WO2012163848A1 - Procédés et compositions pharmaceutiques destinés au traitement de la maladie de crohn - Google Patents

Procédés et compositions pharmaceutiques destinés au traitement de la maladie de crohn Download PDF

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WO2012163848A1
WO2012163848A1 PCT/EP2012/059880 EP2012059880W WO2012163848A1 WO 2012163848 A1 WO2012163848 A1 WO 2012163848A1 EP 2012059880 W EP2012059880 W EP 2012059880W WO 2012163848 A1 WO2012163848 A1 WO 2012163848A1
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cells
mice
ccl3
antibody
ccr1
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PCT/EP2012/059880
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Nadine CERF-BENSUSSAN
Julie SCHULTHESS
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to methods and pharmaceutical compositions for the treatment of Crohn's disease.
  • CD Crohn's disease
  • IBD Inflammatory Bowel Disease
  • CD Crohn's disease
  • the characteristic inflammatory presentation of Crohn's disease is of abdominal pain, diarrhea, fever and weight loss which may be complicated by intestinal fistulization, obstruction, or both. Fistula formation may occur to the adjacent bowel, the skin, the urinary bladder, or other locations. Obstruction, if present, is initially intermittent due to bowel wall edema and spasm; further progression may lead to chronic scarring and stricture formation. Perianal disease is common and may manifest as anal fissure, perianal fistula, or abscess. In the absence of a key diagnostic test, the diagnosis of Crohn's disease is based on endoscopic, radiographic and pathological findings documenting focal, asymmetric transmural or granulomatous features. Laboratory abnormalities include non-specific markers of inflammation such as elevated sedimentation rate and C-reactive protein (CRP). In more severe cases, finding may include hypoalbuminemia, anemia, and leukkocytosis.
  • CRP C-reactive protein
  • CD Crohn's disease
  • Th-1 T-helper 1
  • Thl-cell activity leading to the production of a wide range of proinflammatory cytokines including interleukin IL-1, IL-2 and tumor necrosis factor (TNF)-alpha and an imbalance between proinflammatory and anti- inflammatory reactivity, is a critical component of CD.
  • Interleukin 15 is a pleio tropic cytokine with a large range of functions at the interface between innate and adaptive immunity.
  • An essential role in the differentiation, survival and/or activation of NK, NK/T cells, TCRy5 intraepithelial lymphocytes (IEL) and CD8 + memory T cells has been firmly established in mice lacking IL-15 or IL-15Ra or, conversely, over-expressing IL-15.
  • IEL intraepithelial lymphocytes
  • CD8 + memory T cells has been firmly established in mice lacking IL-15 or IL-15Ra or, conversely, over-expressing IL-15.
  • IL-15 is also thought to participate in the pathogenesis of a spectrum of inflammatory or autoimmune diseases and different mechanisms have been propounded.
  • IL-15 In rheumatoid arthritis, increased IL-15 concentrations were found in the synovium, and IL-15 was suggested to exert direct chemoattractant activity toward synovial T cells, to stimulate their proliferation and their production of TNF-a. In addition IL-15-activated T cells were found to stimulate TNF-a production by peripheral blood monocytes and synovial macrophages via a contact-dependent mechanism. A comparable mechanism was suggested in Crohn's disease (CD) where lamina limba (LP) T cells activated by IL-15 could stimulate macrophage production of TNF-a and IL-12 via a CD40-CD40L dependent mechanism.
  • CD Crohn's disease
  • LP lamina limbal
  • IL-15 activated IEL from celiac disease patients could destroy epithelial cells via an NK-like mechanism involving the NKG2D receptor and a comparable mechanism was evoked to explain epithelial destruction in a mouse model of acute intestinal inflammation induced by intra-peritoneal injection of poly-IC.
  • IL-15 might impair immunoregulatory mechanisms and thereby bolster CD8 + and CD4 + effector responses.
  • IL-15 controlled the recruitment of CCR1+ inflammatory monocytes (IM) by sustaining the expansion and activation of a subset of gut NK cells producing the CCL3 chemokine.
  • the present invention relates to methods and pharmaceutical compositions for the treatment of Crohn's disease. More particularly, the present invention relates to a C-C Chemokine Receptor 1 (CCR1) antagonist for use in the treatment of Crohn's disease.
