WO2001045747A1 - Vaccin base sur un facteur d'induction de l'interferon gamma et utilisation de ce vaccin destine a conferer une immunite de protection contre la sclerose en plaques - Google Patents

Vaccin base sur un facteur d'induction de l'interferon gamma et utilisation de ce vaccin destine a conferer une immunite de protection contre la sclerose en plaques Download PDF

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Publication number
WO2001045747A1
WO2001045747A1 PCT/US1999/026094 US9926094W WO0145747A1 WO 2001045747 A1 WO2001045747 A1 WO 2001045747A1 US 9926094 W US9926094 W US 9926094W WO 0145747 A1 WO0145747 A1 WO 0145747A1
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Prior art keywords
antibody
cells
interferon gamma
animal
inducing factor
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PCT/US1999/026094
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English (en)
Inventor
Nathan Carin
Gizi Wildbaum
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Technion Research & Development Foundation Ltd.
Friedman, Mark, M.
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Application filed by Technion Research & Development Foundation Ltd., Friedman, Mark, M. filed Critical Technion Research & Development Foundation Ltd.
Priority to EP99972428A priority Critical patent/EP1140206A4/fr
Priority to CA002355345A priority patent/CA2355345A1/fr
Priority to AU41640/00A priority patent/AU4164000A/en
Publication of WO2001045747A1 publication Critical patent/WO2001045747A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising 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 present invention relates to the treatment of multiple sclerosis and, more particularly, to interferon gamma inducing factor (IGIF) based vaccines for effecting same.
  • IGIF interferon gamma inducing factor
  • CD4 + T-cells can be divided to Thl cells that produce large amounts of interferon gamma (IFN- ⁇ ) and TNF- ⁇ , and, to a much lesser extent, IL-4 and IL-10; Th2 cells that produce IL-4, IL-10, and IL-13 and, to a much lesser extent, IFN- ⁇ and TNF- ⁇ (1-10), and the newly defined Th3 cells that produce significant amounts of transforming growth factor beta (TGF- ⁇ ) and have been associated with oral tolerance (11).
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ interferon gamma
  • TGF- ⁇ transforming growth factor beta
  • Thl cells selected in response to various auto-antigens transfer T-cell mediated autoimmune diseases, whereas IL-4 secreting Th2 cells, selected in response to these same antigens, either inhibit or exert no profound effect on the inflammatory process (5, 12-24).
  • High levels of IFN- ⁇ and low levels of IL-4 positively select for Thl cells, whereas, low levels of IFN- ⁇ together with high levels of IL-4 mediate Th2 selection (1-6).
  • Interferon gamma inducing factor is a recently described cytokine (25) that shares structural features with the interleukin- 1 (IL-1) family of proteins (26). Activation of IGIF is mediated by interleukin- 1 beta converting enzyme (ICE) (27, 28). Like IL-12, IGIF is a potent inducer of the production of IFN- ⁇ by Thl and natural killer (NK) cells, and acts on Thl cells together with IL-12 in a synergistic manner (25, 29-32).
  • EAE Experimental autoimmune encephalomyelitis
  • CNS central nervous system
  • MS multiple sclerosis
  • Antigen specific T cells are thought to play a pivotal role in the manifestation of both diseases (35-37).
  • Thl cells The role of Thl cells in the manifestation of EAE has been widely studied.
  • Thl but not Th2 cells transfer the disease to normal naive recipients (18). Shifting the Thl/Th2 balance towards Th2 cells by in vivo administration of IL-4 (12), by antibodies to B7-1 (14), by soluble peptide therapy (38), or by administration of neutralizing antibodies to IL-12 (15) markedly suppressed EAE.
  • the present invention disclosed the use of anti interferon gamma inducing factor antibody in the treatment of multiple sclerosis. This use can be effected in a variety of ways as further described and exemplified hereinbelow. According to one aspect of the present invention there is provided an antibody comprising an immunoglobulin capable of binding interferon gamma inducing factor.
  • a pharmaceutical composition for inducing protective immunity against multiple sclerosis comprising a pharmaceutically acceptable carrier and an antibody being capable of binding an interferon gamma inducing factor.
  • a pharmaceutical composition for inducing protective immunity against multiple sclerosis comprising a pharmaceutically acceptable carrier and an interferon gamma inducing factor or an immunogenic portion thereof, thereby eliciting an antibody being capable of binding the interferon gamma inducing factor in vivo.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis comprising the step of administering to the animal cells being capable of producing and secreting an antibody capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis comprising the step of administering to the animal an antibody capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis comprising the step of administering to the animal an antigen including an interferon gamma inducing factor or an immunogenic portion thereof, thereby eliciting an antibody being capable of binding in vivo an interferon gamma inducing factor.
