WO1999025737A1 - Compositions et procedes de traitement de maladies autoimmunes a l'aide d'un anticorps monoclonal se liant a la chaine interleukine-12 beta2 - Google Patents

Compositions et procedes de traitement de maladies autoimmunes a l'aide d'un anticorps monoclonal se liant a la chaine interleukine-12 beta2 Download PDF

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WO1999025737A1
WO1999025737A1 PCT/NL1998/000663 NL9800663W WO9925737A1 WO 1999025737 A1 WO1999025737 A1 WO 1999025737A1 NL 9800663 W NL9800663 W NL 9800663W WO 9925737 A1 WO9925737 A1 WO 9925737A1
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chain
cells
il12r
monoclonal antibody
human
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PCT/NL1998/000663
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English (en)
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Mark De Boer
Marcel Theodorus Den Hartog
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Tanox Pharma B.V.
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates to methods of treating diseases of in which the immune system is involved.
  • this invention relates to methods of treating autoimmune diseases.
  • One of the most interesting characteristics of the immune system is its unlimited specificity. When threatened by potentially dangerous foreign substances (antigens), including pathogens, the immune system mounts a tailor-made response. This tailor- made response is provided by the immune systems antigen specific T and B lymphocytes. The virtually unlimited repertoire provided by these immune cell calls for a tight regulatory system preventing the recognition of our own (self) antigens. For years it was thought that the immune system was able to discriminate between self and non- self. However, with the growing knowledge of immunology, this theory has become more and more unsatisfactory. The sclf/non-self paradigm does not explain why perfectly healthy individuals can have circulating autoreactive T and B cells without any symptoms of autoimmune diseases.
  • Thl and Th2 cells the role of Thl cells in autoimmune diseases
  • Th cells Helper T cells regulate immune responses via cytokines that they produce upon recognition of specific antigen presented by antigen presenting cells.
  • Individual Th cells clones
  • Th cells In response to most antigens, Th cells produce many cytokines simultaneously (type 0 cytokine profile).
  • the Th cell response is biased to low levels of interferon-gamma (IFN- ⁇ ) and high levels of interleukin-4 (IL-4) and interleukin-5 (IL-5) (type 2 cytokine profile, Th2).
  • IFN- ⁇ interferon-gamma
  • IL-4 interleukin-4
  • IL-5 interleukin-5
  • Thl type 1 cytokine profile
  • type 1 and 2 profiles result from modulation of the local cytokine microenvironment (Trinchieri, Immunology Today 13: 379 (1993); Snijdewint et al., J. Immunology 150: 5321 (1993)).
  • Various factors may directly act on the T cells, but they may also act indirectly by affecting antigen-presenting cells, which in turn secrete mediators that skew to Thl or Th2 profiles.
  • Antigen presenting cell-derived factors that skew T cell cytokine production towards Thl and Th2 profiles include interleukin- 12 (IL-12) and prostaglandin E2 (PGE-2).
  • IL-12 interleukin- 12
  • PGE-2 prostaglandin E2
  • EAE Experimental autoimmune encephalomyelitis
  • non-obese diabetic (NOD) mouse model transfer of T cells specific for a pancreatic autoantigen that had been differentiated in the presence of type 1 cytokines in vitro, caused disease, while the same T cells that had been differentiated in the presence of type 2 cytokines did not (Katz et al, Science 268: 1185 (1995)).
  • Thl- type of response a strong inverse relationship between delayed hypersensitivity responses to M. tuberculosis and the presence of asthma, serum IgE levels and Th2-cytokine profiles (Shirakawa et al, Science 275: 77 (1997)).
  • IL-12 a major regulator of type 1 T-cell cytokine responses
  • IL-12 is a heterodimeric glycoprotein composed of two covalently linked peptide chains, called p40 and p35 (Trinchieri Ann. Rev. Immunol. 13: 251 (1995)).
  • IL- 12 is mainly produced by activated monocytes and dendritic cells.
  • IL12 can be produced by monocytes after stimulation with bacterial products such as LPS or after stimulation with activated T cells.
  • bacterial products such as LPS
  • T cells for dendritic cells the ligation of CD40 with CD40L on the surface of activated T cells is the strongest trigger for IL-12 production.
  • the most pronounced effect of IL-12 is the stimulation of IFN- ⁇ by human NK cells and T cells.
