WO2015116852A1 - Methods for treating rheumatoid arthritis by administering an il-6r antibody - Google Patents

Methods for treating rheumatoid arthritis by administering an il-6r antibody Download PDF

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Publication number
WO2015116852A1
WO2015116852A1 PCT/US2015/013588 US2015013588W WO2015116852A1 WO 2015116852 A1 WO2015116852 A1 WO 2015116852A1 US 2015013588 W US2015013588 W US 2015013588W WO 2015116852 A1 WO2015116852 A1 WO 2015116852A1
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subject
antibody
administration
level
pharmaceutical composition
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PCT/US2015/013588
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French (fr)
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WO2015116852A8 (en
Inventor
Anita BOYAPATI
Jennifer Davidson HAMILTON
Jerôme MSIHID
Emmanuelle Cousin
Ling CAI
Stefano FLORE
Janet VAN ADELSBERG
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Regeneron Pharmaceuticals, Inc.
Sanofi Biotechnology
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Publication of WO2015116852A1 publication Critical patent/WO2015116852A1/en
Publication of WO2015116852A8 publication Critical patent/WO2015116852A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the treatment and/or prevention of rheumatoid arthritis and related conditions. More specifically, the invention relates to the administration of interleukin-6 receptor (IL-6R) antagonists to treat or prevent rheumatoid arthritis in a patient in need thereof.
  • IL-6R interleukin-6 receptor
  • Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation of synovial tissue, leading to destruction of the joint architecture. It is recognized that cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1 ) and interleukin-6 (IL-6) play a role in joint inflammation and cartilage damage observed in RA.
  • IL-6 is a pleiotropic cytokine with biological effects on many cell types. IL-6 is often regarded as being downstream of TNF or IL-1 in inflammatory cytokine cascades and may therefore represent a common pathway factor in a wide range of inflammatory processes. Blockade of IL-6 signaling therefore offers the potential to ameliorate multiple pathogenic features of RA and other inflammatory diseases.
  • anti-IL-6R antibodies are described in US 7,582,298; 6,410,691 ; 5,817,790; 5,795,695; 6,670,373; and 7,582,298.
  • RA rheumatoid arthritis
  • the methods of the present invention comprise administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an interleukin-6 receptor (IL-6R) antagonist.
  • IL-6R interleukin-6 receptor
  • the IL- 6R antagonist is an antibody or antigen-binding fragment thereof that specifically binds IL-6R.
  • Exemplary anti-IL-6R antibodies that can be used in the context of the methods of the present invention are described elsewhere herein, including working Example 1.
  • the IL-6R antagonist is an anti-IL-6R antibody having the binding characteristics of the reference antibody referred to herein as "mAbl " (e.g., an antibody or antigen-binding fragment thereof comprising the complementarity determining regions of mAbl ).
  • the present invention provides methods for treating RA in a subject, the methods comprising: (a) selecting a subject who exhibits an elevated level of at least one RA-associated biomarker; and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist.
  • methods for treating RA are provided which comprise administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist, wherein administration of the pharmaceutical composition to the subject results in a decrease in at least one RA -associated biomarker in the subject.
  • RA-associated biomarkers that can be evaluated and/or measured in the context of the present invention include, but are not limited to, matrix metalloprotease (MMP) cleaved collagen type 1 (C1 M), collagen type 2 (C2M), collagen type 3 (C3M), C reactive protein (CRPM), RANKL, and OPG.
  • MMP matrix metalloprotease
  • the present invention also includes an interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits an elevated level of at least one RA-associated biomarker prior to or at the time of treatment.
  • the interleukin-6 receptor (IL-6R) antagonist is used at a dose of about 75 mg to about 300 mg. In one aspect of the invention, the interleukin-6 receptor (IL-6R) antagonist is used once every other week.
  • the present invention also provides methods for decreasing the level of one or more RA-associated biomarker(s) in a subject, or improving one or more RA-associated parameter(s) in a subject, wherein the methods comprise sequentially administering to a subject in need thereof a single initial dose of a pharmaceutical composition comprising an IL-6R antagonist, followed by one or more secondary doses of the pharmaceutical composition comprising the IL- 6R antagonist.
  • the present invention provides methods for decreasing the level of one or more RA-associated biomarker(s) in a subject, or improving one or more RA-associated parameter(s) in a subject, wherein the methods comprise administering to the subject about 75 mg to about 300 mg of a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds IL-6R.
  • the pharmaceutical composition may be administered to the subject at a dosing frequency of, e.g., once every other week.
  • the present invention also includes an IL-6R antagonist as disclosed herein for use in treating or preventing RA, for improving an RA-associated parameter, for decreasing the level of at least one RA-associated biomarker, and/or for treating any of the other indications or conditions disclosed herein.
  • the present invention provides methods for treating RA in a subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein the subject has been diagnosed with RA and has also been selected for treatment on the basis of the subject exhibiting a reduced level of a RA-associated biomarker before treatment, as compared to a reference level of the biomarker (e.g., expression of the biomarker in a subset of subjects diagosed with RA and/or expression of the biomarker in healthy subjects).
  • IL-6R interleukin-6 receptor
  • the present invention also provides methods for treating RA by administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein the subject has been diagnosed with rheumatoid arthritis, has already been treated with the anti-IL6R antibody for a defined period of time, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker (e.g., OPG) after treatment for the defined period of time (e.g., 24 weeks), wherein the reduced expression of the biomarker is determined based on a comparison to the level of expression of the respective biomarker in the subject prior to treatment with the anti-IL-6R antibody (e.g., a reduction of OPG expression that is equal to or greater than about a 9% reduction in expression as compared to pre-treatment expression levels).