  • the antibody is polyclonal.
  • the antibody is monoclonal.
  • the antibody is a neutralizing antibodies to the interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • the antibody is humanized.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis comprising the step of administering to the animal a therapeutic composition including a recombinant construct including an isolated nucleic acid sequence encoding a polypeptide being capable of eliciting antibodies capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis comprising the steps of (a) removing cells of the animal; (b) genetically modifying the cells in vitro with a recombinant construct including an isolated nucleic acid sequence encoding an interferon gamma inducing factor or an immunogenic portion thereof; and (c) reintroducing the genetically modified cells to the animal.
  • a pharmaceutical composition for inducing protective immunity against multiple sclerosis comprising a pharmaceutically acceptable carrier and a recombinant construct including an isolated nucleic acid sequence encoding a polypeptide being capable of eliciting antibodies capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • nucleic acid sequence being operatively linked to one or more transcription control sequences.
  • the transcription control sequences are selected from the group consisting of RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and ⁇ -actin control sequences.
  • the recombinant construct is an eukaryotic expression vector.
  • the recombinant construct is selected from the group consisting of pcDNA3, pcDN A3.1 (+/-), pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV, pBK- CMV, pTRES and their derivatives.
  • the therapeutic composition is administered to the animal parenterally.
  • the animal is a human being.
  • the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous physiologically balanced solution, an artificial lipid-containing substrate, a natural lipid-containing substrate, an oil, an ester, a glycol, a virus and metal particles.
  • the pharmaceutically acceptable carrier comprises a delivery vehicle that delivers the nucleic acid sequences to the animal.
  • the delivery vehicle is selected from the group consisting of liposomes, micelles, and cells.
  • the recombinant construct is an eukaryotic expression vector.
  • the composition is suitable for parenteral administration to a human.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing new horizons to the treatment of the devastating autoimmune disease multiple sclerosis.
  • FIGs. 1A-F demonstrate IGIF mRNA in the inflamed EAE brain.
  • Figures 1A, C, G and E - Rats were injected with 10 cells form L68-86 immunized rats to allow for the development of transferred EAE.
  • day 0 Before adoptive transfer of disease (day 0), and at various time points: before the onset of disease (day 3), at the day of onset (day 5), the peak (day 7), following recovery (day 10), and 10 days after recovery (day 20) mid-brain and brain stem samples from six different rats at each time point were examined.
  • mRNA was isolated from each sample and subjected to RT-PCR analysis using specific oligonucleotide primers constructed for IGIF ( Figure IC) and for IFN- ⁇ ( Figure IE).
  • FIGs. 3A-B demonstrate that neutralizing antibodies to recombinant rat IGIF block the development of both active and transferred EAE.
  • Figure 3A Lewis rats were immunized with p68-86/CFA to induce active EAE and then separated into three groups of six rats each. Eight, ten and eleven days after induction of disease, these groups were injected IV with rabbit anti-rat IGIF (IgG fraction lOO ⁇ g/rat), with IgG fraction purified from non- immunized rabbits (control IgG), or with PBS. The rats were then monitored daily for clinical signs of EAE by an observer blind to the treatment protocol. Results are presented as mean clinical score ⁇ S.E.
  • Figure 3B Transferred EAE was induced as described above ( Figure 1).
  • FIGs. 4A-D demonstrate alteration in IFN- ⁇ and IL-4 production in EAE rats injected with anti-IGIF neutralizing antibodies.
  • Lewis rats were immunized with p68-86/CFA to induce active EAE and separated into three groups. Five and seven days after disease induction these groups were injected IV with either rabbit anti-rat IGIF (IgG fraction 100 ⁇ g/rat), with purified IgG from non-immunized rabbits, or with PBS.
  • splenic T cells from three rats in each group were cultured with 100 ⁇ M MBP p68-86 for 72 hours in stimulation medium that was not ( Figures 4A and 4B) or was ( Figures 4C and 4D) supplemented with recombinant rat IL-4 (5 ng/ml).
  • IFN- ⁇ levels were determined in culture supernatants by an ELISA assay.
  • Results are of triplicate cultures expressed as mean ⁇ S.E.
  • FIG. 5 demonstrates alteration in TNF- ⁇ production in EAE rats injected with anti-IGIF neutralizing antibodies. Levels of TNF- ⁇ were determined in supernatants obtained in experiment described in Figures 4A and 4B, by an ELISA assay. Results are of triplicate cultures expressed as mean ⁇ S.E. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention is of interferon gamma inducing factor (IGIF) based vaccines which can be used in the treatment of multiple sclerosis. Specifically, the present invention can be used to confer protective immunity against multiple sclerosis.