  • CCR1 C-C Chemokine Receptor 1
  • the inventors have used the mouse model of acute ileitis induced by oral infection of C57B1/6 mice with Toxoplasma gondii.
  • the intestinal immune reaction is necessary to eliminate the parasite but is excessive and results in lethal jejuno-ileitis.
  • Intestinal inflammation depends on the activation of CD4 + Thl intestinal LP cells specific of the triggering antigen but several complementary mechanisms are believed to participate in both pathogen exclusion and inflammation, notably the activation of CD8 TCRaP + IEL and that of inflammatory monocytes.
  • IL-15 "7" mice has suggested that IL-15 may be necessary for both protection and inflammation during T. gondii infection.
  • IL-15 although not required to control parasite replication, is necessary for the development of full blown inflammation and tissue destruction.
  • IL-15 pro- inflammatory effect did not depend on IEL activation.
  • IL-15 was dispensable for the development of the CD4 Thl response.
  • IL-15 controlled the recruitment of CCR1 + inflammatory monocytes (IM) by sustaining the expansion and activation of a subset of gut NK cells producing the CCL3 chemokine.
  • CCRl C-C Chemokine Receptor 1
  • CCRl CC chemokine receptor 1
  • CCRl is a member of the seven transmembranespanning GPCR family. In humans, it is found in monocytes/macrophages, osteoclasts and T cells, and its expression on these cells is upregulated in inflammatory conditions. Eleven different chemokines have been shown to bind and activate CCRl in vitro, including CCL3, RANTES and leukotactin-1. Furthermore, CCL3 has been shown to recruit monocytes, neutrophils, eosinophils and lymphocytes to the site of intradermal challenge in human subjects. Like many other chemokine receptors, CCRl is involved in directing leukocyte migration and activation.
  • CCRl has been considered a potential target for the development of therapies for inflammatory and autoimmune disorders (Carson KG, Jaffee BD, Harriman GCB. CCRl antagonists. Annu Rep Med Chem 2004;39: 149-58; Pease JE, Horuk R. CCRl antagonists in clinical development. Expert Opin Investig Drugs 2005;14:785-96), but the implication of CCRl in Cohn's disease, even if suggested, has not yet been demonstrated.
  • CCRl antagonist refers to any compound natural or not that is able to inhibit the activation of CCRl by CCL3 also known as MIP-1 alpha. Typically said antagonist can inhibit binding of CCL3 to CCRl . Accordingly, processes or cellular responses mediated by the binding of CCL3 to CCRl can be inhibited (reduced or prevented, in whole or in part) recruitment of inflammatory monocytes. Antagonistic activities of CCRl may be determined according to any method well known in the art.
  • the assays may consist in a ligand-binding assay (inhibition of 125I-MIP-la binding to membranes prepared from transfected CHO cells) and in a functional assay by measuring inhibition of MIP-1 a induced Ca2+ flux in transfected CHO cells loaded with fluo-4 AM.
  • a commercially- available cell line that stably co-expresses CCRl and Gal 6 may be used. Said cell may be then loaded with calcium dye, pre-incubated with a test compounds and challenged with CCL3. Intracellular calcium flux may then measured and compared to control (CCL3, but no compound).
  • CCRl antagonists according to the invention may for example be selected from the group consisting of small organic molecules, antibodies, aptamers and polypeptides.
  • the CCRl antagonist according to the invention is small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • CCRl antagonists that are currently in clinical trials are BX471 ((2R)- 1- [ [2- [(aminocarbonyl)amino] -4-chlorophenoxy] acetyl] -4- [(4-fluorophenyl)methyl] - 2- methylpiperazine monohydrochloride) AZD-4818, CP-481715, MLN-3897, or CCX634 (Expert Opin. Ther. Patents (2010) 20(11): 1609-1618).
  • a CCR1 antagonist according to the invention is, for example, a compound disclosed in WO2001/062728 or WO2001/098273.
  • a CCR1 antagonist is, for example, N- ⁇ 2- [((21S)-3- ⁇ [l-(4-chlorobenzyl)piperidin-4- yl]amino ⁇ -2-hydroxy-2-methylpropyl)oxy]-4- hydroxyphenyl ⁇ acetamide (see WO 2003/051839), or, 2- ⁇ 2-Chloro-5- ⁇ [(2S)-3-(5-chloro- 1 [Eta],3H-spiro [1 -benzofuran-2,4'- piperidin]-r-yl)-2-hydroxypropyl]oxy ⁇ -4- [(methylamino)carbonyl]phenoxy ⁇ -2- methylpropanoic acid (see PCT publication no.