  • IL-12 exerts its effects through binding to a high affinity receptor.
  • the functional, high- affinity IL-12 receptor consist of a ⁇ l and a ⁇ 2 chain, of which only the latter is involved in signal transduction.
  • the nucleotide and amino acid sequences of the IL-12 receptor ⁇ l chain are disclosed in EP-A-638644.
  • the sequences of the IL-12 receptor ⁇ 2 chain are disclosed in EP-A-759466.
  • autoimmune diseases are primarily directed at the treatment of symptoms. Most, if not all of these drugs are ineffective at stopping the disease process, need to be administered chronically and are often associated with significant side effects. This makes the presently used drugs highly unfavourable.
  • Optimal drugs for the treatment of autoimmune diseases will be able to attenuate the autoimmune process by re-establishing the immune system's self-regulatory mechanisms that have failed and resulted in the autoimmune attack. Treatment during the early phase of the autoimmune process with such drugs have the potential to arrest the disease process. It has been demonstrated that T cells play a central role in the auto-destructive process in autoimmune diseases such as rheumatoid arthritis (Sigall et al., Clin. Exp.
  • Treatments that selectively suppress the activity of such autoreactive T cells can therefore be preferred.
  • Such treatment could consist of the administration of an autoantigen or peptides derived thereof.
  • This type of treatment has been very successful in the suppression of disease symptoms in various experimental autoimmune disease models in laboratory animals and it has been suggested that successful therapy is associated with the up-regulation of Th2 responses and a down- regulation of Thl responses. It is therefore proposed by the present inventors to combine antigen-specific therapy targeting autoreactive T cells with the modulation of the cytokine microenvironment.
  • the current invention is based on the finding that Th2 cell development from naive Th cells is associated with suppression of IL-12R ⁇ 2 chain expression leading to loss of IL-12 responsiveness and, consequently, the inability to promote IFN- ⁇ production. Furthermore, the present invention is based on the finding that allergen- specific Th2 clones generated from atopic patients do not produce IFN- ⁇ . Even upon exposure to IL-12, IFN- ⁇ protein and mRNA expression cannot be induced in such clones. Further analyses revealed the complete lack of signalling via the IL-12R in these
  • Th2 clones as indicated by their inability to phosphorylate STAT4 despite the abundant presence of this selectively IL-12-induced transcription factor. FACS analysis showed normal expression of the IL-12R ⁇ l chain. These findings strongly suggest the absence of functional ⁇ 2 chains on human Th2 cells, similar to mouse Th2 cells. RNAse- protection assays with a human IL-12R ⁇ 2 chain-specific DNA probe indeed indicated the absence of IL-12R ⁇ 2 mRNA in activated Th2 clones.
  • the inventors propose to specifically neutralize the activity of the IL-12R ⁇ 2 chain.
  • Specific neutralization of the IL-12R ⁇ 2 chain can be accomplished by a specific monoclonal antibody that binds to the IL-12R ⁇ 2 chain, but does not stimulate the phosphorylation of STAT4.
  • Such an antagonistic monoclonal antibody to the IL-12R ⁇ 2 chain can be used to prevent or treat diseases in which activated type 1 T cells are involved.
  • Such an antagonistic monoclonal antibody can be used to enhance the effect of antigen-specific therapy of autoimmune diseases targeting Thl -like autoreactive T cells.
  • a primary object of this invention to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody capable of binding to the human IL-12R ⁇ 2 chain, where said binding prevents interleukin- 12 from mediating a signal through said receptor resulting in the phosphorylation of STAT4, in a pharmaceutically acceptable excipient.
  • said administration is combined with the administration of specific autoantigens, modified autoantigens or peptide fragments thereof, or, alternatively, with the administration of other therapeutic monoclonal antibodies, such as monoclonal antibodies to co-stimulatory receptors on T cells or antigen presenting cells, including CD4, CD40, CD40L, CD80 and CD86.
  • the invention pertains to antibodies, preferably monoclonal antibodies, capable of binding to the ⁇ 2 chain of the IL12 receptor.
  • the binding should be such that phosphorylation of a Signal Transducer and Activator of Transcription (STAT), specifically STAT4, is not activated.
  • STAT Signal Transducer and Activator of Transcription
  • STAT4 is the only one that is tyrosine phosphorylated after stimulation of T cells with interleukin 12.