  • IGIF interferon gamma inducing factor
  • interferon gamma inducing factor (IGIF) based vaccines may be better understood with reference to the drawings and accompanying descriptions.
  • IGIF interferon gamma inducing factor
  • T cells reactive to self-antigens escape elimination in the thymus, and are activated in the periphery where they can provoke damage to specific cells and organs. Perturbation of the balance between self-reactive T cells with different cytokine profiles may serve as an effective way of restraining the harmful effect of autoimmune T cells (12, 14-16, 20-22, 38, 41-43).
  • Cytokines present at the initiation of CD4 + T cell responses determine whether a Thl or a Th2 response will predominate (1-6).
  • administration of IL-4 or of antibodies to IL-12 preferentially favors Th2 selection in vivo and thus serves as a powerful way to inhibit two different T cell mediated autoimmune diseases: EAE and IDDM (12, 15, 22)
  • IGIF is a recently described cytokine (25) that shares structural features with the interleukin- 1 (IL-1) family of proteins (26). Activation of IGIF is mediated by interleukin- 1 beta converting enzyme (ICE) (27, 28). Like IL-12, IGIF is a potent inducer of IFN- ⁇ from Thl and NK cells, and acts on Thl cells together with IL-12 in a synergistic manner (25, 29-32). IGIF actually has more potent IFN- ⁇ inducing capabilities than IL-12 and apparently utilizes a distinct signal transduction pathway for its elicitation (25, 31, 32, 51). Little is known about the role of IGIF in T cell mediated autoimmune disease. A recent study used RT PCR to demonstrate that the active stage of autoimmune diabetes in NOD mice is associated with the expression of IGIF (52).
  • oligonucleotide primers were used to identify and isolate interferon gamma inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell mediated autoimmune disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS).
  • IGIF interferon gamma inducing factor
  • IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein which was used to induce anti-IGIF neutralizing antibodies. These antibodies significantly reduced the production of interferon gamma (IFN- ⁇ ) by primed T cells proliferating in response to their target myelin basic protein (MBP) epitope and by Con A activated T cells from naive donors.
  • IFN- ⁇ interferon gamma
  • MBP myelin basic protein
  • Splenic T cells form protected rats were cultured with the encephalitogenic MBP epitope and evaluated for production of IL-4 and IFN- ⁇ . These cells, which proliferated, exhibited a profound increase in IL-4 production, accompanied by a significant decrease in IFN- ⁇ and TNF- ⁇ production.
  • IFN- ⁇ The direct role of IFN- ⁇ in EAE is enigmatic. Grewal et al. have used a CD40L-deficient mice that carry a transgenic T cell receptor specific for MBP to demonstrate that EAE induction is IFN- ⁇ dependent (54). On the other hand not only were mice lacking IFN- ⁇ susceptible to induction of active EAE (55) but also antibodies to IFN- ⁇ were found capable of enhancing this disease (56, 57). A recent study has demonstrated that IL-12 is directly involved in the generation of autoreactive Thl -cells that induce EAE, both in the presence and the absence of IFN- ⁇ (58).
  • Thl/Th2 balance towards IL-4 secreting Th2 cells confers EAE resistance not because it leads to a reduced production of IFN- ⁇ , but rather because it results in a reduced production of TNF- ⁇ accompanied by a marked increase in IL-4 production.
  • IGIF primarily effects IFN- ⁇ production by Thl but not Th2 cells (29). It is possible that immunization with p68-86/CFA induces a substantial selection of antigen specific Th2 cells, albeit not enough to inhibit the subsequent development of a Thl mediated autoimmune disease. Hence, as shown herein, in vivo administration of anti IGIF neutralizing antibodies notably shift the Thl/Th2 balance in antigen specific proliferating T cells towards Th2 response.
  • an anti interferon gamma inducing factor antibody in the treatment of multiple sclerosis. This use can be effected in a variety of ways and applications, some of which are further described and exemplified hereinbelow. According to one aspect of the present invention there is provided an antibody which comprises an immunoglobulin capable of binding interferon gamma inducing factor (IGIF, IL-18).
  • IGIF interferon gamma inducing factor
  • antibody and "immunoglobulin”, which are interchangeably used, refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, which proteins include IgG, IgD, IgE, IgA, IgM and related proteins. These terms further relate to chimeric immunoglobulins which are the expression products of fused genes derived from different species.
  • immunologically active derivatives of the above proteins including, but not limited to, an F(ab')2 fragment, an Fab fragment, an Fv fragment, a heavy chain, a light chain, an unassociated mixture of a heavy chain and a light chain, a heterodimer consisting of a heavy chain and a light chain, a catalytic domain of a heavy chain, a catalytic domain of a light chain, a variable fragment of a light chain, a variable fragment of a heavy chain, and a single chain variant of the antibody.
  • antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent.
  • Antibodies of the IgG class are made up of four polypeptide chains linked together by disulfide bonds.
  • the four chains of intact IgG molecules are two identical heavy chains referred to as H-chains and two identical light chains referred to as L- chains.
  • the immunoglobulin or antibody according to the present invention could also be a "humanized” antibody, in which, for example animal (say murine) variable regions are fused to human constant regions, or in which murine complementarity-determining regions are grafted onto a human antibody structure (Wilder, R.B. et al., J. Clin. Oncol, 14:1383-1400, 1996). Unlike, for example, animal derived antibodies, "humanized” antibodies often do not undergo an undesirable reaction with the immune system of the subject.
  • sFv single chain antigen binding protein
  • sFv CC49 Larson, S.M. et al., Cancer, 80:2458-68, 1997.
  • humanized antibodies also reads on antibodies produced by non-human cells or organisms genetically modified to include nucleic acid sequences encoding a functional portion of the human immune system, wherein the resulting antibodies are substantially identical to human antibodies in that they are encoded by human derived genes.
  • antibody as used herein, further relates to soluble portions of receptors capable of specifically binding their respective protein ligands, which, in that respect, function like immunoglobulins.
  • animal refers to any organism with an immune system.
  • compositions for inducing protective immunity against multiple sclerosis comprising a pharmaceutically acceptable carrier and an antibody being capable of binding an interferon gamma inducing factor.
  • composition according to the present invention comprises a pharmaceutically acceptable carrier and an interferon gamma inducing factor or an immunogenic portion thereof, thereby eliciting an antibody being capable of binding the interferon gamma inducing factor in vivo.
  • immunogenic portion refers to an immunogenic proteinaceous compound, which may include, among optional additional components, a plurality of amino acid residues.
  • amino acid is understood to include the 20 naturally occurring amino acid residues; those amino acid residues often modified post-rranslationally in vivo, including for example hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acid residues including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acid residues.
  • amino acid residues according to the present invention form a peptide.
  • the latter is understood to include native peptides, including degradation products or synthetically synthesized peptides, and further to peptidomimetics, such as peptoids and semipeptoids, which are peptide analogs, which may have, for example, modifications rendering the peptides more stable or less immunogenic while contacting body fluids.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis is effected by administering, to the animal, an antibody capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis is effected administering, to the animal, an antigen including an interferon gamma inducing factor or an immunogenic portion thereof, thereby eliciting an antibody being capable of binding in vivo an interferon gamma inducing factor.
  • an antigen refers to an immunogen including at least one immunogenic epitope, which is represented in the equivalent native peptide in a continuous or discontinuous fashion.
  • the antibody is polyclonal.
  • Preparation of polyclonal antibodies is known in the art and further described in the Examples section hereinunder.
  • the antibody is monoclonal.
  • Methods of producing and identifying monoclonal antibodies are well known in the art.
  • Monoclonal antibodies may be obtained by processes comprising the generation of a plurality of monoclonal antibodies to an antigen and screening the plurality of antibodies so generated to identify a monoclonal antibody that binds and/or neutralizes the peptide of interest, interferon gamma inducing factor in the present case. Monoclonal antibodies may be generated either in vitro or in vivo.
  • an animal is immunized with an antigen thereby generating antibody producing lymphocytes in said animal, antibody producing lymphocytes are removed from the animal, said lymphocytes are fused with myeloma cells to produce a plurality of immortalized hybridoma cells each of which produces monoclonal antibodies, the plurality of monoclonal antibodies is screened to identify a monoclonal antibody that binds the peptide, and the hybridoma producing the antibody is cloned and propagated.
  • Animals are typically immunized with a mixture comprising a solution of the immunogen in a physiologically acceptable vehicle, and any suitable adjuvant, which achieves an enhanced immune response to the immunogen.
  • the primary immunization conveniently may be accomplished with a mixture of a solution of the immunogen and Freund's complete adjuvant, said mixture being prepared in the form of a water in oil emulsion.
  • the immunization may be administered to the animals intramuscularly, intradermally, subcutaneously, intraperitoneally, into the footpads, or by any appropriate route of administration.
  • the immunization schedule of the immunogen may be adapted as required, but customarily involves several subsequent or secondary immunizations using a milder adjuvant such as Freund's incomplete adjuvant.
  • Antibody titers and specificity of binding to the hapten can be determined during the immunization schedule by any convenient method including by way of example radioimmunoassay, or enzyme linked immunoassay.
