WO2020178193A1 - Method of treatment of sarcoidosis - Google Patents

Abstract

During a retrospective study of CSF biomarkers in patients with neurosarcoidosis who underwent CSF analysis, the inventors showed that a higher concentration of IL-6 in the CSF of the patient was positively correlated with the disease severity and the rate of disease progression. Furthermore the inventors showed that IL-6R blockade was efficient to treat 3 patients with refractory sarcoidosis, demonstrating that this molecule is not only a biomarker, but also a suitable therapeutic target. Furthermore, one of the patients treated with IL-6 antagonist did not show any relapse. Thus, blocking IL-6 constitutes a new therapeutic approach for preventing or treating sarcoidosis. The present invention provides an IL-6 antagonists for a novel use in the treatment of sarcoidosis, especially in a patient having a high level of IL-6 in a biological biopsy.

Description

METHOD OF TREATMENT OF SARCOIDOSIS

FIELD OF THE INVENTION:

The present invention relates to a method of preventing and/or treating sarcoidosis in a patient. More specifically, it concerns the use of an interleukin-6 (IL-6) antagonist, for the prevention and treatment of sarcoidosis in a patient, especially in a patient having a high level of IL6 in a biological sample.

BACKGROUND OF THE INVENTION:

Sarcoidosis is a multi -systemic disease characterized by the formation of non-caseating granulomas in various organs [1] Central nervous system (CNS) is clinically involved in about 5% of cases and represents a major cause of disability [2] Neurosarcoidosis is a challenging condition [3] First, CNS localization of sarcoidosis may be the first manifestation of the disease and should be differentiated from a broad range of inflammatory, infectious or neoplastic conditions like multiple sclerosis, infectious meningitis, neoplasia (solid tumours and lymphoma), histiocytic disorders or other autoimmune conditions. Next, the diagnosis of sarcoidosis relies on the documentation of non-caseating granulomas but the accessibility of CNS for a biopsy is low [4] Finally, the course of sarcoidosis is frequently relapsing despite glucocorticosteroids and immunosuppressive drugs [5]

Therefore, there is a need for a new therapy in order to treat sarcoidosis.

Furthermore, biomarkers for sarcoidosis diagnosis and prognosis are lacking. Cerebro spinal fluid (CSF) analysis usually reveals lymphocytic meningitis, sometimes with low glycorachia level and specific oligoclonal bands, in CNS localisations [6] However, all these biological findings have been reported in other inflammatory, infectious or neoplastic conditions. Typical sarcoidosis granulomas are composed of histiocytic cells (epithelioid and multinucleated giant cells) and T CD4 lymphocytes. Therefore, patients with sarcoidosis harbour a blood CD4 lymphopenia together with an enrichment of organs in T CD4 lymphocytes. The CD4/CD8 ratio serves as a biomarker in bronchoalveolar lavage (BAL): a value of this ratio superior to 3.5 has demonstrated is a reliable marker for diagnosis, compared to other interstitial lung diseases [7] The value of CSF CD4/CD8 ratio has been evaluated in a small study of 7 patients: 2 of them had an elevation of the CD4 lymphocyte subpopulation [8] However, this study was too small to allow conclusions. The CSF interleukin (IL)-6 and 10 have been used in various neurological conditions. In particular, these dosages have been evaluated in cerebral lymphoma, multiple sclerosis, and neurological involvement of Behcet disease (BD) [9-13] The CSF IL-6 is elevated in a broad spectrum of neurological diseases, including infectious diseases and neuro-BD. An elevated CSF IL-10 concentration seems to be a reliable marker of lymphoma, but the comparative groups in previous studies did not include patients with neurosarcoidosis [9, 14, 15]

Thus inventors aimed to study the lymphocyte subpopulations phenotype, IL-6 and IL- 10 levels in CSF of patients with neurosarcoidosis, compared to other inflammatory disorders of the CNS and to identify a new strategic therapy of sarcoidosis.

SUMMARY OF THE INVENTION:

During a retrospective study of CSF biomarkers in patients with neurosarcoidosis who underwent CSF analysis between 2012 and 2017, the inventors showed that a higher concentration of IL-6 in the CSF of the patient was positively correlated with the disease severity and the rate of disease progression. Furthermore the inventors showed that IL-6R blockade was efficient to treat 3 patients with refractory sarcoidosis, demonstrating that this molecule is not only a biomarker, but also a suitable therapeutic target. Furthermore, one of the patients treated with IL-6 antagonist did not show at this time, any relapse. Thus, blocking IL- 6 constitutes a new therapeutic approach for preventing or treating sarcoidosis.

The present invention provides an IL-6, antagonists for a novel use in the treatment of sarcoidosis, especially in a patient having a high level of IL-6 in a biological sample.

DETAILED DESCRIPTION OF THE INVENTION:

Therapeutic methods and uses

The present invention provides methods and compositions (such as pharmaceutical compositions) for treating sarcoidosis disease.

In the context of the invention, the term "treatment or prevention" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in reducing the number of granuloma. Most preferably, such treatment leads to the complete depletion of granuloma. Preferably, the individual to be treated is a human or non-human mammal (such as a rodent (mouse, rat), a feline, a canine, or a primate) affected or likely to be affected with cancer. Preferably, the individual is a human.

According to a first aspect, the invention provides an antagonist of Interleukin-6 (IL-6), for use in the prevention or treatment of sarcoidosis.

Sarcoidosis is a chronic disease with infiltration of various tissues/organs by abnormal collections of inflammatory cells (majority of macrophage) that form lumps known as non- caseating granuloma (Iannuzzi M, et al (2007). " New England Journal of Medicine. 357 (21): 2153-2165). Any organ, however, can be affected but the disease usually begins in the lungs, skin, or lymph nodes and less commonly affected are the eyes, liver, heart, and brain. The signs and symptoms depend on the organ involved. Often there are no, or only mild, symptoms. Central nervous system (CNS) is clinically involved in about 5% of cases and represents a major cause of disability [2] CNS localization of sarcoidosis (neurosarcoidosis) may be the first manifestation of the disease.

In a specific embodiment of the invention, sarcoidosis is neurosarcoidosis (CNS localization of sarcoidosis). In another embodiment, the sarcoidosis is refractory sarcoidosis. A “refractory sarcoidosis” means that it not responds to the current therapy glucocorticosteroids and immunosuppressive drugs (Valeyre D, et al Lancet. 2014 Mar 29;383(9923): 1155-67).

In one embodiment, the invention provides an antagonist of Interleukin-6 (IL-6) antagonist, for use in the treatment of sarcoidosis in a patient having a high level of IL-6 in a biological sample.

In another aspect the invention provides a pharmaceutical composition, comprising an antagonist of IL-6, for use in the prevention or the treatment of sarcoidosis in a patient, especially in a patient having a high level of IL6 in a biological sample.

The term“Interleukin-6”, also called“IL-6” has its general meaning in the art and refers to is an interleukin that acts as a pro-inflammatory cytokine and an anti-inflammatory myokine (cytokines produced and released by muscle cells (myocytes) with autocrine, paracrine and/or endocrine effects). In humans, it is encoded by the IL6 gene (Gene ID: 3569). In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation. Smooth muscle cells in the tunica media of many blood vessels also produce IL-6 as a pro-inflammatory cytokine. IL-6 signals through a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL- 6Ra chain (CD126), and the signal-transducing component gpl30 (also called CD130).