  • the molecular cloning of STAT4 based on its homology with STAT1 was described by Yamamoto et al, Molec. Cell Biol. 14: 4342 (1994).
  • Antibodies that result in binding which prevents activation of STAT phosphorylation can be selected in a manner known per se, as exemplified in Example 5.
  • the invention further pertains to antibodies, preferably monoclonal antibodies, capable of binding to the ⁇ 2 chain of the IL12 receptor, especially to an epitope of
  • Antibodies the binding of which prevents (hetero)dimerization of the ⁇ l chain to the ⁇ 2 chain, can be selected e.g. by immunoprecipitation of the antibody-IL12R immuno- complex and comparison of the molecular weight of the immunocomplexes; antibodies resulting in immunocomplexes having the lower molecular weight complex (i.e. the complex which does not contain the heterodimer) are the ones sought according to the invention.
  • antibody refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab, F(ab')2, Fv, and other fragments which retain the antigen binding function of the parent antibody.
  • the term "monoclonal antibody” refers to an antibody composition having a homogeneous antibody population.
  • the term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.
  • the term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab')2, Fv, and others which retain the antigen binding function of the antibody.
  • Monoclonal antibodies of any mammalian species can be used in this invention.
  • chimeric antibodies means that the constant regions of an immunoglobulin are derived from human immunoglobulin sequences.
  • humanized antibodies means that at least a portion of the framework regions of an immunoglobulin are derived from human immunoglobulin sequences.
  • single chain antibodies or ScFv refers to antibodies prepared by determining the binding domains (both heavy and light chains) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function. This forms, in essence, a radically abbreviated antibody, having only that part of the variable domain necessary for binding to the antigen. Determination and construction of single chain antibodies are described in U.S. Patent 4,946,778, incorporated herein by reference.
  • CD80 As used herein, the terms "CD80”, “CD86”, “CD40” and “CD40L” refer to human surface molecules as extensively reviewed in Van Gool et al., Immunol. Rev. 153: 46 (1996), incorporated herein by reference.
  • CD80, CD86, CD40 and CD40L antigen may be prepared by any technique known in the art.
  • Antibodies to human CD80, CD86, CD40 and CD40L are known in the art.
  • the present invention also contemplates a new use for such antibodies as detailed above.
  • autoantigen refers to a human protein that is recognized by autologous T cells, resulting in self-tissue destruction in autoimmune disease patients.
  • autoantigens that are recognized by autologous T cells are myelin basic protein in multiple sclerosis; collagen type II and human cartilage glycoprotein 39 (WO 96/13517) in rheumatoid arthritis; insulin and glutamic acid decarboxylase (diabetes); and alpha-fodrin (Sjogren's syndrome).
  • autoantigens may be administered in there native form, modified by selected amino acid substitutions (WO 96/16085), or in peptide fragments with (Kumar et al., Proc. Natl.
  • interleukin- 12 receptor refers to the human surface molecule capable of binding human interleukin-12 as reviewed above.
  • human interleukin-10 antigen may be prepared by any technique known in the art.
  • the term "antagonistic” refers to the capacity of a soluble ligand to bind to a cell surface receptor, where said binding prevents intracellular signal transduction leading to the activation of said cell surface receptor by the natural ligand for said.
  • compositions of this invention are administered at a concentration that is therapeutically effective to modulate the hosts immune response.
  • the pharmaceutical composition may be formulated using a variety of acceptable excipients known in the art.
  • the pharmaceutical composition is administered by injection, either subcutaneous, intramuscular, intravenous or intra- peritoneal. Methods to accomplish this administration are known to those of ordinary skill in the art. It may also be possible to obtain compositions which may be orally administered, or which may be capable of transmission across mucous membranes. Before administration to patients, formulants may be added to the pharmaceutical composition.
  • IL-12 modulates the production ofIL-4, IL-5 and IFN-y by stimulated CD4+ T cells
  • Naive CD45RA CD4 + T cells were isolated from the heavy fraction of PBMC in a two step protocol. First CD4 + cells were isolated by incubation with CD4 specific
  • Iscove's modified Dulbecco's medium (IMDM; Life Technologies, Paisley, UK), supplemented with 5 % pooled, C-inactivated normal human serum (CLB).