  • Antibody activity assays can performed in vitro as further exemplified in the Examples section that follows. When suitable antibody titers are achieved, antibody producing lymphocytes from the immunized animals are obtained, and these are cultured, selected and cloned, as is known in the art. Typically, lymphocytes may be obtained in large numbers from the spleens of immunized animals, but they may also be retrieved from the circulation, the lymph nodes or other lymphoid organs.
  • Lymphocytes are then fused with any suitable myeloma cell line, to yield hybridomas, as is well known in the art.
  • lymphocytes may also be stimulated to grow in culture, and may be immortalized by methods known in the art including the exposure of these lymphocytes to a virus, a chemical or a nucleic acid such as an oncogene, according to established protocols.
  • the hybridomas are cultured under suitable culture conditions, for example in multiwell plates, and the culture supernatants are screened to identify cultures containing antibodies that recognize the hapten of choice.
  • Hybridomas that secrete antibodies that recognize the hapten of choice are cloned by limiting dilution and expanded, under appropriate culture conditions.
  • Monoclonal antibodies are purified and characterized in terms of immunoglobulin type binding affinity and in vivo or in vitro neutralizing activity.
  • the antibody according to the present invention is a neutralizing antibody to interferon gamma inducing factor in affecting cells to produce interferon gamma, that is to say that the antibody interferes with the functionality of interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis is effected by administering, to the animal, cells capable of producing and secreting an antibody capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • cloned cDNAs encoding antibodies or fragments thereof are used to genetically modify receptive cells, to thereby render the cells antibody producing cells.
  • Cloning of cDNAs encoding antibodies or fragments thereof may be accomplished by several approaches known in the art. In the preferred approach, mRNA from clonal hybridoma cell lines which produce antibodies is employed as starting material.
  • the cells are harvested and mRNA is extracted by standard methods known in the art.
  • the cDNA is prepared by reverse transcription of the mRNA by standard methods known in the art.
  • the cDNA for each chain of the immunoglobulin is cloned separately, and may be amplified by polymerase chain reaction using appropriate primers.
  • the cDNA is then ligated into appropriate vectors by standard methods.
  • the cDNA may be cloned into expression vectors and expressed separately in any convenient expression system, so that the properties of the expressed single chains of the antibodies may be determined. Alternatively, the individual chains may be expressed in the same cells which are then screened for the production of recombinant active antibodies.
  • the method of using the invention will be modified in accordance with the system that is selected according to the current principles that are known in the art of recombinant protein production.
  • the genetic information for the production of the antibody of interest is introduced into the cells by an appropriate vector as is known in the art or by any other acceptable means.
  • the present invention provides information that will enable the skilled artisan to prepare constructs of genetic material comprising an open reading frame that encodes at least one chain of a novel antibody capable of binding IGIF. It will be appreciated that in certain embodiments it will suffice to produce an active fragment of the catalytic activity, for instance a Fab fragment of the intact antibody or even an Fv fragment thereof.
  • the constructs of the invention may comprise the following elements: a selectable marker, an origin of replication, a transcriptional promoter, a translation start site, a signal sequence for secretion of the product.
  • DNA sequences encoding for a variety of chemokines and for the cytokine tumor necrosis factor alpha were shown to elicit protective immunity against both induced and transferred EAE, while the prior art teaches protective immunity against both induced and transferred EAE via passive vaccination (administration of antibodies).
  • DNA vaccination in various forms, some of which are further detailed hereinunder, can be used to confer protective immunity against multiple sclerosis.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis is effected by administering to the animal a therapeutic composition including a recombinant construct including an isolated nucleic acid sequence encoding a polypeptide being capable of eliciting antibodies capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • a method for treating an animal for inducing protective immunity against multiple sclerosis is effected by implementing the following method steps, in which, in a first step, cells are removed from the animal. In a second step, the cells are genetically modified in vitro with a recombinant construct including an isolated nucleic acid sequence encoding an interferon gamma inducing factor or an immunogenic portion thereof. Finally, the genetically modified cells are reinrroduced to the animal.
  • the term "genetically modified” refers to a process of inserting nucleic acids into cells.
  • the insertion may, for example, be effected by transformation, viral infection, injection, transfection, gene bombardment, electroporation or any other means effective in introducing nucleic acids into cells.
  • the nucleic acid is either integrated in all or part, to the cell's genome (DNA), or remains external to the cell's genome, thereby providing stably modified or transiently modified cells.
  • the cells according to this method of the invention may be of any kind.
  • Especially suitable cells are those readily removable, genetically modifiable, and reintroduceable cells, such as, but not limited to, cells of the various blood lineages, derived either from whole blood or from bone marrow, fibroblast cells, etc.