  • a biomarker e.g., OPG
  • the present invention also includes an interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits a lower level of at least one RA-associated biomarker prior to or at the time of treatment (e.g., as compared to a population of RA patients or a subset of the RA patient population), as compared to a reference level of the biomarker.
  • IL-6R interleukin-6 receptor
  • the present invention also includes an interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits a lower level of at least one RA-associated biomarker after treatment with the IL-6R antagonist for a defined period of time (e.g., 24 weeks) as compared to the level of the biomarker prior to treatment.
  • the lower level of at least one RA-associated biomarker after treatment with the IL-6R antagonist for a defined period of time is equal to or greater than about a 9% reduction in the exhibition of the biomarker as compared to pre-treatment levels.
  • the interleukin-6 receptor (IL- 6R) antagonist is used at a dose of about 75 mg to about 300 mg.
  • the interleukin-6 receptor (IL-6R) antagonist is used once every other week.
  • the present invention also includes methods involving the use, quantification, and analysis of RA-associated biomarkers.
  • RA-associated biomarker means any biological response, cell type, parameter, protein, polypeptide, enzyme, enzyme activity, metabolite, nucleic acid, carbohydrate, or other biomolecule which is present or detectable in an RA patient at a level or amount that is different from (e.g., greater than or less than) the level or amount of the marker present or detectable in a non-RA patient.
  • Exemplary RA-associated biomarkers include, but are not limited to, e.g., C1 M (matrix metalloprotease (MMP) cleaved collagen type 1 ), C2M (MMP cleaved collagen type 2), C3M (MMP cleaved collagen type 3), CRPM (MMP cleaved C reactive protein), MMP-3 (matrix metalloproteinase-3), CTX-1 (carboxy-terminal collagen crosslinks/C-terminal telopeptide I), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), and OC (osteocalcin).
  • MMP matrix metalloprotease
  • kits for measuring such RA-associated biomarkers are available from various commercial sources; and various commercial diagnostic laboratories offer services which provide
  • methods for treating RA comprise: (a) selecting a subject who exhibits a level of at least one RA-associated biomarker prior to or at the time of treatment which signifies the disease state, and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist.
  • the subject is selected on the basis of an elevated level of C1 M, C2M, C3M, or CRPM.
  • methods for treating RA comprise administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist, wherein administration of the pharmaceutical composition to the subject results in a decrease in at least one RA-associated biomarker (e.g., C1 M, C2M, C3M, CRPM, etc.) at a time after administration of the pharmaceutical composition, as compared to the level of the biomarker in the subject prior to the administration.
  • RA-associated biomarker e.g., C1 M, C2M, C3M, CRPM, etc.
  • an increase or decrease in an RA-associated biomarker can be determined by comparing (i) the level of the biomarker measured in a subject at a defined time point after administration of the pharmaceutical composition comprising an IL-6R antagonist to (ii) the level of the biomarker measured in the patient prior to the administration of the pharmaceutical composition comprising an IL-6R antagonist (i.e., the "baseline measurement").
  • the defined time point at which the biomarker is measured can be, e.g., at about 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 15 days, 20 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 84 days, 85 days, or more after administration of the of the pharmaceutical composition comprising an IL-6R antagonist.
  • a subject may exhibit a decrease in the level of one or more of C1 M, C2M, C3M, and/or CRPM following administration of a pharmaceutical composition comprising an IL-6R antagonist (e.g., an anti-IL- 6R antibody).
  • an IL-6R antagonist e.g., an anti-IL- 6R antibody
  • a first, second, third or fourth dose of a pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl )
  • the subject may exhibit a decrease in C1 M of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline” is defined as the level of C1 M in the subject just prior to the first administration).
  • a first, second, third or fourth dose of a pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl )
  • the subject may exhibit a decrease in C2M of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline” is defined as the level of C2M in the subject just prior to the first administration).
  • the subject at about day 4, day 8, day 14, day 15, day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day 71 , day 84 or day 85, following administration of a first, second, third or fourth dose of a pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl ), the subject, according to the present invention, may exhibit a decrease in C3M of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline” is defined as the level of C3M in the subject just prior to the first administration).
  • baseline is defined as the level of C3M in the subject just prior to the first administration.
  • composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl ), the subject, according to the present invention, may exhibit a decrease in CRPM of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline” is defined as the level of CRPM in the subject just prior to the first administration).
  • baseline is defined as the level of CRPM in the subject just prior to the first administration.
  • the present invention also includes methods for determining whether a subject is a suitable subject for whom administration of a pharmaceutical composition comprising an IL-6R antagonist would be beneficial. For example, if an individual, prior to receiving a pharmaceutical composition comprising an IL-6R antagonist, exhibits a level of an RA-associated biomarker which signifies the disease state, the individual is therefore identified as a suitable patient for whom administration of a pharmaceutical composition of the invention (a composition
  • an individual may be identified as a good candidate for anti-IL-6R therapy if the individual exhibits one or more of the following: (i) a C1 M level greater than about 180 ng/mL, greater than about 190 ng/mL, greater than about 200 ng/mL, greater than about 210 ng/mL, greater than about 220 ng/mL, greater than about 230 ng/mL, greater than about 240 ng/mL, greater than about 250 ng/mL, greater than about 300 ng/mL, greater than about 350 ng/mL, greater than about 400 ng/mL, greater than about 450 ng/mL, or greater than about 500 ng/mL; (ii) a C2M level greater than about 0.25 ng/mL, greater than about 0.26 ng/mL, greater than about 0.26 ng/mL, greater than about 0.27 ng/mL, greater than about
  • RA erythrocyte sedimentation rate
  • hsCRP high-sensitivity C-reactive protein
  • SAA serum amyloid A
  • ESR erythrocyte sedimentation rate
  • serum hepcidin serum hepcidin
  • biomarker levels and/or changes in biomarker levels with treatment may have predictive value for efficacy of anti-IL6R therapy with particular anti- IL6R agents.
