was carried out.
  • the plasmid pCI-flL-12 was hernized.
  • the DNA was transcribed on the one hand in vitro using the T7 Cab Scribe Kit (Boehnnger Mannheim) and on the other hand used directly for transfection.
  • the resulting RNA was used for short-term transfection of BHK-21 cells.
  • RNA transfection was carried out under routine conditions.
  • BHK-21 cells were treated with water under the same conditions for negative control transfected.
  • SP2 / 0 cells were used and grown on G418 medium 24 hours after transfection.
  • cells were transfected with water for control purposes.
  • Untransfected cells died within 7 days because, due to the lack of the neomycin resistance gene, they were unable to protect themselves from the toxic effects of the neomycin.
  • the supernatants of the cells transfected with RNA or DNA, in which the IL-12 was suspected were used for the culture of lymphocytes freshly obtained from specified pathogen-free (SPF) cats. Prior to their use, these lymphocytes were incubated with the cell culture supernatant containing IL-12 for 72 hours at 37 ° C.
  • Example 2c In vivo induction of IFN gamma in cats by injection of IL-12 protein
  • Example 2d Checking the functionality of the complete IL-12 or the individual chains p35 and p40 after ballistic transfer into the feline cell line 3201
  • 3201 cells were bombarded with gold beads (diameter 1 micron). The gold beads had previously been coated with the gene coding for p35 and p40 or with the gene coding for the green fluorescing protein (GFP). In parallel, 3201 cells were also transfected electrically with that for p35 alone, that for p40 alone, that for p35 and p40, and with the gene coding for GFP. Aliquots of the 3201 cells were then cocultured with SPF lymphocytes for 24 hours.
  • GFP green fluorescing protein
  • the lymphocytes were harvested periodically and examined for the expression of IFN gamma as described in Example 2c. The results are shown in Figure 4. It becomes clear that the transfection by means of gold beads and by means of electric current leads to the production of IFN gamma 16 to 24 hours after cocultivation of the transfected cells with the lymphocytes. Ballistic or electrical transfection with GFP or the p35 or p40 genes alone does not lead to IFN gamma synthesis.
  • Example 3 Immunization of the cat against FIV using IL-12 as an adjuvant.
  • IL-12 as an adjuvant can improve the effectiveness of a vaccine
  • a vaccination test was carried out in which 3 groups of 4 cats were used.
  • the basic antigen in all groups (with the exception of the control group) was the gene which codes for the gp140-SU antigen.
  • the gene construct mentioned here should generally be referred to as gp140-DNA.
  • Vaccination by direct injection of naked DNA is disclosed in US 5,580,859, US 5,589,466 and US 5,593,972.
  • the DNA constructs were according to Wittig et al. (WO 98/21322); they contained minimalistic expression constructs consisting only of the coding sequence, in front of which the sequence of the cytomegalo virus promoter (CMV) had been placed.
  • CMV cytomegalo virus promoter
  • the coding sequence and the CMV promoter were used as linear double-stranded molecules which were covalently terminated on both sides in order to prevent extracellular or intracellular degradation by exonucleases.
  • the DNA constructs were adsorbed onto small gold particles, which were shot directly into the skin of the test animals. The animals were bombarded three times three weeks apart with the appropriate constructs, the DNA being raised to 1 mg gold per shot.
  • a helios gene gun Biorad, Kunststoff, Germany
  • a pressure of 500 psi were used for the immunization.
  • the entire DNA Dose was approx. 2 ⁇ g per animal per vaccination.
  • Vaccine contained: Research question no. Negative controls, none of these cats
  • the protective effect of the different vaccine preparations was determined by measuring the following parameters at weekly intervals (exception: measurement of the RNA load, only in week 5)
  • the amount of FIV-RNA in the plasma of these cats was determined using a TaqMan ® -PCR method
  • FIV-RNA virus load in plasma The results of the FIV-RNA quantification in the plasma of the cats are shown in FIG. 5. The results can be commented as follows:
  • Group 1 The RNA virus load was highest here.
  • Group 2 The cats vaccinated with the gp140 DNA showed a significantly lower RNA virus load than the animals of the controls; From this it can be deduced that the gp140 construct alone can induce partial protection. These results are in agreement with the serology.
  • Amount of proviral DNA The results of the quantification of the proviral amount of DNA of all cats are summarized in Table 6. The results can be commented as follows:
  • Group 1 As with serology and RNA measurement, the animals in Group 1 also proved to be fully susceptible to the test infection.
  • Group 2 The animals in group 2 were also provirus positive without exception, but the mean amount of FIV provirus was only marginally lower than that of the control group.
  • IL-12 DNA together with gp140-DNA can induce a high protective effect.
  • the protective effect manifests itself on the one hand in less virus replication, which leads to little or no absence of seroconversion and / or to less integration of the viral DNA into the host cell DNA.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne une interleukine-12 féline (IL-12) utilisée comme adjuvant ou comme stimulant immunitaire, aussi bien lors de la vaccination contre des maladies infectieuses chez les félidés que lors de la thérapie desdites maladies. L'invention concerne en outre des procédés permettant d'exprimer les deux sous-unités de l'IL-12 dans les rapports quantitatifs requis.
EP00954321A 1999-07-08 2000-07-08 Interleukine-12 feline utilisee comme stimulant immunitaire Withdrawn EP1208115A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CH125999 1999-07-08
CH125999 1999-07-08
PCT/DE2000/002263 WO2001004155A2 (fr) 1999-07-08 2000-07-08 Interleukine-12 feline utilisee comme stimulant immunitaire

Publications (1)

Publication Number Publication Date
EP1208115A2 true EP1208115A2 (fr) 2002-05-29

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP00954321A Withdrawn EP1208115A2 (fr) 1999-07-08 2000-07-08 Interleukine-12 feline utilisee comme stimulant immunitaire

Country Status (4)

Country Link
US (1) US20030157059A1 (fr)
EP (1) EP1208115A2 (fr)
AU (1) AU6683500A (fr)
WO (1) WO2001004155A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5571515A (en) * 1994-04-18 1996-11-05 The Wistar Institute Of Anatomy & Biology Compositions and methods for use of IL-12 as an adjuvant
CA2265505C (fr) * 1997-05-16 2009-09-08 Toray Industries, Inc. Medicament immunologique, methode de traitement et agent preventif, et methode applicable aux chiens et aux chats

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0104155A2 *

Also Published As

Publication number Publication date
WO2001004155A2 (fr) 2001-01-18
WO2001004155A3 (fr) 2001-08-02
US20030157059A1 (en) 2003-08-21
AU6683500A (en) 2001-01-30

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