  • the Patent cooperation treaty (PCT) application WO 2009/137338 also describes pyrazole compounds as CCR1 antagonists.
  • the PCT applications WO2009/134666 and WO 2010/036632 describe indazole and azaindazole compounds, respectively.
  • the Patent cooperation treaty (PCT) application WO2005080336 describes trans-dimethylpiperazine derivatives.
  • the PCT applications WO2008103126 and WO2009011653 describe spirocyclic piperidine chemokine CCR1 antagonists.
  • the CCR1 antagonist according to the invention is an antibody or a portion thereof. Said antibody may be directed against CCR1 or CCL3.
  • the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody.
  • the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.
  • antibody includes both naturally occurring and non-naturally occurring antibodies.
  • antibody includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
  • Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of CCRl or CCL3. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.
  • Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the recombinant CCRl proteins may be provided by surface expression on recombinant cell lines.
  • CCRl may be provided in the form of human cells that express CCRl .
  • Recombinant forms of CCRl may be provided using any previously described method.
  • lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma.
  • cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
  • cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
  • Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
  • the Fc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDRS complementarity determining regions
  • compositions and methods that include humanized forms of antibodies.
  • humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules.
  • Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567,5,225,539,5,585,089, 5,693,761 , 5,693,762 and 5,859,205, which are hereby incorporated by reference.
  • the above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies.
  • the first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies.
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
  • One of ordinary skill in the art will be familiar with other methods for antibody humanization.
  • humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
  • Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
  • a "humanized" antibody retains a similar antigenic specificity as the original antibody.
  • the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591 ,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the antibody according to the invention is a single domain antibody.
  • the term "single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the CCR1 antagonist according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA.
  • each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).
  • a further object of the invention relates to an inhibitor of CCR1 or CCL3 gene expression for use in the treatment of intestinal inflammation in particular for use in the treatment of Crohn's disease.
  • Inhibitors of expression for use in the present invention may be based on anti-sense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of CCR1 (or CCL3) mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of CCR1 (or CCL3), and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding CCR1 (or CCL3) can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs can also function as inhibitors of expression for use in the present invention.
  • CCR1 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that CCR1 (or CCL3) gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • phosphodiester bonds of the siRNAs of the invention are advantageously protected. This protection is generally implemented via the chemical route using methods that are known by art.
  • the phosphodiester bonds can be protected, for example, by a thiol or amine functional group or by a phenyl group.
  • the 5'- and/or 3'- ends of the siRNAs of the invention are also advantageously protected, for example, using the technique described above for protecting the phosphodiester bonds.
  • the siRNAs sequences advantageously comprises at least twelve contiguous dinucleotides or their derivatives.
  • RNA derivatives with respect to the present nucleic acid sequences refers to a nucleic acid having a percentage of identity of at least 90% with erythropoietin or fragment thereof, preferably of at least 95%, as an example of at least 98%, and more preferably of at least 98%.
  • percentage of identity between two nucleic acid sequences, means the percentage of identical nucleic acid, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the nucleic acid acids sequences.
  • best alignment or “optimal alignment” means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two nucleic acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity.
  • the identity percentage between two sequences of nucleic acids is determined by comparing these two sequences optimally aligned, the nucleic acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
  • shRNAs short hairpin RNA
  • shRNAs can also function as inhibitors of expression for use in the present invention.
  • Ribozymes can also function as inhibitors of expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of CCR1 (OR CCL3) mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.
  • antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and preferably cells expressing CCR1 (OR CCL3).
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • AAV adeno-associated virus
  • 12 different AAV serotypes AAVl to 12
  • Recombinant AAV are derived from the dependent parvovirus AAV2 (Choi, VW J Virol 2005; 79:6801-07).
  • the adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316- 27).
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • Another object of the invention relates to a method for treating intestinal inflammation (in particular Crohn's disease) comprising administering a subject in need thereof with a CCR1 antagonist or inhibitor of CCR1 (or CCL3) gene expression, as above described.
  • the CCR1 antagonist or inhibitor of CCR1 (or CCL3) gene expression may be administered in the form of a pharmaceutical composition, as defined below.
  • said inhibitor is administered in a therapeutically effective amount.