  • RA patients having higher baseline levels of OPG before therapy and/or smaller reductions in OPG levels after 24 weeks of treatment of RA using a particular anti-IL6R therapeutic i.e., sarilumab
  • were found to have worse treatment outcomes in terms of radiographic progression e.g., modified Total Sharp Score and erosion score
  • additional aspects of the invention are methods of treating rheumatoid arthritis in a subject comprising administration of an anti-IL6R antibody (e.g., sarilumab), wherein the subject has been diagnosed with rheumatoid arthritis and has been selected for treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker (e.g., OPG) pre- treatment, wherein the reduced expression of the biomarker is determined based on a comparison to a reference level of expression of the respective biomarker.
  • an anti-IL6R antibody e.g., sarilumab
  • a biomarker e.g., OPG
  • An additional aspect of the invention features methods of treating rheumatoid arthritis in a subject comprising administration of an anti-IL6R antibody (e.g., sarilumab), wherein the subject has been diagnosed with rheumatoid arthritis, has been treated with the anti-IL6R antibody, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker (e.g., OPG), wherein the reduced expression of the biomarker is determined based on a comparison to the level of expression of the respective biomarker in the subject prior to treatment with the anti-IL-6R antibody.
  • an anti-IL6R antibody e.g., sarilumab
  • a biomarker e.g., OPG
  • a particular aspect of the invention features methods of treating rheumatoid arthritis in a subject comprising administration of an anti-IL6R antibody (e.g., sarilumab), wherein the subject has been diagnosed with rheumatoid arthritis, has been treated with the anti-IL6R antibody, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of the OPG biomarker, wherein the reduced expression of the OPG biomarker in comparison to pre- treatment expression levels is equal to or greater than a 9% reduction in OPG expression.
  • an anti-IL6R antibody e.g., sarilumab
  • the present invention includes methods which comprise administering to a subject in need thereof a therapeutic composition comprising an interleukin-6 receptor (IL-6R) antagonist.
  • an "IL-6R antagonist” is any agent which binds to or interacts with IL-6R and inhibits the normal biological signaling function of IL-6R when IL-6R is expressed on a cell in vitro or in vivo.
  • Non-limiting examples of categories of IL-6R antagonists include small molecule IL-6R antagonists, anti-IL-6R aptamers, peptide-based IL-6R antagonists (e.g., "peptibody” molecules), and antibodies or antigen-binding fragments of antibodies that specifically bind human IL-6R.
  • the present invention includes methods that comprise administering to a patient a human antibody, or an antigen-binding fragment thereof, that binds specifically to hlL-6R.
  • hlL-6R means a human cytokine receptor that specifically binds human interleukin-6 (IL-6).
  • the antibody that is administered to the patient binds specifically to the extracellular domain of hlL-6R.
  • the extracellular domain of hlL-6R is shown in the amino acid sequence of SEQ ID NO:1
  • antibody is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, C H 1 , C H 2 and C H 3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region.
  • the light chain constant region comprises one domain (C L 1 ).
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed
  • each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the anti-IL- 6R antibody may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
  • SMIPs small modular immunopharmaceuticals
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the V H and V L domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain V H -V H , V H -V L or V L -V L dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen- binding fragment of an antibody of the present invention include: (i) V H -C H 1 ; (ii) V H -C H 2; (iii) V H - C H 3; (iv) VH-CH1 -C h 2; (V) V H -CH1 -C h 2-CH3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1 ; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1 -C H 2; (xii) V L -C h 1 -C h 2-CH3; (xiii) V L -C H 2-C
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V H or V L domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format may be adapted for use in the context of an antigen- binding fragment of an antibody of the present invention using routine techniques available in the art.
  • the constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity.
  • the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term "human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • an immunoglobulin molecule comprises a stable four chain construct of
  • the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular
  • the instant invention encompasses antibodies having one or more mutations in the hinge, C H 2 or C H 3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
  • an "isolated antibody,” as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment.
  • an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced is an “isolated antibody” for purposes of the present invention.
  • An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • the term "specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions.
  • Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • an antibody that "specifically binds" IL-6R includes antibodies that bind IL-6R or portion thereof with a K D of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less than about 0.5 nM, as measured in a surface plasmon resonance assay.
  • An isolated antibody that specifically binds human IL-6R may, however, have cross-reactivity to other antigens, such as IL-6R molecules
  • the anti-IL-6R antibodies useful for the methods of the present invention may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the present invention includes methods involving the use of antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the
  • germline mutations corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations").
  • a person of ordinary skill in the art starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof.
  • all of the framework and/or CDR residues within the V H and/or V L domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • the use of antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
  • the present invention also includes methods involving the use of anti-IL-6R antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • the present invention includes the use of anti-IL-6R antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
  • K D is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to human IL-6R.