An " IL-6 antagonist" refers to a molecule (natural or synthetic) capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of IL-6 including, for example, reduction or blocking of IL-6 receptor (CD 126 or CD 130) activation, reduction or blocking of IL-6 receptor (CD 126 or CD 130) downstream molecular signalling (such as LMT- 28 compound). IL6 antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Antagonists also include small molecule inhibitors of a protein and receptor molecules and derivatives which bind specifically to IL-6 thereby sequestering its binding to its IL-6 receptor (CD126 or CD130), such as soluble IL-6 receptors or fusions proteins, antagonist variants of the protein, siRNA molecules directed to a protein, antisense molecules directed to a protein, aptamers, and ribozymes against a protein. For instance, the IL-6 antagonist may be a molecule which binds to IL-6 or to IL-6 receptor and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of IL-6 (such as inducing inflammation). Alternatively, the IL-6 antagonist may be a molecule which binds to IL-6 and neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of IL-6. More particularly, the IL-6 antagonist according to the invention is an anti- IL-6 antibody or anti- IL-6 receptor antibody. More preferably, this IL6 receptor antibody is tocilizumab.

The term“Interleukin-6 receptor” has its general meaning in the art and refers to a type I cytokine receptor (transmembrane receptors expressed on the surface of cells that recognize and respond to cytokines with four a-helical strands). The IL-6 receptor is a receptor complex consisting of the ligand-binding IL-6Ra chain (also known as “CD126” or“Cluster of Differentiation 126” / Gene ID: 3570 for human CD126), and the signal-transducing component gpl30 (also called CD130 / Gene ID: 3572 for human gpl30).

The term "biological activity" of Interleukin-6 means stimulation of inflammation. Interleukin-6 stimulates the inflammatory and auto-immune processes in many diseases such as diabetes (Kristiansen OP, et al (2005) Diabetes. 54 Suppl 2: SI 14-2), systemic lupus erythematosus, (Tackey E, et al (2004). Lupus. 13 (5): 339-43) and rheumatoid arthritis (Nishimoto N (May 2006). Current Opinion in Rheumatology. 18 (3): 277-81). C-reactive protein (CRP) blood testing is commonly used as marker of inflammation. The inhibition of C- reactive protein (CRP) production is a surrogate marker of anti-IL-6 therapy efficacy: (CRP) production by human hepatocytes is completely under the control of IL6 in vivo, as evidenced by the loss of circulating CRP throughout anti-IL6 treatment and a quick loss reversal at treatment discontinuation (Klein B, et al. Blood 1991;78: 1198-204). In the context of sarcoidosis, the "biological activity" of IL-6 could also be associated with non-caseating granuloma formed during inflammation, and which contains inflammatory cells (macrophage or neutrophils), in involved organs.

Tests for determining the capacity of a compound to be Interleukin-6 antagonist are well known to the person skilled in the art. In a preferred embodiment, the antagonist specifically binds to Interleukin-6 in a sufficient manner to inhibit the biological activity of Interleukin-6. Binding to Interleukin -6 and inhibition of the biological activity of Interleukin -6 may be determined by any competing assays well known in the art. For example the assay may consist in determining the ability of the agent to be tested as Interleukin-6 antagonist to bind to Interleukin-6 or Interleukin-6 receptor. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an antagonist that "specifically binds to Interleukin -6" is intended to refer to an inhibitor that binds to human Interleukin -6 polypeptide with a KD of 1 mM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of Interleukin -6. The functional assays may be envisaged such evaluating the ability to inhibit the induction/stimulation of inflammation (for instance with C-reactive protein (CRP) blood tests) or to reduce the amount of granuloma (for instance by imagery analysis such computed tomography, magnetic resonance imaging, and/or 18 fluorodeoxyglucose positron emission tomography as described in the“Example” section).

The skilled in the art can easily determine whether an Interleukin-6 antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of Interleukin-6. To check whether the Interleukin-6 antagonist bind to Interleukin-6 and/or inhibit the induction/stimulation of inflammation in the same way than the initially characterized blocking Interleukin-6 antibody and/or binding assay and/or a inflammation assay may be performed with each antagonist. For instance inflammation linked to IL6 can be measured with C-reactive protein (CRP) blood tests and/or the amount of granuloma can be measured by imagery analysis.

In one embodiment, the IL-6 antagonist is an inhibitor of the interaction between IL-6 and -6 receptor such as IL6 antibody and IL-6R antibody.

The terms "blocking the interaction", "inhibiting the interaction" or "inhibitor of the interaction" are used herein to mean preventing or reducing the direct or indirect association of one or more molecules, peptides, proteins, enzymes or receptors; or preventing or reducing the normal activity of one or more molecules, peptides, proteins, enzymes, or receptors.

Thus, the term "inhibitor of the interaction between Interleukin-6 and Interleukin-6 receptor" refers to a molecule which can prevent the interaction between Interleukin-6 and Interleukin-6 receptor (IL-6 antibody and IL6-R antibody) by competition or by fixing to one of the molecules.

Accordingly, the Interleukin-6 antagonist may be a molecule which binds to Interleukin- 6 or Interleukin-6 receptor selected from the group consisting of antibodies, aptamers, polypeptides and small organic molecules.

The skilled in the art can easily determine whether an Interleukin-6 antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of Interleukin-6: (i) binding to Interleukin -6 or Interleukin -6 receptor and/or (ii) inducing /stimulation of inflammation.

Examples of IL6 antagonists include but are not limited to any of the IL-6 or IL-6R antagonists described in Ferry JF. et al. (Clin Cancer Res; 21(6) March 15, 2015) and Jones SA, et al (The Journal of Clinical Investigation. 121 (9): 3375-83. (2011) all of which are herein incorporated by reference.

In particular embodiment, the IL-6 antagonist is a IL-6 activity inhibitor or a IL-6 expression inhibitor.

In some embodiment, the IL-6 activity inhibitor is a IL-6 antibody, a IL-6 receptor antibody, a IL-6 receptor polypeptide or a IL-6 receptor antagonist small molecule.

In some embodiment, the IL-6 antibody is selected from the group consisting of siltuximab, olokizumab, sirukumab, elsilimomab, clazakizumab, gerilimzumab VX30, EB-007, and FM101.

In some embodiment, the IL-6 receptor (IL-6R) antibody is selected from the group consisting of tocilizumab, sarilumab vobarilizumab.

In some embodiment, the IL-6 receptor polypeptide is olamkicept.

In some embodiment, the IL-6 receptor antagonist small molecule is LMT-28 or derived compounds.

In some embodiment, the IL-6 expression inhibitor is an antisense, oligonucleotides, a ribozymes or a siRNA to directly block the translation of IL-6 mRNA.