  • a “therapeutically effective amount” is meant a sufficient amount of the CCR1 antagonist or inhibitor of CCR1 (or CCL3) gene expression to treat Crohn's disease at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the CCR1 antagonist or inhibitor of CCR1 (or CCL3) gene expression may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, aminoethyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-pheny
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the CCR1 antagonist or inhibitor of CCR1 (or CCL3) gene expression of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the CCRl antagonist or inhibitor of CCRl (or CCL3) gene expression of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • parenteral administration such as intravenous or intramuscular injection
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration ; liposomal formulations ; time release capsules ; and any other form currently used.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 The recruitment of inflammatory monocytes during T. gondii-mdnc d ileitis depends on CCR1/CCL3 interactions
  • A qRT-PCR quantification in jejunal biopsies of ccl2,-4,-5 and cxcllO transcripts from IL-15 "7" and WT mice naive or on day 7 PI.
  • B qRT-PCR quantification of cxcllO transcripts in LP cells from IL-15 "7” and WT mice naive or on day 7 PI.
  • C qRT-PCR quantification of ccl3 transcripts from IL-15 " and WT mice naive on days 3, 5 and 7 PI in jejunal biopsies (left) or in LP cells (right).
  • E,H Spontaneous secretion of IL- ⁇ , TNFa evaluated by ELISA in 24h supernatants of jejunal biopsies from WT mice treated or not with anti-CCL3 mAb (E) and from IL-15 "7 , WT and CCR1 "7” mice (H) either naive or on day 7 PI .
  • IEL immunodeficiency EL
  • WT CD45.1
  • IL-15 "7" CD45.2
  • recipient mice were lethally irradiated at 900 rads with a Ce 137 source and intravenously injected with 5.10 6 bone marrow cells from either WT or IL-15 "7" donor mice.
  • mice were intragastrically gavaged with 35 cysts from T. gondii 76K strain and their weight and survival were monitored. Histology was evaluated on paraffin sections and parasitic load determined by quantitative PCR on day 7 PI (Burg et al, 1988).
  • WT mice received intraperitoneal injections of 100 ⁇ g of control isotypes or of anti-NKG2D (clone CX5, IgGl gift from Novo Nordisk, Denmark) on days -1 and 5 or of anti-CCL3 (MAB450, IgG2a, R&D systems) on days 4 and 5 PI, or anti-NKl .
  • l (clone PK136, IgG2a, AbDserotec) on days-1, 1 and 5.
  • Peripheral blood was obtained from healthy adult volunteers. Intestinal biopsies were obtained from 19 patients with Crohn's disease and 15 controls (age: 7-15 years) according to protocol AOM08087 approved by Ethical Committee He de France II.
  • Mouse lymphocytes were isolated from blood, spleen, MLN, small intestinal epithelium and LP as previously described (Mennechet et al, 2004). Peritoneal mononuclear cells were harvested in cold PBS. Human PBL were isolated on Ficoll Hypaque gradient according to standard procedures.
  • IL- ⁇ , IL-6, TNFa, IFNy or CCL3 was measured in culture supernatants by ELISA (R&D Systems). Stimulation of human PBL was performed using human IL-15 and/or coated anti-CD3 (5 ⁇ g/well) and soluble anti-CD28 (0.25 ⁇ g/ml) antibodies (BD Biosciences).
  • Phenotyping and lymphocyte subset isolation by flow cytometry Isolated cells were incubated with anti-FcgRII/III mAb (Fcblock, 2.4G2 ; BD Pharmingen) for 10 min and then stained with various mixes of directly coupled mAbs : Ly6C-FITC, Ly6G-PE, F4/80-PECy5, CD3-PE, CD3-APCCy7, NK1.1-APC, CD127-PECy7, NKP46-PECY5, CDl lb-APCH7, CDl lc-PECY7, TCRaP-APC (BD Pharmingen), CD45- eFluor450 (eBiosciences), Aqua view blue (Invitrogen) or control isotypes.
  • Ly6C-FITC Ly6G-PE
  • F4/80-PECy5 CD3-PE, CD3-APCCy7, NK1.1-APC, CD127-PECy7, NKP46-
  • LPL suspensions were incubated for 4 h in culture medium added with 2 ⁇ 1/ ⁇ 1 Brefeldin A (Sigma-Aldrich) at 37°C. After surface staining, cells were fixed with 2% paraformaldhehyde (Euromedex), permeabilized using 0.5% saponin (Sigma) and stained intracellularly with anti-IFNy-APC, anti-TNFa-APC (BD Pharmingen) or anti-CCL3-PE mAbs (R&D systems). Cells were analysed on a FACScanto II or sorted using a FACSAria cell sorter III (BD Biosciences).