  • VELOCIMMUNETM technology see, for example, US 6,596,541 , Regeneron Pharmaceuticals or any other known method for generating monoclonal antibodies
  • high affinity chimeric antibodies to IL-6R are initially isolated having a human variable region and a mouse constant region.
  • the VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation.
  • the DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions.
  • the DNA is then expressed in a cell capable of expressing the fully human antibody.
  • lymphatic cells such as B-cells
  • the lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest.
  • DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain.
  • Such an antibody protein may be produced in a cell, such as a CHO cell.
  • DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific
  • lymphocytes are lymphocytes.
  • high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region.
  • the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc, using standard procedures known to those skilled in the art.
  • the mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified lgG1 or lgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
  • the antibodies that can be used in the methods of the present invention possess high affinities, as described above, when measured by binding to antigen either immobilized on solid phase or in solution phase.
  • the mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
  • human antibodies or antigen-binding fragments of antibodies that specifically bind IL-6R which can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment which comprises the three heavy chain CDRs (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 227, 19, 231 , 35, 51 , 67, 83, 99, 1 15, 131 , 147, 239, 241 , 163, 179, 235, 195 and 21 1.
  • HCVR heavy chain variable region
  • the antibody or antigen- binding fragment may comprise the three light chain CDRs (LCVR1 , LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 1 , 229, 27, 233, 43, 59, 75, 91 , 107, 123, 139, 155, 171 , 187, 203 and 219.
  • LCVR light chain variable region
  • Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991 ); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
  • the antibody or antigen-binding fragment thereof comprises the six CDRs (HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3) from the heavy and light chain variable region amino acid sequence pairs
  • HCVR/LCVR selected from the group consisting of SEQ ID NOs: 3/1 1 ; 227/229; 19/27;
  • the antibody or antigen-binding fragment thereof comprises HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 3/1 1 ; 227/229; 19/27; 231/233; 35/43; 51/59; 67/75; 83/91 ; 99/107; 1 15/123; 131/139; 147/155; 239/155; 241 ;155; 163/171 ; 179/187; 235/237; 195/203; and 21 1/219.
  • mAb1 anti-hlL-6R antibody
  • This antibody is also referred to herein as VQ8F1 1 -21.
  • mAbl comprises an HCVR/LCVR amino acid sequence pair having SEQ ID NOs:19/27, and HCDR1 - HCDR2-HCDR3 / LCDR1 -LCDR2-LCDR3 domains represented by SEQ ID NOs:21 - 23 - 25 / SEQ ID NOs:29 - 31 - 33.
  • the methods of the present invention can be practiced using any anti-IL-6R antibody disclosed herein, as well as variants and antigen-binding fragments of such antibody.
  • the present invention includes methods which comprise administering an IL-6R antagonist to a patient, wherein the IL-6R antagonist is contained within a pharmaceutical composition.
  • the pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-31 1.
  • the dose of antibody administered to a patient according to the methods of the present invention may vary depending upon the age and the size of the patient, symptoms, conditions, route of administration, and the like.
  • the dose is typically calculated according to body weight or body surface area.
  • Effective dosages and schedules for administering pharmaceutical compositions comprising anti-IL-6R antibodies may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Pharmaceut. Res. 8:1351 ).
  • Specific exemplary doses of anti-IL6R antibodies, and administration regimens involving the same, that can be used in the context of the present invention are disclosed elsewhere herein.
  • compositions of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or
  • mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • a pharmaceutical composition of the present invention can be delivered
  • a pen delivery device readily has applications in delivering a
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK),
  • DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ),
  • OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM are examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention.
  • disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active
  • Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • compositions comprising an anti-IL-6R antibody that can be used in the context of the present invention are disclosed, e.g., in US Patent Application
  • the amount of IL-6R antagonist (e.g., anti-IL-6R antibody) administered to a subject according to the methods of the present invention is, generally, a therapeutically effective amount.
  • a therapeutically effective amount means an amount of IL- 6R antagonist that results in a detectable improvement in one or more symptoms or indicia of rheumatoid arthritis.
  • a “therapeutically effective amount” also includes an amount of IL-6R antagonist that inhibits, prevents, lessens, or delays the progression of RA in a subject.
  • a therapeutically effective amount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg, about 1 .0 mg, about 1 .5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 1 10 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg
  • the amount of IL-6R antagonist contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg/kg).
  • the IL-6R antagonist may be administered to a patient at a dose of about 0.0001 to about 10 mg/kg of patient body weight.
  • the methods of the present invention comprise administering to the subject one or more additional therapeutic agents in combination with the IL-6R antagonist.
  • additional therapeutic agents in combination with the IL-6R antagonist.
  • composition comprising the IL-6R antagonist.
  • the additional therapeutic agent when administered "before" the pharmaceutical composition comprising the IL-6R antagonist, may be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes or about 10 minutes prior to the administration of the pharmaceutical composition comprising the IL-6R antagonist.
  • the additional therapeutic agent when administered "after” the pharmaceutical composition comprising the IL-6R antagonist, the additional therapeutic agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours after the
  • Administration "concurrent" with the pharmaceutical composition comprising the IL-6R antagonist means that the additional therapeutic agent is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of
  • the present invention includes methods of treating rheumatoid arthritis which comprise administering to a patient in need of such treatment an anti-hlL-6R antibody in combination with at least one additional therapeutic agent.
  • additional therapeutic agents which can be administered in combination with an anti-hlL-6R antibody in the practice of the methods of the present invention include, but are not limited to NSAIDs, DMARDs, TNFa antagonists, T-cell blockers, CD-20 antagonists (e.g., anti-CD-20 antibodies), IL-1 antagonists, JAK antagonists, IL- 17 antagonists, and any other compound known to treat, prevent, or ameliorate rheumatoid arthritis in a human subject.