Typically, an IL6 antagonist according to the invention includes but is not limited to: i. IL-6 antibody such as siltuximab, olokizumab, sirukumab, elsilimomab, clazakizumab, gerilimzumab VX30, EB-007, and FMIOI_^

ii. IL-6R antibody such as tocilizumab, sarilumab vobarilizumab

iii. IL6R antagonists small molecule such as LMT-28 and derived compounds iv. Inhibitor of Interleukin-6 gene expression such as siRNA. an antisense oligonucleotide, a nuclease or a ribozyme.

v. IL6 receptor polypeptide such as olamkicept (FE 999301, FE301, TJ301) (Soluble gpl30-Fc fusion protein)

In a preferred embodiment the IL-6 antagonist is an IL-6 or IL-6R antibody such as tocilizumab.

• Antibody

In one embodiment, the Interleukin-6 antagonist is an antibody (the term including antibody fragment or portion) that can block the interaction of Interleukin-6 receptor with Interleukin-6.

In preferred embodiment, the Interleukin-6 antagonist may consist in an antibody directed against the Interleukin-6 receptor or Interleukin-6, in such a way that said antibody impairs the binding of an Interleukin-6 to Interleukin-6 receptor ("neutralizing antibody").

Then, for this invention, neutralizing antibody of Interleukin-6 or the Interleukin-6 receptor are selected as above described (for their capacity to (i) bind to Interleukin-6 or Interleukin-6 receptor and/or (ii) reduce of inflammation and/or (iii) the capacity to reduce the amount of granuloma.

In one embodiment of the antibodies or portions thereof described herein, 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. In one embodiment of the antibodies or portions thereof described herein, 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.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "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 Interleukin-6. 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.

Briefly, the recombinant Interleukin-6 may be provided by expression with recombinant cell lines. Recombinant form of Interleukin-6 may be provided using any previously described method. Following the immunization regimen, 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. Following fusion, 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). Following culture of the hybridomas, 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 immunoprecipitati on .

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, 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.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "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.

In one embodiment of the 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. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et ah, /. 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. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), 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.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, 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 CDR1 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 CDR1 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 CDR1 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 CDR1 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.

In another embodiment, 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. Examples of anti-IL6 antibodies are IL-6 antibody or IL-6 receptor antibody.

In one embodiment of the invention, the IL6 antagonist may be an IL6 antibody, such as siltuximab (CNTO 328 or cCLB8/ Janssen)), olokizumab ((CDP6038/UCB Pharma), sirukumab (CNTO 136 / GlaxoSmithKline), elsilimomab (B-E8 / Creative Biolaps) and is full human equivalent (mAb 1339 or OP-R003-1), clazakizumab (ALD518 and BMS-945429 / Bristol Myers Squib), VX30 (V0P-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), gerilimzumab (ARGX-109 / Argenx N. V) and FM101 (Femta Pharmaceuticals) which are lead IL6 antibody antagonists. Such antibodies are described, for example, in: US7612182 (siltuximab), Genovese MC et al Annals of the Rheumatic Diseases 2014;73 : 1607-1615 (Olokizumab), US7560112 (sirukumab), EP0430193 (elsilimomab), Fulciniti; M. et al. (2009). Clinical Cancer Research. 15 (23): 7144-52 (OP-R003-1, 1339 IL6 antibody), US8062864 (clazakizumab), EP2310413 (gerilimzumab)

In one embodiment of the invention, the IL6 antagonist may be an IL6 receptor antibody, such as tocilizumab (or atlizumab / Hoffman Roche), sarilumab (Kevzara/Regeneron) vobarilizumab (ALX0061 nanobody / Ablynx) , which are lead IL-6 receptor antibody antagonists. Such antibody are described, for example, in: EP0783893 (tocilizumab), US7582298 (sarilumab), US8748581 (vobarilizumab).

The skilled artisan can use routine technologies to use the antigen-binding sequences of these antibodies (e.g., the CDRs) and generate humanized antibodies for treatment of sarcoidosis as disclosed herein.

In such embodiment, the anti-IL-6 antibody comprises the six CDRs from an antibody selected from the group consisting of siltuximab, olokizumab, sirukumab, elsilimomab, clazakizumab, gerilimzumab, VX30, EB-007, ARGX-109 (ArGEN-X), FM101. In certain embodiments, the anti-IL-6 antibody comprises the heavy chain region and light chain region from an antibody selected from the group consisting of siltuximab, olokizumab, sirukumab, elsilimomab, clazakizumab, gerilimzumab, VX30, EB-007, ARGX-109 (ArGEN-X), FM101.

• Aptamer

In another embodiment, the Interleukin-6 antagonist is an aptamer directed against Interleukin-6 receptor or Interleukin-6. 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. In this library, 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. cob Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then, for this invention, neutralizing aptamers of Interleukin-6 are selected as above described for their capacity to (i) bind to Interleukin-6 or Interleukin-6 receptor and/or (ii) inhibit inflammation and/or (iii) the capacity to reduce the amount of granuloma).

• Small chemical entity

In another embodiment, the Interleukin-6 antagonist is a small chemical entity. As used herein, the term " small chemical entity " refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000 Da, and most preferably up to about 1000 Da.

In one embodiment, the IL6 antagonist may be a small chemical entity such as the following compounds: LMT-28 ((4S)-3-[(2S,3S)-3-Hydroxy- 2-methyl-4-methylene-l- oxononyl]-4-(l-methylethyl)-2-oxazolidinone) and derived compounds described, for example, in. Hong SS. Et al J Immunol. 2015 Jul l; 195(l):237-45 and which have the following structure :

• Inhibitor of Interleukin-6 gene expression

In still another embodiment, the Interleukin-6 antagonist is an inhibitor of Interleukin-6 gene expression or an inhibitor of Interleukin-6 receptor gene expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an "inhibitor of IL6 gene expression" denotes a natural or synthetic compound that has a biological effect to inhibit the expression of Interleukin-6 gene. In a preferred embodiment of the invention, said inhibitor of Interleukin-6 gene expression is a siRNA, an antisense oligonucleotide, a nuclease or a ribozyme.

Inhibitors of Interleukin-6 (or Interleukin-6 receptor) gene expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of Interleukin-6 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Interleukin-6, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding IL6 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 (siRNAs) can also function as inhibitors of Interleukin-6 gene expression for use in the present invention. Interleukin-6 (or Interleukin-6 receptor) gene expression can be reduced by using small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Interleukin-6 gene expression is specifically inhibited (i.e. RNA interference or RNAi). 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 Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Examples of said siRNAs against human Interleukin-6 or against Interleukin-6 receptor include, but are not limited to, those described, for example, in:. Bjorck P. et al (Immunology Letters (1998), ISSN: 0165-2478, Vol: 61, Issue: 1, Page: 1-5) Levy Y. et al (J Clin Invest. 1991 Aug; 88(2): 696-699), Bran G et al (In Vivo. 2011 Jul-Aug;25(4):579-84), Jiang XP et al (Anticancer Research September 2011 vol. 31 no. 9 2899-2906) Kong B et al (Gynecologic Oncology 1996Volume 63, Issue 1, Pages 78-84), EP0747386, WO9221380, JP5300338, US- 5716846.

Inhibitors of IL-6 gene expression for use in the present invention may be based nuclease therapy (like Talen or Crispr). The term“nuclease” or“endonuclease” means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which are used for gene targeting efforts. The synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALE or CRISPR recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented.