  • the genetically modified cells are preferably reinrroduced to the animal parenterally.
  • a pharmaceutical composition for inducing protective immunity against multiple sclerosis comprises a pharmaceutically acceptable carrier and a recombinant construct including an isolated nucleic acid sequence encoding a polypeptide being capable of eliciting antibodies capable of in vivo neutralizing an interferon gamma inducing factor in affecting cells to produce interferon gamma.
  • the nucleic acid sequence is operatively linked to one or more transcription control sequences, such as, but not limited to, RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and/or ⁇ -actin control sequences.
  • RSV control sequences such as, but not limited to, RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and/or ⁇ -actin control sequences.
  • the recombinant construct is an eukaryotic expression vector, such as, but not limited to, pcDNA3, pcDNA3.1 (+/-), pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV, pBK-CMV, pTRES, and their derivatives.
  • the therapeutic composition is administered to the animal parenterally.
  • the animal is a human being.
  • the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous physiologically balanced solution, an artificial lipid-containing substrate, a natural lipid-containing substrate, an oil, an ester, a glycol, a virus and metal particles.
  • the pharmaceutically acceptable carrier comprises a delivery vehicle that delivers the nucleic acid sequences to the animal.
  • the delivery vehicle can be effected by liposomes, micelles or cells.
  • Rats Female Lewis rats, six weeks old, were purchased from Harlan (Israel) and maintained under SPF conditions in an animal facility.
  • Peptide antigens Myelin Basic Protein ( MBP) p68-86, Y G S L P Q K S Q R S Q D E N P V (SEQ ID NO: 1), was synthesized on a MilliGen 9050 peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by high performance liquid chromatography. Structure was confirmed by amino acid analysis and mass spectroscopy. Only peptides that were greater than 95 % pure were used in our study.
  • Rats were immunized subcutaneously in the hind foot pads with 0.1 ml of MBP epitope 68-86 (p68-86) dissolved in PBS (1.5 mg/ml) and emulsified with an equal volume of CFA (incomplete Freund's adjuvant supplemented with 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra in oil (Difco laboratories, Inc., Detroit, MI). Rats were then monitored for clinical signs daily by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; 3, front and hind limb paralysis. Induction of transferred EAE: EAE was induced by immunizing
  • Lewis rats Intraperitoneally with 10 ⁇ activated spleen cells from EAE donors obtained as follows:
  • splenic cells were cultured (12 x 10 /ml) at 37 °C in humidified air containing 7.5 % CO2 for two days in stimulation medium that includes Dulbecco's modified Eagle's medium (Gibco) supplemented with 2-mercaptoefhanol (5 x 10" ⁇ M), L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 ⁇ g/ml), streptomycin (100 ⁇ g/ml), 1 % syngeneic serum and 20-30 ⁇ g/ml of the immunizing epitope. Then, cells were separated on a Ficoll gradient (Sigma), resuspended in PBS and injected to naive recipients.
  • stimulation medium includes Dulbecco's modified Eagle's medium (Gibco) supplemented with 2-mercaptoefhanol (5 x
  • Antigen-specific T cell proliferation assays Lewis rats were immunized with MBP p68-86/CFA as described above. Nine to ten days later spleen cells were suspended in stimulation medium and cultured in U- shape 96-well microculture plates (2 x 10 5 cells/well) for 72 hours, at 37 °C in humidified air containing 7.5 % CU2- Each well was pulsed with 2 ⁇ Ci of [ 3 H]-Thymidine (specific activity 10 Ci/mmol) for the final six hours. The cultures were then harvested on fiberglass filters and the proliferative response expressed as CPM ⁇ S.E. or as stimulation index (SI) (mean CPM of test cultures divided by mean CPM of control cultures).
  • SI stimulation index
  • RT-PCR analysis verified by Southern blotting, was utilized on brain samples according to the protocol described elsewhere with some modifications (39). Rats were euthanized by CO2 narcosis. Brain samples containing mainly the midbrain and brain stem were obtained after perfusion of the rat with 160-180 ml of ice-cold phosphate buffered saline (PBS) injected into the left ventricle following an incision in the right atrium. Each sample was homogenized. Total RNA was extracted using the Tri-Zol procedure (Gibco BRL) according to the manufacturer's protocol.
  • PBS ice-cold phosphate buffered saline
  • mRNA was then isolated using a mRNA isolation kit (#1741985, Boheringer Mannheim, Germany), and reverse transcribed into first strand cDNA as described in detail elsewhere (39).