  • Specific, non-limiting examples of additional therapeutic agents that may be administered in combination with an anti-hlL-6R antibody in the context of a method of the present invention include, but are not limited to methotrexate, sulfasalazine,
  • the additional therapeutic agent(s) can be administered concurrently or sequentially with the anti-hlL-6R antibody.
  • a pharmaceutical formulation can be made which contains both an anti-hlL-6R antibody and at least one additional therapeutic agent.
  • the amount of the additional therapeutic agent that is administered in combination with the anti- hlL-6R antibody in the practice of the methods of the present invention can be easily determined using routine methods known and readily available in the art.
  • the present invention includes methods comprising administering to a subject a pharmaceutical composition comprising an IL-6R antagonist at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved.
  • a pharmaceutical composition comprising an anti- IL-6R antibody once a week dosing at an amount of about 75 mg, 150 mg, 200 mg, or 300 mg, can be employed.
  • multiple doses of an IL-6R antagonist may be administered to a subject over a defined time course.
  • the methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an IL-6R antagonist.
  • sequentially administering means that each dose of IL-6R antagonist is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the present invention includes methods which comprise sequentially administering to the patient a single initial dose of an IL-6R antagonist, followed by one or more secondary doses of the IL-6R antagonist, and optionally followed by one or more tertiary doses of the IL-6R antagonist.
  • the terms "initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the IL-6R antagonist.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose;
  • the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of IL-6R antagonist, but generally may differ from one another in terms of frequency of administration.
  • the amount of IL-6R antagonist contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • two or more (e.g., 2, 3, 4, or 5) doses area at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").
  • each secondary and/or tertiary dose is administered 1 to 14 (e.g., 1 , 1 1 ⁇ 2, 2, 21 ⁇ 2, 3, 31 ⁇ 2, 4, 41 ⁇ 2, 5, 51 ⁇ 2, 6, 61 ⁇ 2, 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 1 1 , 1 1 1 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, or more) weeks after the immediately preceding dose.
  • the phrase "the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of IL-6R antagonist which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an IL-6R antagonist.
  • any number of secondary and/or tertiary doses of an IL-6R antagonist may comprise administering to a patient any number of secondary and/or tertiary doses of an IL-6R antagonist.
  • only a single secondary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
  • only a single tertiary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are
  • administered to a patient can vary over the course of the treatment regimen.
  • the frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • the present invention includes administration regimens comprising an up-titration option (also referred to herein as "dose modification").
  • an "up-titration option” means that, after receiving a particular number of doses of an IL-6R inhibitor, if an RA patient has not achieved a specified improvement in one or more defined therapeutic parameters (e.g., at least a 20% improvement in swollen joint count [SJC] and/or tender joint count [TJC]), or otherwise exhibits a clear lack of efficacy in the opinion of a physician or health care provider, the dose of the IL-6R inhibitor is thereafter increased.
  • SJC swollen joint count
  • TJC tender joint count
  • an anti-IL-6R antibody e.g., sarilumab, tocilizumab, etc.
  • the dose of anti-IL-6R antibody is increased to e.g., 200 mg, 300 mg, or more, administered once every two weeks thereafter (e.g., starting at week 10, 12, 14, 16, 18, or later).
  • the present invention includes methods which comprise administering to a subject in need thereof a therapeutic composition comprising an IL-6R antagonist.
  • a subject in need thereof means a human or non-human animal that exhibits one or more symptoms or indicia of rheumatoid arthritis (e.g., chronic disease anemia, fever, depression, fatigue, rheumatoid nodules, vasculitis, neuropathy, scleritis, pericarditis, Felty's syndrome and/or joint destruction) and/or who has been diagnosed with rheumatoid arthritis.
  • rheumatoid arthritis e.g., chronic disease anemia, fever, depression, fatigue, rheumatoid nodules, vasculitis, neuropathy, scleritis, pericarditis, Felty's syndrome and/or joint destruction
  • a variety of clinical indices, patient-reported outcomes, and/or other techniques for evaluating RA patients pre- or post-treatment may be used with the present invention.
  • Exemplary evaluation techniques for use with the present invention include, but are not limited to, modified Total Sharp Score (mTSS), erosion score (ES), and joint space narrowing (JSN).
  • mTSS modified Total Sharp Score
  • ES erosion score
  • JSN joint space narrowing
  • Example 1 Generation of Human Antibodies to Human IL-6 Receptor.
  • hlL-6R antigen is administered directly to mice which comprise DNA loci encoding both human Ig heavy chain variable region and Kappa light chain variable region (VeloclmmuneTM, Regeneron Pharmaceuticals, Inc.; US 6,596,541 ), with an adjuvant to stimulate the immune response.
  • Such an adjuvant includes complete and incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptides) (see O'Hagan, Vaccine Adjuvant, by Human Press, 2000, NJ).
  • Such an adjuvant can prevent rapid dispersal of polypeptide by sequestering the antigen in a local depot, and may contain factors that can stimulate host immune response.
  • hlL-6R is administered indirectly as DNA plasmid that contains hlL-6R gene and expresses hlL-6R using the host cellular protein expression machinery to produce antigen polypeptide in vivo. In both approaches, the immunization schedule requires several administrations spaced by a few weeks. The antibody immune response is monitored by standard antigen-specific
  • the antibody expressing B cells were harvested and fused with mouse myeloma cells to preserve their viability, forming hybridoma cells.