Restriction endonucleases (also called restriction enzymes) as referred to herein in accordance with the present invention are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides. In contrast, some endonucleases such as for example Fokl comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. Therefore, preferably the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical. Moreover, also preferably the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease with reduced DNA binding and/or reduced catalytic activity when compared to the wildtype restriction endonuclease.

According to the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind as a homodimer to DNA regularly, the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonucleases subunits. Preferably, in accordance with the present invention the cleavage modules referred to herein have a reduced capability of forming homodimers in the absence of the DNA recognition site, thereby preventing unspecific DNA binding. Therefore, a functional homodimer is only formed upon recruitment of chimeric nucleases monomers to the specific DNA recognition sites. Preferably, the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease. The preferably palindromic DNA recognition sites of these restriction endonucleases consist of at least four or up to eight contiguous nucleotides. Preferably, the type IIP restriction endonucleases cleave the DNA within the recognition site which occurs rather frequently in the genome, or immediately adjacent thereto, and have no or a reduced star activity. The type IIP restriction endonucleases as referred to herein are preferably selected from the group consisting of: Pvull, EcoRV, BamHl, Bcnl, BfaSORF1835P, Bfil, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, CfirlOl, Ecll8kl, EcoO1091, EcoRl, EcoRll, EcoRV, EcoR1241, EcoR12411, HinPl l, Hindi, Hindlll, Hpy991, Hpyl881, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pad, PspGl, Sau3Al, Sdal, Sfil, SgrAl, Thai, VvuYORF266P, Ddel, Eco571, Haelll, Hhall, Hindll, and Ndel. Other nuclease for use in the present invention are disclosed in WO 2010/079430, WO2011072246, W02013045480, Mussolino C, et al (Curr Opin Biotechnol. 2012 Oct;23(5):644-50) and Papaioannou I. et al (Expert Opinion on Biological Therapy, March 2012, Vol. 12, No. 3 : 329-342) all of which are herein incorporated by reference.

Ribozymes can also function as inhibitors of Interleukin-6 gene 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 endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Interleukin-6 mRNA sequences are thereby useful within the scope of the present invention. Specific 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. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Antisense oligonucleotides, siRNAs and ribozymes useful as inhibitors of Interleukin-6 gene 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 and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA or ribozyme nucleic acid to the cells and preferably cells expressing IL6. Preferably, 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. In general, 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 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 rouse 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. One can readily employ other vectors not named but known to the art.

Preferred 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). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, 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. In addition, 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.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et ah, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 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. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, 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. It may also be administered into the epidermis or a mucosal surface using a gene-gun. 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 mi croencap sul ati on .

• IL6 receptor polypeptide

In one embodiment, the IL6 antagonist is an isolated IL6 receptor polypeptide such as CD126 or CD130 (also called gpl30) Soluble gpl30-Fc fusion protein).

As used herein, the term "IL6 receptor polypeptide" refers to a polypeptide that specifically bind to IL6 can be used as IL6 antagonists that bind to and sequester the IL6 protein (IL6 Trap) , thereby preventing it from signalling.

In a particular embodiment, the IL6 receptor polypeptide is soluble. A soluble IL6 receptor polypeptide exerts an inhibitory effect on the biological activity of the IL6 protein by binding to the protein, thereby preventing it from binding to IL6 receptor present on the surface of target cells. It is undesirable for an IL6 receptor polypeptide not to become associated with the cell membrane. In a preferred embodiment, the soluble IL6 receptor polypeptide lacks any amino acid sequences corresponding to the transmembrane and intracellular domains from the IL6 receptor from which it is derived.

In a preferred embodiment, said polypeptide is a soluble IL6 receptor (s IL6 receptor) or a functional equivalent thereof.

The terms "soluble IL6 receptor" or "sIL6 receptor", as used herein, refer to a polypeptide comprising or consisting of the extracellular region of the IL6 receptor or a fragment thereof. For example, sIL6 receptor, particularly CD 130, may include all the extracellular domain of human CD130, polypeptides (i.e. a polypeptide comprising or consisting of the amino acid sequence of human CD130 polypeptide (interleukin-6 receptor subunit beta isoform 1 precursor : NM_002184 NP_002175).

A "functional equivalent of sIL6 receptor" is a molecule which is capable of binding to IL6, preferably which is capable of specifically binding to IL6 such as CD130. The term "functional equivalent" includes fragments and variants of sIL6 receptor as above described. As used herein, "binding specifically" means that the biologically active fragment has high affinity for IL6 but not for control proteins. Specific binding may be measured by a number of techniques such as ELISA, flow cytometry, western blotting, or immunoprecipitation. Preferably, the functionally equivalent specifically binds to IL6 at nanomolar or picomolar levels.

By "biological activity" of a functional equivalent of the extracellular region of the IL6 receptor such as CD130 is meant i) the capacity to bind to IL6; and/or (ii) the capacity to inhibit inflammation and/or (iii) the capacity to reduce the amount of granuloma.

The skilled in the art can easily determine whether a functional equivalent of the extracellular region of the IL6 receptor is biologically active. To check whether the newly generated polypeptides (i) bind to IL6 and/or (ii) to inhibit inflammation, a binding assay, a inflammation assay (ie. inflammation linked to IL6 can be measured with C-reactive protein (CRP) blood tests) may be performed with each polypeptide.

Thus, the polypeptide according to the invention encompasses polypeptides comprising or consisting of fragments of the extracellular region of the IL6 receptor, provided the fragments are biologically active. In the frame of the invention, the biologically active fragment may for example comprise at least 15, 20, 25, 50, 75, 100, 150 or 200 consecutive amino acids of the extracellular region of the IL6 receptor (such as CD126 or CD130).

In preferred embodiments, IL6 antagonists comprise part of the extracellular domain of an IL-6 receptor, such as CD126 or CD130, e.g., human CD126 or CD130. More specifically, such IL6 antagonists can be polypeptides comprising the IL6-binding domain, such as CD126 or CD130. Without being bound by theory, such IL6-binding domain comprising polypeptides sequester IL6 and thereby prevent IL6 signaling. These IL6-binding domain comprising polypeptides may comprise all or a portion of the extracellular domain of an IL6 receptor (i.e., all or a portion of the extracellular domain of CD 126 or CD 130). In specific embodiments, the extracellular domain of an IL-6 receptor is soluble.

In certain embodiments, the IL-6-binding, extracellular domain of an Il-6receptor is mutated relative to the wild-type receptor such that the IL6-binding, extracellular domain of an IL-6receptor binds with higher affinity to IL-6. In particular, the IL-6-binding, extracellular domain of an IL-6 receptor is mutated relative to the wild-type receptor such that the IL-6- binding, extracellular domain of an IL-6 receptor binds with higher affinity to IL6. Such higher affinity can be at least 10%, 25%, 50%, 75%, 100%, 250%, 500%, or 1000% higher than the affinity to the next highest affinity ligand.

In certain embodiments, the IL6-binding domain comprising polypeptides are linked to an Fc portion of an antibody (i.e., a conjugate comprising an activin-binding domain comprising polypeptide of an IL-6 receptor and an Fc portion of an antibody is generated).