  • First strand cDNA was then subjected to 35 cycles of PCR amplification using specific oligonucleotide primers to rat IGIF and IFN- ⁇ which were designed based on the published sequence of each cytokine (NCBI accession number for rat IGIF - U77777; and for rat IFN- ⁇ - M29315 ) as follows (Table 1):
  • PCR products described above was cloned into a pUC57/T vector (T- cloning Kit #K1212, MBI Fermentas, Lithuania) and transformed to E. coli according to the manufacturer's protocol. Each clone was then sequenced (Sequenase version 2, USB, Cleveland, Ohio) according to the manufacturer's protocol.
  • Rabbit anti-rat IGIF IgG Rabbit anti-rat IGIF antibodies were generated as described (40) and IgG fraction was purified using a HiTrap protein G kit (Pharmacia, Piscataway, NJ, Kit #17-040-01). Antibody titer was determined by a direct ELISA assay: ELISA plates (Nunc, Denmark) were coated with recombinant rat IGIF(50 ng/well). Rabbit anti-rat IGIF (IgG fraction) was added in serial dilutions
  • Cytokine determination Spleen cells from EAE donors were stimulated in vitro ( ⁇ cells/ml) in 24 well plates (Nunc) with 100 ⁇ M p68-86. Spleen cells from naive donors were cultured cells/ml, 24 well plates) with 2 ⁇ g/ml Con A (Sigma).
  • rat recombinant IFN- ⁇ as a standard (Cat No 328 ISA, Gibco BRL); TNF- ⁇ , commercial semi- ELISA kit for the detection of rat TNF- ⁇ , (Cat No 80-3807-00, Genzyme, Cambridge, MA); IL-4, mouse anti-rat IL-4 monoclonal antibody (24050D OX-81 , PharMingen, San Diego, CA) as a capture antibody, and rabbit anti- rat IL-4 biotin-conjugated polyclonal antibody (241 1-2D, PharMingen) as second antibody.
  • Recombinant rat IL-4 purchased from R&D (504-RL) was used as a standard.
  • IGIF mRNA is transcribed in the inflamed EAE brain: Midbrain- brain stem samples were obtained from rats with developing transferred EAE (Figure 1A) before adoptive transfer of disease (day 0), and at various time points: before the onset of disease (day 3), at the day of onset (day 5), the peak (day 7), following recovery (day 10), and 10 days after recovery (day 20). For each time point, samples from six different brains were subjected to RT-PCR analysis using specific oligonucleotide primers which constructed to IGIF and IFN- ⁇ . Each amplification was calibrated to ⁇ - actin and verified by Southern blot analysis.
  • Recombinant rat IGIF and its neutralizing antibodies affect IFN-f production by activated T cells from naive donors more significantly than by antigen specific primed T cells: PCR products encoding rat IGIF were used to generate the recombinant protein which was used to produce anti- IGIF neutralizing antibodies.
  • IGIF primarily affects IFN- ⁇ production by Thl not Th2 cells (29). It is possible that immunization with p68-86/CFA induces a substantial selection of antigen specific Th2 cells, albeit not enough to inhibit the subsequent development of a Thl mediated autoimmune disease.
  • splenic T cells were cultured with MBP p68-86 in stimulation medium that was ( Figures 4C and 4D) or was not supplemented with recombinant rat IL-4 ( Figures 4A and 4B).
  • IFN- ⁇ was produced only when the priming antigen was added to the culture ( Figures 4A, 0.3 ⁇ 0.1 ng/ml without addition of MBP 68-86 versus 13.5 ⁇ 0.7 in cells proliferating to p68-86).
  • Spleen T cells from anti-IGIF treated rats produced markedly reduced levels of IFN- ⁇ in response to antigenic stimulation in cultures that were or were not supplemented with IL-4 (Figure 4A, 4.7 ⁇ 0.4 ng/ml in spleen cells from anti-IGIF treated rat versus 9.7 ⁇ 0.8 in spleen cells from rats treated with normal rabbit IgG and 13.5 ⁇ 0.7 in PBS treated rats, with backgrounds of 0.4, 0.8 and 0.7, p ⁇ 0.001, when comparing anti-IGIF treatment to each control group).
  • TNF- ⁇ production was then evaluated in spleen cells from the above groups.
  • the above spleen cells from anti-IGIF treated rats produced markedly reduced levels of TNF- ⁇ in response to antigenic stimulation (Figure 5, 850 ⁇ 45 pg/ml in spleen cells from anti-IGIF treated rats versus 1975 ⁇ 80 in spleen cells from rats treated with normal rabbit IgG and 2100 ⁇ 110 in PBS treated rats, with backgrounds of 230, 210 and 270, respectively, p ⁇ 0.001, when comparing anti-IGIF treatment to each control group).
  • perturbation of the Thl/Th2 balance in anti-IGIF treated rats is associated with a marked reduction in TNF- ⁇ production.