  • conditioned media of the hybridoma cells or transfected cells were screened for specificity, antigen-binding affinity, and potency in blocking hlL-6 binding to hlL-6R (described below).
  • DNA encoding VH and VL domains may be isolated directly from a single antigen positive B cell. Briefly, the hlL-6Ra immunized transgenic mouse was terminated and splenocytes were harvested. Red blood cells were removed by lysis followed by pelleting the harvested splenocytes. Resuspended splenocytes were first incubated with a cocktail of human IgG, FITC-anti-mFc, and biotin-IL6Ra for 1 hour. The stained cells were washed twice with PBS, then stained with a cocktail of human and rat IgG, APC-anti-mlgM, and SA-PE for one hour.
  • the stained cells were washed once with PBS and were analyzed by flow cytometry on a MoFlo (Cytomation). Each IgG positive, IgM negative, and antigen positive B cell was sorted and plated into a separate well on a 96-well plate. RT-PCR of antibody genes from these B cells was performed according to a method described by Wang et al. (2000) (J Immunol Methods
  • cDNAs for each single B cell were synthesized via RT-PCR. Each resulting RT product was then split and transferred into two corresponding wells on two 96-well plates. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human IgG heavy chain variable region leader sequence and a 3' primer specific for mouse heavy chain constant region, to form an amplicon. The amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human IgG heavy chain variable region sequence and a nested 3' primer specific for mouse heavy chain constant region.
  • the other set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human kappa light chain variable region leader sequence and a 3' primer specific for mouse kappa light chain constant region to form an amplicon.
  • the amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human kappa light chain variable region sequence and a nested 3' primer specific for mouse kappa light chain constant region.
  • the heavy chain and light chain PCR products were cloned into Sap l-linearized antibody vectors containing lgG1 heavy chain constant region and kappa light chain constant region, respectively.
  • the heavy chain plasmid has a lox2272 site and a lox51 1 site flanking the heavy chain expression cassettes.
  • immediately downstream of the lox2272 in the heavy chain plasmid there is a hygromycin- resistance gene that lacks a promoter and an initiating ATG.
  • the hygromycin-resistance gene is also transcriptionally linked to a downstream eGFP gene via an IRES sequence.
  • the light chain plasmid has a loxP site and lox2272 site flanking the light chain expression cassette.
  • the light chain plasmid has a SV40 promoter immediately before an ATG at the lox2272 site, such that upon integration into an appropriate host cell the lox2272-proximal SV40 promoter and initiating ATG from the light chain plasmid is brought adjacent to the hygromycin- resistance gene in the heavy chain plasmid in the proper reading frame to allow transcription and translation of the hygromycin-resistance and eGFP genes.
  • Purified recombinant plasmids having a heavy chain variable region sequence and plasmids having a light chain variable region sequence from the same B cell were then combined and transfected, together with a plasmid that expresses the Cre recombinase, into a modified CHO host cell line.
  • the modified CHO host cell line contains, from 5' to 3', a loxP site, an eCFP, a lox2272 site, DsRed, and a lox51 1 site at a transcriptionally active locus. Consequently, the host CHO cell can be isolated by flow cytometry as a blue-positive, red-positive, and green-negative cell.
  • CHO cells transfected with recombinant plasmids having a heavy chain variable region sequence and plasmids having a light chain variable region sequence from the same B cell were sorted by flow cytometry, and proper recombinants that show the blue-negative, red-negative, and green- positive phenotype were isolated, and stable recombinant antibody-expressing CHO cell lines were established from isolated clones.
  • K D of the antigen binding to the selected antibodies described above were determined by surface kinetics on a real-time biosensor surface plasmon resonance assay (BIAcoreTM). More specifically, the affinity of the antibodies for human IL-6R was measured using a BIAcore® 2000 or BIAcore® 3000. The antibody was captured on an anti-mouse IgG surface and exposed to various concentrations of recombinant hlL-6R protein either in monomeric or dimeric form. Kinetic analysis using BIAevaluationTM software was performed to obtain the association and dissociation rate constants.
  • BIAcoreTM real-time biosensor surface plasmon resonance assay
  • Binding affinities of the antibodies to hlL-6R was also measured for either hybridoma- conditioned media or purified proteins by plate-based competition immunoassay.
  • the antibody proteins were purified using Protein G affinity chromatography from hybridoma cell conditioning medium that was bovine IgG-depleted (Invitrogen).
  • competition ELISA briefly, constant amounts of antibody at different levels were premixed with serial dilutions of antigen protein, hlL- 6R-hFc, ranging from 0 to 10 ⁇ g ml, and incubated for two hours at room temperature to reach pseudo-binding equilibrium between the antibody and antigen.
  • Results are shown in Table 1 (control: humanized monoclonal antibody to human IL-6R (U.S. Patent No. 5,817,790 SEQ ID NO:69 and 71 ).
  • Monomer and dimer K D determined by BIAcoreTM; solution K D
  • hlL-6 blocking activities of the anti-hlL-6R antibodies of the invention were screened by hlL-6 blocking immunoassays, in vitro hlL-6 dependent cell growth bioassays, and surface plasmon resonance (BIAcoreTM).
  • the immunoassay was used to screen ability of the tested antibody to block hlL-6 binding to hlL-6R, and the in vitro bioassay was used to determine the potency of the antibodies in neutralizing hll_-6R-mediated cellular signal transduction.