  • qRT-PCR was performed using TaqMan gene expression assays (hprt, ifng, Mb, tnfa, 6, ccl2, ccl3, ccl4, ccl5, ccrl, ccr2, ccrS) and TaqMan Universal PCR master mix andGeneAmp 7000 machine (Applied Biosystems). CDNA samples were assayed in duplicates and expression levels were normalized relative to HPRT with Ct calculation.
  • IL-15 controls the severity of intestinal inflammation induced by T. gondii in C57BL/6 mice: In wild type male C57BL/6 mice, oral gavage with 35 cysts of T. gondii strain 76 K induced a severe jejuno-ileitis leading to animal death between days 10 and 13. In IL-15 "7" mice, oral infection was associated with a significant weight loss, but all animals survived the early phase of infection and the severity of ileal lesions was markedly attenuated. In contrast, no significant increase in parasite load was observed in the intestine, mesenteric lymph nodes (MLN), liver and spleen of IL-15 " " mice indicating that, in this model, IL-15 is not necessary to control parasite replication but rather contributes to intestinal inflammation. These results differentiate the role of IL-15 from that of IFNy which is necessary not only to induce intestinal inflammation (Liesenfeld et al., 1996) but also to control local and systemic replication of the parasite.
  • IL-15 can be produced by many cell types including enterocytes, dendritic cells (DC) and macrophages. Accordingly, up-regulation of U15 mR A following T. gondii infection was observed in enterocytes as well as in CDl lc + CDl lb +/" DC isolated from LP and MLN. To define which cellular source of IL-15 determines the severity of the ileitis, the outcome of T. gondii infection was compared in hematopoietic chimeras.
  • IL-15 The enhancing effect of IL-15 on intestinal inflammation does not depend on IEL during T. gondii-mduced ileitis:
  • a stromal source of IL-15 is necessary for the expansion of CD8aa IEL and more particularly of TCRy5 IEL (Schluns et al, 2004b).
  • TCRy5 IEL were almost absent in IL-15 "7" mice reconstituted or not with WT bone marrow which developed an attenuated form of ileitis.
  • WT chimeras reconstituted with IL-15 "7" bone marrow, in which T. gondii induced a lethal ileitis, suggesting their possible contribution to IL-15-driven inflammation.
  • Epithelium-derived IL-15 might also activate cytotoxic CD8 + TCRa,p + IEL. After oral infection with T. gondii, this subset of IEL can in vitro kill T. gondii- fectGd targets (Chardes et al, 1994). Moreovever, IL-15 can stimulate IEL cytotoxicity by inducing perforin and granzymes (Mention et al, 2003) or by enhancing expression of and/or signaling via the activating NKG2D receptor (Hue et al, 2003; Meresse et al, 2004).
  • NKG2D expression increased significantly in LP CD8 TCRa- ⁇ lymphocytes from WT but not from IL- 15 " mice but IEL remained largely NKG2D negative during T.gondii ileitis.
  • intravenous treatment with a blocking (but non-depleting) anti-NKG2D antibody did not modify the course of the disease in WT mice.
  • the ratio of granzyme B and perforin to CD3s mRNA expression increased during infection in WT but not in IL-15 "7" mice, so that a significant difference was observed on day 7.
  • IL-15 is dispensable for the induction of T. goni/M-specific Thl response: A previous study has suggested that IL-15 promotes specific Thl response during T. gondii infection (Combe et al, 2006). This response is thought to be initiated by mucosal CDl lc + DC cells which are infected by the parasite in the mucosa within the first two days of infection and then emigrate to MLN where they can be detected between days 3 to 7 postinfection (Courret et al., 2006). In MLN, DC elicit T. gondii-specific CD4 + Thl cells which then home into LP.