Without being bound by theory, the antibody portion confers increased stability on the conjugate and/or reduces the patient's immune response against the IL6 antagonist. In certain embodiments, the IL6-binding domain is linked to an Fc portion of an antibody via a linker, e.g., a peptide linker.

In one embodiment, the IL6 antagonist may be an IL-6 receptor polypeptide such as the following compounds: olamkicept (FE 999301, FE301, TJ301/ Ferring Pharmaceuticals) (Soluble gpl30-Fc fusion protein). A splice variant of the cDNA encoding a soluble form of gpl30 has been found expressed in blastocysts (Sharkey et ak, (1995). Biol Reprod Oct;53(4):974-81). Such variant lacks the intracellular signaling domain. Olamkicept is a fusion molecules (Soluble gpl30-Fc fusion protein) comprising two of such sgpl30 variants

Olamkicept (Soluble gpl30-Fc fusion protein) is described, for example, in. EP1148065, Atreya R, et ak. (2000) Nat. Med., 6 (5): 583-8., Kallen KJ. (2002) Biochim. Biophys. Acta, 1592 (3): 323-43, Rose-John S. (2017) . Clin Pharmacol Ther. 2017 Oct; 102(4) : 591 -598

Alternatively, a nucleic acid encoding a polypeptide of the invention (such as sIL6 receptor) or a vector comprising such nucleic acid or a host cell comprising such expression vector may be used in the prevention or treatment of a malignant haematological disease. Nucleic acids of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination(s).

Expression vectors of the invention are well known in the art (since they are easily constructed using conventional methods or are commercially available) and are disclosed below (see the section "Inhibitors of IL6 gene expression").

In another particular embodiment, the polypeptide is a IL6 receptor fusion protein.

As used herein, "IL6 receptor fusion protein" means a protein comprising a soluble IL6 receptor polypeptide fused to a heterologous polypeptide (i.e. polypeptide derived from an unrelated protein, for example, from an immunoglobulin protein).

As used herein, the terms "fused" and "fusion" are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence.

As used herein, the term "fusion protein" means a protein comprising a first polypeptide linearly connected, via peptide bonds, to a second, polypeptide.

As used herein, the term "IL6 receptor fusion protein" refers to a polypeptide comprising the extracellular region of the IL6 receptor or a fragment thereof fused to heterologous polypeptide. The IL6 receptor fusion protein will generally share at least one biological property in common with s IL6 receptor (as described above).

An example of an IL6 receptor fusion protein is a IL6 receptor immunoadhesin.

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

The term "IL6 receptor immunoadhesin" is used interchangeably with the term " IL6 receptor- 1 -immunoglobulin chimera", and refers to a chimeric molecule that combines at least a fragment of an IL6 receptor molecule (native or variant) with an immunoglobulin sequence. For instance, the IL6 receptor immunoadhesin comprises the extracellular domain (ECD) of IL6 receptor or a fragment thereof sufficient to bind to IL6.

The immunoglobulin sequence preferably, but not necessarily, is an immunoglobulin constant domain (Fc region). Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to the patient, and are safe for chronic or repeated use. In one embodiment, the Fc region is a native sequence Fc region. In another embodiment, the Fc region is a variant Fc region. In still another embodiment, the Fc region is a functional Fc region. The IL6 receptor portion and the immunoglobulin sequence portion of the IL6 receptor immunoadhesin may be linked by a minimal linker. The immunoglobulin sequence preferably, but not necessarily, is an immunoglobulin constant domain. The immunoglobulin moiety in the chimeras of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgGl or IgG3.

As used herein, the term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.

A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc.

A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGi Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of a sIL6 receptor or functional equivalents thereof, or a IL6 receptor fusion protein such as a IL6 receptor immunoadhesin for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

Another object of the invention relates to a method for treating sarcoidosis in a patient comprising administering a subject in need thereof with a therapeutically effective amount of a IL-6 antagonist as described above.

By a "therapeutically effective amount" of a IL-6 antagonist as above described is meant a sufficient amount of the antagonist to prevent or treat sarcoidosis disease. It will be understood, however, that 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 subject 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 subject; 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. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, 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 subject 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 10 mg/kg of body weight per day.

• Treatment of sarcoidosis in a patient having a high level of IL6

The inventors discovered that Interleukin-6 (IL-6) is an excellent and specific biomarker of the severity and rate of disease progression of sarcoidosis in humans and a therapeutic target to treat sarcoidosis, especially in patient having a high level of IL6 in a biological sample, especially Cerebro-spinal fluid (CRF) regarding neurosarcoidosis (see experimental data).

Accordingly in one embodiment, the invention provides an Interleukin-6 (IL-6) antagonist, for use in the treatment of sarcoidosis in a patient having a high level of IL6 in a biological sample.

In one embodiment, the invention provides an Interleukin-6 (IL-6) antagonist, for use in the treatment of sarcoidosis in a patient having a high level of IL6 in a biological sample, whereas said treatment comprising a prior step of measuring the level of Interleukin-6 in a biological sample from said patient and a step of comparing the level of Interleukin-6 with a control reference value. In another aspect the invention provides a pharmaceutical composition, comprising an antagonist of IL6R, for use in the prevention or the treatment of sarcoidosis in a patient, especially in a patient having a high level of IL-6 in a biological sample.

As used herein, the term“biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a patient, as well as tissues, cells and fluids present within a patient. Accordingly he term“biological sample” refers to any sample obtained from a subject, such as a fluid sample or a tissue biopsy. In a specific embodiment the biological sample is a fluid sample for use in the methods of the invention, said fluid sample is for instance a blood sample, urine sample, saliva sample, cerebro-spinal fluid (CSF) sample, lymph sample or any other bodily secretion or derivative thereof.

Preferably if the pathology is neurosarcoidosis, the fluid sample is cerebro-spinal fluid sample.

The term“patient” refers to a human being suffering from sarcoidosis. Preferably, the patient suffers of neurosarcoidosis.

The inventors observed that IL-6 concentration was directly correlated with sarcoidosis and severity of the disease. As such, an increased IL-6 concentration in the fluid sample of the patient compared to a corresponding control value is positively correlated with the disease severity and/or the rate of disease progression in said patient.

The control value may be a value obtained my measurement of the IL-6 concentration in a fluid sample from the patient at an earlier time point or a reference control value.

Typically a reference control value can be a mean value obtained from a mean population of healthy subjects (i.e. : subjects who are not suffering from sarcoidosis), or who are suffering from another inflammatory disease (ie multiple sclerosis).

The method for assessing the disease severity and/or the rate of disease progression in a patient suffering from sarcoidosis as per the present invention typically include the steps of (a) measuring the concentration of IL-6 in a biological sample from said patient to obtain concentration value(s), and (b) comparing the obtained concentration value of said IL-6 biomarker to corresponding control values, wherein the difference in the concentration value(s) compared to the respective control value is indicative of the disease severity and/or the rate of disease progression.

As used herein, the term“decrease” or“increase” means a statistically significant decrease or increase of a control value, preferably, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 90%, or at least 99% decrease or increase of the control value. The quantification may be relative (by comparing the amount of the biomarker to a control with known amount of biomarker for example and detecting“higher” or“lower” amount compared to that control) or more precise (i.e. : quantitative), at least to determine the specific amount relative to a known control amount (i.e.: to determine the difference between the concentration value and the control value).