  • C-inactivated normal human serum C-inactivated normal human serum
  • T cells (2 x 10 4 /well) were stimulated in the absence of accessory cells (AC) with a combination of immobilized anti-CD3 mAb (1 / Kg/ml), soluble anti-CD28 mAb (1 ⁇ g/ml) and with IL-2 (5 U/ml).
  • IL-12 200
  • the human IL12 receptor consist out of two chains, called ⁇ l and ⁇ 2. These form a heterodimer in order to act as a functional molecule on the cell-membrane.
  • the ⁇ 2 chain is responsible for the transduction of signals into the IL12R expressing cells.
  • the cDNA encoding the (32 chain of the human IL12R was generated by PCR from RNA isolated from PBMC's. Briefly, the PBMC's were separated from red blood cells by gradient centrifuge using Ficoll, after which the lymphocyte fraction was stimulated for 2 - 20 h with PMA (lng ml) and ionomycin (l ⁇ g/ml) in IMDM/FCS at 37°C with 5% CO2.
  • the cells were washed twice with phosphate buffered saline (PBS pH 7.4) and lysed in 5M guanidinium thiocyanate in the presence of 0.7 M 2-mercaptoethanol.
  • the RNA was bound on a Qiagen spin column, washed according to manufactures protocol and eluted in DEPC treated water. RNA was stored in -70°C.
  • First strand cDNA was synthesized by incubation at 37°C for 1 hour of 1-5 ⁇ g total RNA in a 50 ⁇ l mix, consisting of lx synthesis buffer (USB), 0.5 mM dNTP, 5 ⁇ M random hexamers and 5 U M-MLV reverse transciptase (USB). This was followed by incubation at 70°C for 10 min. After cooling on ice from this mixture 2.5 ⁇ l was used as template in a PCR reaction using primers specific for respectively the IL12R ⁇ 2 chain. These primers (SEQ ID NO 1 and 2)were based on the published cDNA coding sequences for IL12R ⁇ 2 (Presky D.H. et al., Proc. Natl. Acad. Sci. USA 93: 14002 (1996)). SEQ ID NO 1 Sense primer: 5' - gcgcgaattc ttgttgatgg cacatacttt tag - 3'
  • Antisense primer 5' - gcgccccggg tcagagcatg agggagtcac ace - 3'
  • Both the sense and the anti-sense primers start with GCGC followed by a restriction site for cloning purpose.
  • the sense primers carriers the ATG start codon, while the anti-sense primers contains a stop codon.
  • the amplified cDNA will encode for the full-length IL12R ⁇ 2 chain including the naturally occurring signal peptide.
  • To amplify the IL12R ⁇ 2 chain a standard PCR was done.
  • the PCR mixture of 100 ⁇ l contained lx PCR buffer, 2.5U Taq polymerase, 0.25 mM dNTPs, 25 pmole of each primer and 2.5 ⁇ l cDNA template.
  • the mixture was run in Perkin Elmer thermocycler for 20 - 40 cycles of 1 min 95°C, 1 min 55°C, and 2 min 72° C followed by 1 step for 7 min at 72°C as extension of the PCR product.
  • the obtained PCR product was gel purified and cloned in pCR Script using the Stratagene cloning kit. Briefly, the PCR product was incubated with plasmid together with T4 ligase and Srfl for lh at KT, after which the entire sample was transformed in XllBlue E.coli cells. The cells were plated on LB plates containing 100 ⁇ g ampicillin/ml, 20 ⁇ g IPTG/ ml and 20 ⁇ g Xgal/ml. After incubation over night at 37°C putative white clones were analysed for having an insert.
  • Clones containing inserts were analysed by cycle sequencing using M13 and M13 reverse primers. Several clones were identified containing a DNA sequence encoding for the IL12R ⁇ 2 chain. By further sequencing a correct cDNA clone encoding full-length II12R ⁇ 2 chain was found without PCR induced mutations.
  • To express the IL12R 132 chain on the cell surface of Sf9 insect cells the obtained cDNA was re-cloned in the baculovirus transfer vector pVL1392.
  • the pVL1392 vector and the IL12R ⁇ 2 chain cDNA cloned in pCR Script were digested with EcoRI and Smal.
  • the IL12R ⁇ 2 chain insert and the linear pVL1392 were gel purified, after which the insert was ligated in pVL1392.