  • T.gondii-inducQd intestinal Thl response To analyze the contribution of IL-15 to T.gondii-inducQd intestinal Thl response, ifng mRNA and IFNy protein expression were compared on days 0, 3, 5 and 7 in jejunal biopsies and in cells isolated from MLN and LP of IL-15 "7" and WT mice and stimulated or not with soluble T.gondii antigen (STAg). A slight delay was observed in the up-regulation of ifng mRNA in MLN lymphocytes and of IFNy secretion by LPL cultured with STAg. Yet, on day 7, LPL from both groups of animals spontaneously produced massive and comparable amounts of IFNy which were not further increased in the presence of STAg.
  • STAg T.gondii antigen
  • mice during T. gondii-induced ileitis Inflammation in WT and IL-15 "7" mice was next assessed by comparing intestinal IL- ⁇ ⁇ , TNFa, IL-6 production during infection. Up-regulation of illb, tnfa, U6 mRNA was first observed in intestinal biopsies and in isolated LP cells on day 5 postinfection and increased markedly on day 7 when copious amounts of IL- ⁇ , TNFa, IL-6 were spontaneously released in the supernatants of either biopsies or isolated LP. Up-regulation of inflammatory cytokines was observed in both animal groups but was significantly higher in WT than in IL-15 "7" mice.
  • LP cells were isolated from infected WT and IL-15 "7" mice on day 7 and sorted into CD3 + , CD19 + , CD3 " NK1.1 + , CD45 + CD3 CD19 NK1.1 " cells.
  • RT-PCR analysis of sorted cells showed that illb, tnfa, U6 mRNA were only up-regulated in CD45 + CD3 " CD 19 " ⁇ 1. ⁇ cells and confirmed the difference between IL-15 "7" and WT mice ELISA authenticated the secretion of IL- ⁇ ⁇ and TNFa by LP CD45 CD3 CD19 NK1.1 " cells isolated from WT mice and showed that the secretion was below the detection limit in the same fraction isolated from IL-15 "7" mice.
  • LP cells from WT mice were isolated at day 7 post-infection (PI) and analyzed by flow cytometry after staining with a panel of surface markers and intracellular TNFa.
  • TNF-a was absent in CD3 + and CDl lc + LP cells and was exclusively detected in Ly6C hi F4/80 + Ly6G " CD1 lb + CD1 lc " LP cells, a phenotype typical of inflammatory monocytes (IM) (Auffray et al, 2009; Dunay et al, 2008).
  • IM inflammatory monocytes
  • IL-15 was necessary to promote the activation and/or the recruitment of IM.
  • IL-15 alone did not induce the production of TNFa and/or IL-1- ⁇ in peritoneal macrophages from WT mice either naive or infected peritoneally with T. gondii for 3 days and did not synergize with LPS.
  • IL-15 even at high concentrations, also failed to stimulate the production of inflammatory cytokines by LP cells from naive WT mice even after stimulation by anti-CD3 and anti-CD28 antibodies.
  • CCL3 concentrations were significantly reduced in supernatants of LP cells from infected IL-15 "7" mice.
  • WT mice were injected on days 4 and 5 with a blocking anti-CCL3 antibody. This treatment strongly reduced the percentage and absolute numbers of Ly6C hl F4/80 + LP cells as well as the production of IL- ⁇ and TNFa ( Figures IB, 1C, 2E), confirming the role of CCL3.
  • CCL3 binds two distinct receptors, CCR1 and CCR5, both of which can be expressed by IM.
  • CCR1 and CCR5 both of which can be expressed by IM.
  • ccr5 mRNA levels were similarly increased in jejunal biopsies and in LP cells from WT and IL-15 "7" mice.
  • ccrl mRNA was significantly lower in IL-15 "7” than in WT mice ( Figure ID) and were almost undetectable in mice treated by anti-CCL3 antibody ( Figure 2F).
  • the role of CCR1 in the recruitment of IM was therefore assessed using CCR1 "7" mice. Contrasting with their recruitment in infected WT mice, no increase in Ly6C hl F4/80 LP cells was observed in CCR1 " mice during T.
  • CCL3 production during T. gondii-induced ileitis depends on intestinal IL-15- dependent NKP46 + ⁇ 1.
  • CD127 cells CCL3 can be produced by cells of hematopoietic or epithelial origin. Accordingly, an increase in ccl3 transcripts was observed in both enterocyte and LP cell fractions isolated from WT mice on day 7 PI. Yet, cc/J transcripts was 100-200 fold more increased in WT LP cells than in enterocytes, indicating that, during T. gondii infection, CCL3 was mainly produced by LP cells. Analysis of sorted LP cell subsets showed that, on day 7 PI, ccl3 mRNA was strongly expressed by a minor subset of NKl .