These quantification assays of a biomarker can be conducted in a variety of ways. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

The level of the IL-6 may be determined by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction such as immunohistochemistry, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

For example, determination of the IL-6 level can be performed by a variety of techniques and method any well known method in the art: RIA kits (DiaSorin; IDS, Diasource) Elisa kits (IDS (manual) IDS (adapted on open analyzers) Immunochemiluminescent automated methods (DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (Janssen MJ, Steroids, nov 2012).

Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of IL-6 in fluid samples previously collected from the patient under testing.

A“control reference value” can be a“threshold value” or a“cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the IL-6 levels with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the IL-6 level (or ratio, or score) determined in a fluid sample derived from one or more subjects who are responders to sarcoidosis disease treatment. In one embodiment of the present invention, the threshold value may also be derived from IL-6 level (or ratio, or score) determined in a blood sample derived from one or more subjects who are non-responders to sarcoidosis disease treatment. Furthermore, retrospective measurement of the IL-6 levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of IL-6 in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER S AS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

Typically in the context of the present study, the control reference value is 20pg/ml in a CSF sample. Accordingly when the level value found for the IL-6 in the patient tested is inferior to said value it is concluded that the patient tested could be a non (or bad) responder to the treatment with IL-6 antagonist. And when the level value found for the IL-6 in the patient tested is superior to said value it is concluded that the patient tested could be of a responder to the treatment with IL-6 antagonist.

Accordingly, in some embodiment a patient having a high level of IL-6 is a patient having a level of IL-6 superior to the reference value.

In particular embodiment, the control reference value is 20 pg/ml in a CSF sample.

In one embodiment, the method of the invention thus may comprise the steps of

- (a) quantifying the concentration of IL-6 in a fluid sample of a patient, and optionally comparing the obtained value with a reference control value and determining whether the obtained value is increased, decreased or is stable with regard to the reference control value,

- (b) providing a treatment to the patient, typically when said obtained value is increased with regard to the reference control value,

- (c) quantifying again the concentration of IL-6 in a fluid sample of said patient and comparing the obtained concentration with the value prior to treatment and optionally to another corresponding control value or to the reference control value.

Typically the treatment is antagonist of IL-6 (an inhibitor of the function or of the expression of IL-6 see above).

Pharmaceutical compositions:

The IL-6 antagonist of the invention as above defined may be combined with pharmaceutically acceptable excipients, to form therapeutic compositions for use in treating sarcoidosis.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, may be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. 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.

Preferably, the pharmaceutical compositions contain 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. In all cases, 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 may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may 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 IL6 antagonist of the invention may 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 may 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 may 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 may 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 may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may 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 several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.

Upon formulation, solutions are 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 may also be employed.

For parenteral administration in an aqueous solution, for example, the solution is suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that may be employed will be known to those of skill in the art in light of the present disclosure. For example, 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 IL-6 antagonist 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 may also be administered.

In the present study, neurosarcoidosis patient were treated at the dose of 8 mg/kg every 4 weeks.

In addition to the compounds of the invention formulated for 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.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Relapse/progressi on-free survival in neurosarcoidosis patients depending on their cerebrospinal fluid interleukin-6 level.

Figure 2: Magnetic resonance imaging (T1 -weighted imaging with gadolinium) before and 3 months after tocilizumab treatment (steroid dosage was not increased). The disappearance of multiple leptomeningeal gadolinium-enhanced lesions is shown.

Figure 3: ROC curve of cerebro-spinal fluid interleukin 6 in neurosarcoidosis patients (controls: multiple sclerosis).

Figure 4: ePOST and SDAI at baseline (before the first injection of tocilizumab) and at the last visit after tocilizumab infusions. A. ePOST (extrapulmonary organ assessment tool) and B SDAI (sarcoidosis disease activity index).

EXAMPLE 1:

Material & Methods

Patient selection

The lymphocyte population counts and IL-6 and 10 levels were analyzed from backup CSF samples obtained from patients with an inflammatory CNS disorder between 2012 and 2017 in one internal medicine department and 2 neurology departments at the Pitie-Salpetriere Hospital (Paris, France). The demographic and clinical characteristics of the patients were retrospectively collected from medical records. Only patients with a definite diagnosis were included. The diagnostic criteria for neurosarcoidosis were as follows: a definite diagnosis of sarcoidosis (compatible clinical and radiological presentations, histological documentation and exclusion of other causes of granulomatous disease) and a CNS localization of sarcoidosis[f 6, 17] The diagnostic criteria for neuro-BD[f 8], optical neuromyelitis spectrum disorder (NMO- SD)[19], multiple sclerosis (MS)[20], neurolupus[2f ], and primary Sjogren syndrome (pSS)[22] were those of international standards. Patients with neurosarcoidosis were followed, and successive IL concentrations were collected when available. Outcomes of neurosarcoidosis were defined with the neurological extrapulmonary physician organ severity tool (ePOST) score, which is a 6-scale score from no activity (ePOST=0) to the highest activity (ePOST=6)

[23] Remission was defined by an ePOST=0. A relapse or progression was defined by an increase in the ePOST score of at least 1 point compared to the previous evaluation. The study was approved by the ethics committee Comite de Protection des Personnes lie de France VI, and was conducted in accordance with the Declaration of Helsinki.

Lymphocyte immunophenotyping and IL concentrations

IL-10 and IL-6 cytokines were assessed for thawed CSF samples by the quantitative Cytometric Bead Array® technique (human IL-10 CBA kit and human IL-6 CBA kit; BD BiosciencesTM, Pont de Claix, France) on a FACSCanto II flow cytometer (BD BiosciencesTM) following the manufacturer’s recommendations and with a quantification cutoff at 2.5 pg/ml. Data were analyzed with FACSDiva and FCAP software (BD BiosciencesTM). This technique is correlated with the standard ELISA, as previously described

[24] Due to the cutoff value for IL-6 and IL-10 determinations, we arbitrarily recorded patients with a value < 2.5 pg/mL as 2 pg/mL.

Immunophenotyping by flow cytometry was performed either within 1 h of lumbar puncture or the cells were stabilized using TransFix® (Caltag Medsystems, UK) to prevent cell mortality. An 8-color panel (anti-CD19, anti-kappa, anti-lambda, anti-CD5, anti-CD3, anti- CD4, anti-CD8 and anti-CD45 antibodies) was analyzed on a FACSCanto II cytometer (BD Biosciences). We determined total T lymphocytes (CD3+), T cell subsets (CD3+CD4+ and CD3+CD8+) and B cells (CD 19+) in the cerebrospinal fluid. The results were expressed as percentages of total lymphocytes, except for the CD4/CD8 ratio.

Treatments

Treatments were administered according to the physician’s decision. One patient, who had both neurosarcoidosis and MS and progressed despite cyclophosphamide treatment, was treated with tocilizumab, an anti-IL-6 receptor monoclonal antibody, at a dosage of 8 mg/kg every 4 weeks.