  • hlL-6 recombinant protein was coated on a 96-well plate in PBS buffer overnight at 4°C. This plate was used to capture free hlL-6R-hFc from antibody sample solutions, and the amount of captured hlL-6R-hFc was quantified according to the standard curve.
  • the sample solutions were composed of a constant amount of hlL-6R-hFc recombinant protein (100 pM) and varying amounts of antibody, either in crude hybridoma condition medium or as purified antibody protein, ranging from 0 to about 50 nM in serial dilutions.
  • the antibody- antigen mixtures were incubated at room temperature for -2 hours to allow antibody-antigen binding to reach equilibrium.
  • test antibody to block hlL-6 binding to the hlL-6R receptor was determined using surface plasmon resonance.
  • Purified antigen hlL-6R-hFc molecules were captured by goat anti-human IgG polyclonal antibodies immobilized on CM-5 surface through amine coupling to a density of 250 RU.
  • hlL-6 solution (0.25ml, 50 nM) was injected over the receptor surface and bound hlL-6 recorded (first injection of IL-6). Bound hlL-6 was then removed with a pulse of 3 M MgCI 2 following by conditioning buffer.
  • Anti-hlL6R antibody in hybridoma conditioned medium was injected over the captured receptor surface followed by second injection of hlL-6. The percent reduction in hL-6 binding resulting from preformed antibody and receptor complex was used as a score to define hlL-6 blockers from non-blockers (second column, Table 2).
  • hlL-6R antibodies to block hlL-6 activity in vitro was measured in the hlL- 6-dependent myeloma line XG-1.
  • XG-1 cells maintained in hlL-6-containing medium were washed twice with hl L-6-free media and cultured for -24 hours in hlL-6-free medium to deplete residual hlL-6.
  • the starved cells were then spun down and re-suspended in the medium at 4 x 10 5 cells per ml and plated 20,000 cells per well in a 96-well tissue culture plate.
  • the purified antibody proteins were serially diluted in medium and added to the plated cells at concentrations ranging from 0 to 50 nM.
  • hlL-6 was added to the wells to a final concentration of 8 pM.
  • Cells were allowed to grow for -72 hours at 37°C in a humidified 5% C0 2 incubator. At the end of growth period, live cells were measured using CCK-8 kit (Dojindo, Japan). IC 50 s were determined as described above, and reported in the third column of Table 2.
  • hlL-6R antibodies to block hlL-6 activity was also measured in vitro in the hlL-6-responsive human hepatoma cell line, HepG2.
  • HepG2 cells were transfected with a reporter plasmid containing a STAT3 (Signal Transducer and activator of Transcription 3) response element linked to a luciferase gene.
  • STAT3 Signal Transducer and activator of Transcription 3
  • the purified antibody proteins were serially diluted in medium and added to the plated cells at concentrations ranging from 0 to 100 nM. Subsequently, recombinant hlL-6 was added to the wells to a final concentration of 50 pM. The response was measured after incubating the cells for 6 hours at 37°C in a humidified 5% C0 2 incubator. Luciferase activity was measured with the Steady-GloTM luciferase assay system (Promega). IC 50 s were determined as described above, and reported in the fourth column of Table 2.
  • An antibody binding competition immunoassay was performed using as a control humanized antibody to human IL-6R. Briefly, a 96-well immunosorbent plate was coated with 20 ng per well hlL-6R recombinant protein overnight at 4°C. After blocking non-specific binding with BSA, the hlL-6R binding sites on one half of the plate were saturated with binding of the control antibody by addition of 500 ng of the control per well, and to the other half of the plate was added binding buffer only. After three hours binding at room temperature, the purified antibodies were spiked in at a final concentration of 50 ng/ml with and without the preexisting control antibody in the well.
  • Antibodies VQ8F1 1 , W3D8, VV6A9, VV6C10-1 bound epitopes overlapping with the control antibody; while antibodies VQ8A9, W1 G4, VV6F12, VV7G4, W9A6, and W6C10-3 appeared to bind distinct epitopes as antigen binding was not blocked by the control antibody. Partial competition may result from steric hindrance from the first antibody bound, even though epitopes may not be overlapping.
  • VQ8F1 1 , VV6A9, and VQ8A9 strongly reacted to monkey receptor with K D values that differed by about 1.5- to about 3-fold from human receptor binding, respectively.
  • W1 G4 which was not blocked by the control antibody (Table 3), showed no binding to monkey receptor despite strong binding to the human receptor with K D of 241 pM.
  • the binding affinity to monomeric hlL-6R of four antibodies having mouse IgG, human lgG1 or human lgG4 (wild-type and modified) were determined using BIAcoreTM as described above except a goat anti-human Fc polyclonal antibody surface was used to capture hlgG antibodies.
  • Monomeric hlL-6R was injected at concentrations of 12.5, 6.25, 3.12, and 1.56 nM.
  • the ability of the antibodies to neutralize hlL-6-dependent HepG2/STAT3 signal transduction was also determined in a luciferase assay (IC 50 ). IC 50 s for different IgG isotypes were similar, suggesting no effect of isotype on antibody affinity for antigen.
  • Biomarker analysis was conducted on samples taken from subjects who participated in clinical trials of mAbl .
  • C1 M, C2M, C3M, and CRPM levels were measured in samples from patients at baseline and at different time points following initiation of study treatment(s).
  • Clinical trials of mAbl are further described below and in U.S. Patent Application Publication 2013/0149310.
  • a dose-ranging phase 2 portion of a clinical trial of subcutaneous mAb1 in patients with active RA treated with concomitant methotrexate (MTX) was conducted.