  • l + CD3 " cells Consistent with the hypothesis that the IL-15 dependent source of CCL3 may be NK cells, absolute numbers of NKP46 + CD3 " cells were significantly decreased in naive and infected IL-15 "7" mice compared to WT mice. Recent work indicates that LP contains two main subsets of NK cells. A first subset of NKP46 + NKl .l " CD127 + cells depends on IL-7 and secretes IL-22, a cytokine that strengthens the intestinal barrier. A second subset of NKP46 + NKl .l + CD 127 " cells depends on IL-15 and thus resembles classical splenic NK cells.
  • a stromal source of IL-15 presumably epithelial cells, was necessary for the optimal differentiation and/or local homeostasis of NKP46 + NK1.1 + CD127 " cells.
  • efficient reconstitution of LP CD3 " NKl . l cells was observed in irradiated WT mice reconstituted with IL-15 "7" bone marrow (WT+BM IL-15 "7” ) but not in irradiated IL-15 "7” mice receiving WT bone marrow (IL-15 "7” +BM WT).
  • LP NKP46 + NK1.1 + CD127 cells depend on a stromal source of IL-15 for their differentiation/homeostasis and are an important source of CCL3 during T. gondii infection that might stimulate the recruitment of CCR1 + IM.
  • mice were treated with a depleting anti-NK.1.1 antibody on day -1 before infection, and on days 1 and 4 PI.
  • this treatment had efficiently depleted LP NKP46 + NK1.1 + CD127 " cells and resulted in a significant decrease in CCL3 production and ccrl mRNA levels. Accordingly, the numbers of LP Ly6C hl F4/80 + IM and the spontaneous production of IL- ⁇ and TFNa by isolated LP cells or whole intestinal tissue were also significantly reduced.
  • CCL3 and CCR1 expression are up-regulated in Crohn's disease: In Crohn's disease (CD), TNFa-producing inflammatory macrophages play a central role in gut inflammation attested by the efficiency of therapeutic administration of anti-TNFa antibodies.
  • CD33 + CD14 + phenotype Recent work has identified their CD33 + CD14 + phenotype and shown their expression of several chemokine receptors, including CCR1 which is absent on gut resident CD33 + CD14 " myeloid cells (Kamada et al, 2008). In keeping with these data, illb, tnfa, U6 and ccrl mRNA levels were significantly up-regulated in inflamed biopsies from CD patients. ( Figures 3 A, 3C).
  • IL15Ra a receptor necessary for transpresentation of IL-15 to T and NK cells expressing IL15Rp/y c (CD122/CD132), the signaling module activated by IL-15 in lymphocytes (Sandau et al, 2004; Schluns et al, 2004a).
  • IL-15 transcription was up-regulated in both in epithelial and dendritic cells.
  • the dramatic course of the ileitis in irradiated WT mice reconstituted with IL-15 "7" bone marrow and in IL-15Tg E mice over-expressing IL-15 in the gut epithelium contrasted with the attenuated ileitis observed in IL-15 "7" mice either or not irradiated and reconstituted with WT bone marrow, altogether pointing to a central role of epithelium-derived IL-15 in shaping the severity of intestinal inflammation.
  • IL-15 stimulated in vivo recruitment of IM, as their numbers increased significantly less in the LP of IL-15 "7" than of WT infected mice.
  • a central role of IM in oral toxoplasmosis was recently demonstrated in CCR2 "7" mice which displayed normal Thl response but were unable to recruit IM and to control parasite replication.
  • CCR2 was dispensable for IM migration into the inflamed gut and it was suggested that CCR2 might rather control monocyte emigration from bone marrow (Dunay et al, 2008).
  • the lesser levels of ccrl and ccl3 mRNA in the intestine of infected IL-15 "7" than WT mice pointed to their possible role in the local recruitment of IM.
  • Increased susceptibility to T. gondii has already been reported in CCR1 "7" mice and ascribed to the impaired recruitment of cells defined as neutrophils on the basis of their staining with the anti-Ly6 RB6-8C5 antibody (Khan et al, 2001).