Statistical analyses

Continuous variables are reported as the mean (standard deviation - SD) or median (interquartile range - IQR) and were compared using a Mann-Whitney test, Kruskal-Wallis test or Wilcoxon matched-pairs signed-rank test. Categorical variables are reported as the count (percentage) and compared using Fisher’s exact test. Survival curves were built using the Kaplan-Meier method considering the time from the first IL-6 concentration measurement to relapse, progression or last follow-up. All of the tests were two-sided, and a p-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad V6.0 (GraphPad, La Jolla, CA, USA).

Data availability statement

The dataset used and analyzed during the current study is available from the corresponding author on reasonable request.

Results

Clinical characteristics

Out of 192 patients who had at least one determination of CSF lymphocyte population counts or IL concentrations, 83 were excluded because they had no definite diagnosis of CNS involvement. Thus, we analyzed the results of 47 patients with neurosarcoidosis, 14 with MS and 48 with various CNS inflammatory disorders (including 10 L-group histiocytosis [25], 8 neurolupus, 7 R-group histiocytosis [25], 6 pSS, 4 neuro-BD, and 4 tuberculosis with CNS localization patients). In the neurosarcoidosis group, 3 patients were further excluded because CNS localization was not confirmed and 1 additional patient was excluded because she had both MS and neurosarcoidosis; therefore, 43 patients were ultimately analyzed in this group. Among these patients, at the time of the first CSF lymphocyte population counts/IL concentration measurement, 26 patients had an ePOST score >0 and were considered“active neurosarcoidosis”, whereas 17 were“non-active”.

CSF lymphocyte population counts

The results of the CSF lymphocytes population counts analysis are shown in Table 1. The CD4/CD8 ratio significantly differed between the 3 groups. Moreover, the CD4/CD8 ratio was higher in neurosarcoidosis patients than in MS patients. Eleven patients had a CD4/CD8 ratio >5: 9 patients with neurosarcoidosis (8 with active and 1 with non-active disease), 1 patient with pSS and 1 patient with neuro-BD. No patient with MS had a CD4/CD8 ratio >5 (the highest ratio in this group was 3.90).

CSF CD 19 percentages were low in all groups, except in 4 patients with R-group histiocytosis. CSF CD19 percentages did not differ between the groups. We observed the presence of CD19 lymphocytes in the CSF of 6/12 (50%) MS, 19/29 (66%) neurosarcoidosis, and 15/29 (52%) other patients.

CSF IL concentrations

The CSF IL concentrations are shown in Table 1. The CSF IL-6 concentration differed between the 3 groups. The IL-6 level was higher in the neurosarcoidosis and other inflammatory disorder groups than in the MS group. Sixteen patients had an IL-6 level > 20 pg/mL: 13 patients with neurosarcoidosis and 3 patients with R-group histiocytosis. The IL-6 concentration was higher in neurosarcoidosis patients with active disease compared with those with non-active disease (Table 2). All MS patients had IL-6 levels < 20 pg/mL. The IL-10 concentration was generally low but sometimes elevated in neurosarcoidosis and other inflammatory disorders, although never in MS.

Outcomes of neurosarcoidosis

We studied the outcomes of the 43 patients with neurosarcoidosis (19 women and 24 men, mean age at the time of the first concentration measurement was 41 years - range: 19-58). The median duration of their disease since the diagnosis of neurosarcoidosis was 15 months (range: 0-141). At the time of the first CSF lymphocyte population counts and/or IL determination, 28 patients were receiving treatment for neurosarcoidosis (corticosteroids for 27 and/or immunosuppressive drugs for 19, including 8 patients treated with infliximab, an anti tumor necrosis factor (TNF)-a monoclonal antibody). The IL-6 concentration was measured repeatedly in 28 patients and decreased under treatment (median at baseline 10, at last determination 6 pg/mL, p=0.015). The patients with an IL-6 concentration at baseline > 50 pg/mL had a higher risk of relapse and worse progression-free survival than those with IL-6 < 50 pg/mL (Figure 1, p=0.0054, hazard ratio 3.60; 95% confidence interval 1.78-23.14).

Additionally, a 43-year-old woman who had a past history of multiple sclerosis received a diagnosis of multiorgan sarcoidosis with CNS involvement. She was treated with tocilizumab, an anti-IL-6 receptor monoclonal antibody, because of the progression of the neurological localization of sarcoidosis despite treatment with steroids and cyclophosphamide. Additionally, the anti-TNF-a monoclonal antibody, which can be used in refractory neurosarcoidosis, was contraindicated because of her history of multiple sclerosis. The oral prednisone daily dose was not increased, and the patient received the first infusion of tocilizumab in December 2016. After 3 administrations, her brain MRI showed a disappearance of all gadolinium-enhanced lesions (Figure 2). The treatment was maintained until July 2018 without side effects. Her prednisone dosage was tapered to 5 milligrams/day.

Discussion

In this study, we report the results of CSF biomarkers in neurosarcoidosis, MS and other inflammatory CNS disorders. We showed that the CD4/CD8 ratio and IL-6 levels were significantly higher in sarcoidosis than MS. We also showed that the IL-6 concentration was significantly higher in neurosarcoidosis patients with active disease compared to those with non-active disease. A high CSF IL-6 concentration (>50 pg/mL) was associated with a shorter time to relapse or progression of neurosarcoidosis. Moreover, a patient with refractory neurosarcoidosis improved after treatment with tocilizumab, which is an anti-IL-6 monoclonal antibody.

CSF CD4/CD8 ratio as a diagnostic marker in neurosarcoidosis

Neurosarcoidosis is challenging to diagnosis and can mimic a broad range of inflammatory diseases. Various CSF biomarkers, e.g., CSF angiotensin conversion enzyme (ACE), have been studied for diagnostic and prognostic purposes but yield low reliability [26] In our study, a CD4/CD8 ratio >5 was highly suggestive of neurosarcoidosis (9 out of 11 patients) and was never observed in MS patients. Moreover, a CSF CD4/CD8 ratio>5 was suggestive of active neurosarcoidosis (8 patients out of 9). The CD4/CD8 ratio has been previously evaluated in the lung for pulmonary manifestations of sarcoidosis[27], in the aqueous humor of sarcoid uveitis patients [28, 29], and in the CSF in a small study of neurosarcoidosis and yielded variable reliability [30] In our larger study, we demonstrated that CSF CD4/CD8 ratio could be useful for diagnostic and prognostic purposes in neurosarcoidosis patients.

CSF IL-6 level as a prognostic marker in neurosarcoidosis

The CSF IL-6 concentration was elevated in neurosarcoidosis and other inflammatory disorders. In neurosarcoidosis patients, the CSF IL-6 concentration significantly decreased with treatment. Moreover, a CSF IL-6 concentration > 50 pg/mL was associated with a higher risk of relapse and progression. IL-6 expression has been found to be upregulated in granulomas [31] Moreover, IL-6 is essential for the differentiation of Thl7 cells, an IL-17-producing helper CD4+ T cell subset that is involved in sarcoidosis pathogenesis [32] The CSF IL-6 concentration may be elevated in other inflammatory disorders. In neurosarcoidosis, an elevated CSF IL-6 level seems to be associated with a higher risk of relapse and worse progression-free survival.