  • the ligation mixture contained 100 ng plasmid,
  • ligation mixture 100 ng insert, lx ligase buffer and T4 DNA ligase (Promega).
  • the ligation mixture was transformed to DH5a, and plated on LB plates containing 100 ⁇ g ampicillin/ml. After incubation over night at 37°C the clones were screened for having the correct plasmid.
  • a pVL1392 plasmid containing the IL12R ⁇ 2 chain insert was selected and large scale plasmid preparation was done using the midi-prep system from Qiagen.
  • the sequence was recombined into the Autographa californica baculovirus (AcNPV). Briefly, using the BaculoGold system from Pharmingen the recombinant plasmid was cotransfected at a 4 to 1 ratio with wild-type baculoviral DNA containing a lethal deletion into Sf9 (Spodoptera frugiperda) insect cells. Recombinant baculovirus was plaque purified, followed by several rounds of amplification to obtain a high titer recombinant virus stock.
  • AcNPV Autographa californica baculovirus
  • Sf9 cells were infected with the IL12R ⁇ 2 chain carrying virus.
  • Sf9 cells were infected with recombinant virus at a MOI of 10.
  • the cells were harvested after 48-72 hours of culture in TC100 FCS at 28°C under standard conditions.
  • the cells were washed with PBS twice followed by injection intraperitoneally in female BALB/c mice (5 x 10 6 Sf9 IL12R ⁇ 2 + cells/ mouse).
  • TC100 FCS TC100 FCS at 28°C under standard conditions.
  • the cells were washed with PBS twice followed by injection intraperitoneally in female BALB/c mice (5 x 10 6 Sf9 IL12R ⁇ 2 + cells/ mouse).
  • mice received a new booster injection with Sf9 IL12R ⁇ 2 + cells.
  • spleen cells from one mouse were isolated and used for cell fusion at a ratio of 10 : 1 with Sp2/0 murine myeloma cells using 38% polyethylene glycol.
  • the fused cells were resuspended in IMDM/FCS supplemented with HAT, followed by plating on ten 96 wells plates. After 10 - 14 days the fusion was screened for grow and antibody production. Therefore the supernatants of each row (1- 12) and separately the supernatants of each column (A-H) of a 96 well plate were pooled, resulting in 200 samples.
  • T cells 0.1 - 0.2 x 10E6/sample
  • FACS buffer PBS pH 7.4 1% BSA 0.1% NaN 3
  • FITC fluorescein isothiocyanate
  • Lymphocytes were fused with Sp2/0 cells, plated on ten 96 wells plates and screened or grow. After FACS analysis 14 IgM and 1 IgG producing hybridoma clones were selected.
  • Hybridoma clone 3H4 was subcloned three times by limiting dilution in IMDM FCS + IL6 (lOOU/ml). The selected positive hybridoma 3H4 was scaled up for production of the antibody. The obtained monoclonal antibody was initially characterize by doing FACS analysis on various type of cells (B- and T cells; dendritic cells; monocytes).
  • the purified monoclonal antibody was titrated in FACS analysis on a non stimulated T-cell line Hut78.8. This cell line was selected based on the constitutively presence of mRNA coding for the IL12R ⁇ 2 chain.
  • the Hut78.8 cells (100,000 cells) were incubated for 20 min. at 4°C with Mab 3H4 ranging from 400 ng/100,000 cells to zero in 100 ⁇ l FACS buffer (PBS pH 7.4 1% BSA 0.1% NaN 3 ).
  • CD45RA + CD4 + T-cells were isolated as described in example 1, followed by an incubation for 3 days with the stimuli as indicated in the legend of figure 4. Subsequently the cells were washed and analyzed by FACS using the 3H4 Mab as described above. In the same experiment freshly isolated CD45RA + CD4 + T-cells were used as control cells. As clearly is shown in figure 4, stimulation of the T cells upregulate the IL12R ⁇ 2 chain which can be recognized by Mab 3H4.
  • IPB immunoprecipitation buffer
  • Lysates are precleared by three incubations with 50 ⁇ l of a 10% (v/v) suspension of non-immune mouse Ig-coated protein A-CL4B Sepharose beads (Pharmacia, Uppsala, SE), and once with uncoated protein A-Sepharose beads. Precleared lysates are then incubated with anti-STAT4 (C20, Santa Cruz) for 30 min followed by protein A- Sepharose beads for 2 h.