  • mice Furthermore, the production of IL- ⁇ ⁇ and TNFa by LP cells was abolished in the latter mice and the lack of IM was associated with a markedly increased parasitic load and a rapid death of infected animals (data not shown). In IL-15 " " mice, the recruitment of IM may be sufficient to control parasite replication but insufficient to induce irreversible intestinal lesions.
  • NK cells are an important source of CCL3 during T. gondii infection. Indeed, it was recently shown that this subset of gut innate cells, alike peripheral NK cells, depends on IL-15 for its differentiation and maturation (Satoh- Takayama et al). Previous work has shown that NK depletion using the anti-asialoGMl antibody markedly reduced intestinal inflammation in WT mice but the role of gut NK cells was not studied (Khan et al, 2006).
  • NK cells contribute, via their production of CCL3, to the recruitment of IM and to the severity of the ileitis after oral infection with T. gondii.
  • the analysis of hematopoietic chimeras further indicates that a stromal source of IL-15, presumably epithelial cells, is necessary to drive their differentiation and/or maintain their survival. Whether IL-15 participates in the local induction of CCL3 during infection is unclear.
  • IL-15 could stimulate CCL3 synthesis by spleen NKP46 + NK1.1 + CD127 " NK cells.
  • CCL3 induction was maximal at late time points (after day 5 PI), when the level of IL-15 mRNA had returned to the baseline.
  • administration of a blocking anti-IL-15 antibody prevented the death of only 50% of infected WT mice (data not shown).
  • IL- 15 is not indispensable for the induction of CCL3 and that other stimuli, yet to be identified, participate in the local induction of CCL3 secretion by NK cells.
  • NK cells are selectively increased in the intestine of Crohn's disease patients (Takayama et al.) and that serum detection of IL-15 in CD patients can predict their response to anti-TNFa antibodies (Bouchaud et al).
  • IL-15 selectively and strongly stimulated the transcription of ccl3 in human peripheral NKP46 + CD56 + NK cells.
  • ccl3 the levels of ccl3 and of ccrl mRNA in intestinal biopsies from CD patients.
  • IL-15-dependent gut NK cells may be activated in response to intracellular pathogens and can, via CCL3, participate in the recruitment of IM.
  • IL-15-dependent NK cells may thereby participate in gut defense but also induce deleterious inflammation in and perhaps beyond the gut.
  • IL-15 renders conventional lymphocytes resistant to suppressive functions of regulatory T cells through activation of the phosphatidylinositol 3- kinase pathway. J Immunol 182, 6763-6770.
  • Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes. J Immunol 153, 4596- 4603.
  • Grl(+) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. Immunity 29, 306-317.
  • Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells. J Exp Med 193, 219-231.
  • CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection.
  • PLoS Pathog 2 e49.
  • IL-15 is highly expressed in inflammatory bowel disease and regulates local T cell-dependent cytokine production. J Immunol 164, 3608-3615.
  • IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9, 669-676. Malamut, G., El Machhour, R., Montcuquet, N., Martin-Lanneree, S., Dusanter-Fourt, I., Verkarre, V., Mention, J.J., Rahmi, G., Kiyono, H., Butz, E.A., et al. IL-15 triggers an antiapoptotic pathway in human intraepithelial lymphocytes that is a potential new target in celiac disease-associated inflammation and lymphomagenesis. J Clin Invest 120, 2131-2143.
  • Lamina limbal synergize with murine intestinal epithelial cells to enhance proinflammatory response against an intracellular pathogen. J Immunol 168, 2988-2996.
  • Interleukin 15 a key to disrupted intraepithelial lymphocyte homeostasis and lymphomagenesis in celiac disease. Gastroenterology 125, 730-745.
  • NKG2D blockade prevents autoimmune diabetes in NOD mice. Immunity 20, 757-767.
  • Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity 29, 958-970.
  • NKG2D recognition mediates Toll-like receptor 3 signaling-induced breakdown of epithelial homeostasis in the small intestines of mice. Proc Natl Acad Sci U S A 104, 7512-7515.

Abstract

La présente invention concerne des procédés et des compositions pharmaceutiques destinés au traitement de la maladie de Crohn. La présente invention concerne plus particulièrement un antagoniste du récepteur 1 aux chimiokines C-C (CCR1) destiné à être utilisé dans le traitement de la maladie de Crohn.
PCT/EP2012/059880 2011-05-27 2012-05-25 Procédés et compositions pharmaceutiques destinés au traitement de la maladie de crohn WO2012163848A1 (fr)

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