Targeting IL-6 in neurosarcoidosis

Neurosarcoidosis is a challenging condition. Glucocorticoids are the cornerstone of treatment for neurosarcoidosis, but they have cumulative toxicity. Immunosuppressive drugs have been used with variable efficacy [5] Infliximab, a chimeric monoclonal antibody directed against tumor necrosis factor-a, has emerged as a therapeutic option [33, 34] However, infliximab may have serious adverse effects, and patients have high relapse rates (36-50%) after treatment interruptions. Thus, there is a need for new therapeutic options in neurosarcoidosis. Here, we found that tocilizumab, an anti-IL-6 receptor monoclonal antibody, which was used without increasing the daily steroid dose, was efficacious for treating one neurosarcoidosis patient for whom infliximab was contraindicated. Tocilizumab has been used to treat uveitis, an inflammatory condition that shares pathogenic mechanisms with sarcoidosis [35] Tocilizumab should probably be investigated in patients with sarcoidosis who are refractory to conventional therapy.

Limitations

Our study has several limitations. The clinical characteristics of the patients were assessed retrospectively. However, this allowed definite diagnoses, since in the setting of neuroinflammatory disorders, the diagnoses might be modified after several months of evolution. The number of samples did not allow multivariate analyses.

In conclusion, in this multicenter comparative study, we showed that the CSF lymphocyte population counts and IL-6/IL-10 concentrations were useful diagnostic and prognostic markers. Moreover, IL-6 was found to be an interesting target for the treatment of sarcoidosis especially CNS localization of sarcoidosis (neurosarcoidosis).

NeuroMultiple

Others

sarcoidosis sclerosis

n=48

n=43 n=13 n=44

IL-10 (pg/mL)

2.50 (2.00- 2.00 (2.00- 0.043* 2.50 (2.00- 0.009* median (range)

132.00) 2.50) 436.00)

n=29 n=12

CD19 median n=29

1.00 (0.00- 1.00 (0.00- 0.80 0.89 (range) 2.00 (0.00-70)

15 00) 12.00)

Table 1. Cerebrospinal fluid lymphocyte phenotypes and interleukin (IL) dosages

* statistically significant ** comparison between neurosarcoidosis and multiple sclerosis patients *** comparison between neurosarcoidosis, multiple sclerosis, and other patients

ePost>0 ePost=0 P

n=21 n=8

CD4/CD8 0.12

3.60 (1 34-13 83) 2.54 (1.49-5.21)

Table 2. Cerebrospinal fluid CD4/CD8 ratio and interleukin-6 level in neurosarcoidosis patients.

Comparison between active (ePost>0) and inactive (ePost=0) patients. EXAMPLE 2:

Material & Methods

In this retrospective and multicenter study, patients were included if they met the following criteria: sarcoidosis documented histologically according to the ATS/WASOG criteria, and administration of at least one biotherapy infusion excluding anti -TNF -alpha. The non-selection criteria were: infusion performed for an indication other than sarcoidosis, data not sufficient. The evaluation of the effectiveness was carried out by the ePOST and SDAI scores. Tolerance data was collected from medical records.

Results

A total of 6 sarcoidosis patients were treated with tocilizumab, an IL-6 antagonist, with a median number of injections of 4 (1-27). The indication of treatment was central nervous system involvement in 4 (3 of them had also uveitis), and multisystemic disease with skin and articular involvement in 1, and lung, ear-nose-throat, skin, and articular in the other 1. During the treatment with tocilizumab, 4 patients also received corticosteroids, 4 had methotrexate, and 1 had azathioprine. Tolerance was generally good with no serious adverse events. All over, 3 patients had partial response, 2 were stable (including 1 who received only 1 injection), and 1 had a progression of the disease. The evaluation scores are displayed in the Figures 4A and 4B.

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Claims

CLAIMS:

1. An Interleukine 6 (IL-6) antagonist for use in treatment of sarcoidosis.

2. The IL-6 antagonist for use according to Claim 1, wherein sarcoidosis is neurosarcoidosis.

3. The IL-6 antagonist for use according to Claim 1 or 2, wherein sarcoidosis is a refractory sarcoidosis.

4. The IL-6 antagonist for use according to Claim 1 or 3, for use in a patient having a high level of IL-6 in a biological sample.

5. The IL-6 antagonist for use according to any one of Claims 1 to 4, wherein the IL-6 antagonist is selected from the group consisting of: i. IL-6 antibody; ii. IL-6 receptor antibody; iii. IL-6R antagonists small molecule; iv. Inhibitor of Interleukin-6 gene expression such as siRNA, an antisense oligonucleotide, a nuclease or a ribozyme; v. IL-6 receptor polypeptide.

6. The IL-6 antagonist for use according to any one of Claim 1 to 5, wherein the IL-6 antagonist is an antibody selected from the list consisting of IL-6 antibody or IL-6 receptor antibody.

7. The IL-6 antagonist for use according to Claim 6, wherein

IL-6 antibody is selected from the list consisting of siltuximab, olokizumab, sirukumab, elsilimomab, clazakizumab, gerilimzumab VX30, EB-007 or FMlOl;

IL-6 receptor antibody is selected from the list consisting of tocilizumab, sarilumab vobarilizumab.

8. The IL-6 antagonist for use according to Claim 6, wherein the IL-6 receptor antibody is tocilizumab.

9. A pharmaceutical composition, comprising an IL-6 antagonist according to any one of Claims 1-8, for use in treatment of sarcoidosis.

10. The pharmaceutical composition for use according of Claim 9, wherein sarcoidosis is neurosarcoidosis.

11. The pharmaceutical composition for use according to Claim 9 to 10 wherein sarcoidosis is a refractory sarcoidosis.

12. The pharmaceutical composition for use according to any one of Claim 9 to 11, wherein the pharmaceutical composition is for use in a patient having a high level of IL-6 in fluid biopsy.

13. The pharmaceutical composition for use according to any one of Claim 9 to 12, wherein the IL-6 antagonist is selected from the group consisting of : i. IL-6 antibody such as siltuximab, olokizumab, sirukumab, elsilimomab, clazakizumab, gerilimzumab VX30, EB-007 or FM101; ii. IL-6 receptor antibody such as tocilizumab, sarilumab vobarilizumab; iii. IL-6R antagonists small molecule such as LMT-28 compound; iv. Inhibitor of Interleukin-6 gene expression such as siRNA, an antisense oligonucleotide, a nuclease or a ribozyme; v. IL-6 receptor polypeptide such as olamkicept (Soluble gpl30-Fc fusion protein).

14. The pharmaceutical composition for use according to Claim 13, wherein the IL-6 antagonist is an antibody selected from the list consisting of IL-6 antibody or IL-6 receptor antibody

15. The pharmaceutical composition for use according to Claim 14, wherein the IL-6 receptor antibody is tocilizumab.

16. A method for treating sarcoidosis in a patient comprising administering a subj ect in need thereof with a therapeutically effective amount of a IL-6 antagonist

17. A method for treating sarcoidosis according to claim 16, wherein the patient have a high level of IL6 in a biological sample, whereas said method for treating comprises a prior step of measuring the level of Interleukin-6 in a biological sample from said patient and a step of comparing the level of Interleukin-6 with a control reference value.