  • anti-STAT4 C20, Santa Cruz
  • the STAT4 immunoprecipitates are resuspended in sample buffer, separated by SDS-PAGE under reducing condition, and transferred to Hybond C nitrocellulose membrane (Amersham Co., Aylesbury, UK), employing a semidry electroblotting chamber (Multiphore II, Pharmacia, SE). Blots are saturated with blocking buffer [50 mM Tris, 150 M NaCl (pH 7.5) containing 0.2 % Tween and 1% BSA] and incubated with horseradish peroxidase-labelled anti- phosphotyrosine (RC20; Signal Transduction Laboratories, Lexington, KY) for 1 h.
  • blocking buffer [50 mM Tris, 150 M NaCl (pH 7.5) containing 0.2 % Tween and 1% BSA] and incubated with horseradish peroxidase-labelled anti- phosphotyrosine (RC20; Signal Transduction Laboratories, Lexington, KY) for 1 h.
  • Phosphorylated tyrosine residues are visualized using enhanced chemiluminescence (ECL, Amersham).
  • ECL enhanced chemiluminescence
  • deprobing of the blots is performed according to the manufacturer's instructions. Blots are then incubated with anti-STAT4 (C20, Santa Cruz Biotechnology) for 1 h, washed, incubated for 1 h with horseradish peroxidase-labelled horse anti-rabbit Ig (CLB), and visualized as described above. It is demonstrated that the specific anti-IL12R B2 monoclonal antibodies can prevent the phosphorylation of STAT4 and thus are potent inhibitors of the signal transduction cascade in lymphocytes leading to a strong type-1 pro-inflammatory cytokine production.
  • Figure 1 shows the effect of addition of IL-12 during the priming of naive T cells.
  • IL- 12 stimulates the production of the type 1 T-cell cytokine IFN- ⁇ , but inhibits the production of the type 2 T-cell cytokines IL-4 and IL-5.
  • Figure 2 shows an FACS analysis of Mab 3H4 on Hut78.8 cells. 100,000 cells were incubated with Mab 3H4 in titration followed by detection with an anti-mouse FITC- labelled antibody.
  • Figure 3 shows an FACS analysis of Mab 3H4 on Hut78.8, JY and Jurkat cells. 100,000 cells were incubated with Mab 3H4 (500 ng) followed by detection with an anti-mouse
  • the overlay is the control incubation in which only the secondary antibody was used in the FACS analysis.
  • Figure 4 shows an FACS analysis of Mab 3H4 on naive T cells (control), naive T cells after stimulation for 3 days with PMA (1 ng/ml) and ionomycin (1 ⁇ g/ml) and on naive T cells after stimulation for 3 days with PHA (1 ng ml), IL12 and anti-IL4.
  • 100,000 T cells were incubated with Mab 3H4 (500 ng) followed by detection with an anti-mouse FITC-labelled antibody.
  • the overlay is the control incubation in which only the secondary antibody was used in the FACS analysis.

Abstract

L'invention porte sur des anticorps monoclonaux pouvant se lier à la chaîne IL12R β2 exprimée sur la surface des lymphocytes T humains. Cette liaison empêche la phosphorylation de STAT4 induite par la chaîne IL12R β2, et/ou empêche la chaîne IL12R β2 de former un complexe avec d'autres protéines associées à une membrane, et/ou empêche la chaîne IL12R β2 de se dimériser par rapport à la chaîne IL12R β1. Les anticorps peuvent être combinés avec des auto-antigènes ou avec des anticorps à des récepteurs costimulateurs sur des lymphocytes T ou des cellules présentant des antigènes. Ces anticorps et leurs combinaisons sont également utilisés comme compositions pharmaceutiques dans le traitement des maladies autoimmunes.
PCT/NL1998/000663 1997-11-19 1998-11-19 Compositions et procedes de traitement de maladies autoimmunes a l'aide d'un anticorps monoclonal se liant a la chaine interleukine-12 beta2 WO1999025737A1 (fr)

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Cited By (1)

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CN104655850A (zh) * 2013-11-18 2015-05-27 李捷 一种细胞早期不可逆损伤程度的检测方法及检测试剂盒

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