  • IL-12 acts directly on CD4 + T cells to enhance priming for IFN- ⁇ production and diminishes IL-4 inhibition of such priming. Proc. Natl. Acad. Sci. USA. 90:10188-10192.
  • IL-10 acts on the antigen presenting cell to inhibit cytokine production by Thl cells. J. Immunol. 146:3444-3451.
  • B7-1 and B7-2 costimulatory molecules activate differentially the Thl/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707-18.
  • Encephalitogenic T cells in the B10.PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype.
  • Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386:619-23.
  • Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin- 12 for interferon-gamma production. European Journal of Immunology 26:1647-51.
  • Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells.
  • IFN-gamma-inducing factor is a costimulatory factor on the activation of Thl but not Th2 cells and exerts its effect independently of IL-12. J Immunol 158:1541-50.
  • CREAE Chronic relapsing experimental autoimmune encephalomyelitis

Abstract

L'invention concerne l'utilisation d'un anticorps neutralisant un facteur d'induction de l'interféron gamma dans le traitement de la sclérose en plaques.
PCT/US1999/026094 1998-11-27 1999-11-04 Vaccin base sur un facteur d'induction de l'interferon gamma et utilisation de ce vaccin destine a conferer une immunite de protection contre la sclerose en plaques WO2001045747A1 (fr)

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EP99972428A EP1140206A4 (fr) 1998-11-27 1999-11-04 Vaccin base sur un facteur d'induction de l'interferon gamma et utilisation de ce vaccin destine a conferer une immunite de protection contre la sclerose multiple
CA002355345A CA2355345A1 (fr) 1998-11-27 1999-11-04 Vaccin base sur un facteur d'induction de l'interferon gamma et utilisation de ce vaccin destine a conferer une immunite de protection contre la sclerose multiple
AU41640/00A AU4164000A (en) 1998-11-27 2000-02-28 Interferon gamma inducing factor based vaccine and use of same for protective immunity against multiple sclerosis

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US09/200,716 1998-11-27

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EP0974600A2 (fr) * 1998-06-24 2000-01-26 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Anticorps dirigé contre l'interleukine-18
WO2006099701A1 (fr) 2005-03-21 2006-09-28 Ivan Ivanov Inhibiteur de l’interferon-gamma humain endogene
WO2009124362A1 (fr) 2008-04-08 2009-10-15 Tigo Gmbh Inhibiteur de gamma interférons endogènes
EP3381935A1 (fr) 2017-03-29 2018-10-03 Tigo GmbH Inhibiteur de l' interferon-gamma humain endogene

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AU1341797A (en) * 1995-12-29 1997-07-28 Incyte Pharmaceuticals, Inc. Nucleic acids encoding interferon gamma inducing factor-2
CA2282845A1 (fr) * 1997-03-18 1998-09-24 Basf Aktiengesellschaft Procedes et compositions permettant de moduler la sensibilite aux corticosteroides

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DATABASE BIOSIS [online] REVOLTELLA. R. P.: "Natural and therapeutically-induced antibodies to cytokines", XP002954931, accession no. STN Database accession no. PREV199800272214 *
KIM. J. J. ET. AL.: "Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens", EUR. J. IMMUNOL., vol. 28, no. 3, March 1998 (1998-03-01), pages 1089 - 1103, XP002118496 *
OKAMURA, H. ET. AL.: "Cloning of a new cyotkine that induces INF-gamma production by T cells", NATURE, vol. 378, 2 November 1995 (1995-11-02), pages 88 - 91, XP002024313 *
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TANIGUCHI, M. ET. AL.: "Characterization of anti-human interleuken-18/interferon-gamma-inducing factor (IGIF) monoclonal antibodies and their application in the measurement of human IL-18 by ELISA", J. IMMUNOL. METH., vol. 206, no. 1-2, 1997, pages 107 - 113, XP002929440 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0974600A2 (fr) * 1998-06-24 2000-01-26 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Anticorps dirigé contre l'interleukine-18
EP0974600A3 (fr) * 1998-06-24 2001-09-19 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Anticorps dirigé contre l'interleukine-18
EP1705191A3 (fr) * 1998-06-24 2008-08-27 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Anticorps dirigé contre l'interleukine-18
WO2006099701A1 (fr) 2005-03-21 2006-09-28 Ivan Ivanov Inhibiteur de l’interferon-gamma humain endogene
WO2009124362A1 (fr) 2008-04-08 2009-10-15 Tigo Gmbh Inhibiteur de gamma interférons endogènes
EP3381935A1 (fr) 2017-03-29 2018-10-03 Tigo GmbH Inhibiteur de l' interferon-gamma humain endogene

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