  • Sera were collected at baseline, 2 weeks, and 12 weeks from patients randomized to treatment with methotrexate (MTX)+placebo (Pbo), MTX+mAb1 150 mg q2w (150mg of mAbl every other week), or MTX+mAb1 200 mg q2w (200 mg of mAbl every other week).
  • C1 M, C2M, C3M and CRPM were analyzed by ELISA.
  • RA rheumatoid arthritis
  • RANKL receptor activated NF kB ligand
  • MMPs matrix metalloproteinases
  • Sera were analyzed from patients randomized to either Placebo (Pbo) + methotrexate (MTX), or subcutaneous administration of 200 mg sarilumab every other week (q2w) + MTX.
  • Levels of serum biomarkers [CTX-1 , C1 M, osteocalcin (OC), C3M, MMP-3, C2M, P2NP, RANKL and OPG] were measured by ELISA. All biomarkers were analyzed at baseline, and post- treatment at week 2 and 24, with the exceptions of CTX-1 and OC, which were analyzed at baseline, week 24 and week 52 post-treatment. Week 52 samples were assessed for patients who completed study; samples collected at or after week 40 were evaluated if patients discontinued treatment prior to week 52.
  • the ratios of RANKL to OPG were also calculated for weeks 0, 2, and 24. As shown in table 9 below, a greater decrease in the log RANKL/OPG ratio occurred in subjects receiving of 200 mg sarilumab every other week (q2w) + MTX, as opposed to placebo + MTX.
  • Sarilumab (also referred to herein as "mAbl ”) is a human monoclonal antibody directed against IL-6R and has been shown to be effective with subcutaneous (SC) dose regimens of 150 mg and 200 mg once every two weeks (q2w) in rheumatoid arthritis (RA) patients with active disease despite treatment with methotrexate (MTX).
  • SC subcutaneous
  • q2w rheumatoid arthritis
  • MTX methotrexate
  • the objective of the present Example was to assess the impact on efficacy and safety of increasing sarilumab dosing from 150 mg to 200 mg q2w in patients who showed poor response to the 150 mg dose.
  • This Example demonstrates that increasing anti-IL-6R antibody dose to 200 mg q2w may have therapeutic value for RA patients with a suboptimal response at Week 16 to the 150mg q2w dose. Patients rescued from the 200mg q2w regimen exhibited minimal radiographic progression . The safety profile observed in the post-rescue period was generally consistent with that observed in the double-blind period.

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Abstract

The present invention provides methods for treating rheumatoid arthritis (RA). Also provided are methods for improving one or more RA-associated parameter(s), methods for decreasing the level of at least one RA-associated biomarker in a subject in need thereof, and methods for treating RA according to the expression levels of one or more RA-associated biomarkers. The methods of the present invention comprise administering to a subject in need thereof a pharmaceutical composition comprising an interleukin-6 receptor (IL-6R) antagonist such as an anti-IL-6R antibody.

Description

METHODS FOR TREATING RHEUMATOID ARTHRITIS BY ADMINISTERING AN IL-6R
ANTIBODY
FIELD OF THE INVENTION
[0001] The present invention relates to the treatment and/or prevention of rheumatoid arthritis and related conditions. More specifically, the invention relates to the administration of interleukin-6 receptor (IL-6R) antagonists to treat or prevent rheumatoid arthritis in a patient in need thereof.
BACKGROUND
[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial tissue, leading to destruction of the joint architecture. It is recognized that cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1 ) and interleukin-6 (IL-6) play a role in joint inflammation and cartilage damage observed in RA. IL-6 is a pleiotropic cytokine with biological effects on many cell types. IL-6 is often regarded as being downstream of TNF or IL-1 in inflammatory cytokine cascades and may therefore represent a common pathway factor in a wide range of inflammatory processes. Blockade of IL-6 signaling therefore offers the potential to ameliorate multiple pathogenic features of RA and other inflammatory diseases.
[0003] Therapeutic methods using IL-6R antagonists are mentioned in US 5,888,510;
6,723,319; and 2001/0001663. Exemplary anti-IL-6R antibodies are described in US 7,582,298; 6,410,691 ; 5,817,790; 5,795,695; 6,670,373; and 7,582,298.
BRIEF SUMMARY OF THE INVENTION
[0004] According to certain aspects of the present invention, methods are provided for treating, preventing and/or reducing the severity of symptoms of rheumatoid arthritis (RA). The methods of the present invention comprise administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an interleukin-6 receptor (IL-6R) antagonist. According to certain embodiments of the present invention, the IL- 6R antagonist is an antibody or antigen-binding fragment thereof that specifically binds IL-6R. Exemplary anti-IL-6R antibodies that can be used in the context of the methods of the present invention are described elsewhere herein, including working Example 1. In certain
embodiments, the IL-6R antagonist is an anti-IL-6R antibody having the binding characteristics of the reference antibody referred to herein as "mAbl " (e.g., an antibody or antigen-binding fragment thereof comprising the complementarity determining regions of mAbl ).
[0005] According to certain exemplary embodiments, the present invention provides methods for treating RA in a subject, the methods comprising: (a) selecting a subject who exhibits an elevated level of at least one RA-associated biomarker; and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist. According to a related aspect of the present invention, methods for treating RA are provided which comprise administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist, wherein administration of the pharmaceutical composition to the subject results in a decrease in at least one RA -associated biomarker in the subject. Exemplary RA-associated biomarkers that can be evaluated and/or measured in the context of the present invention include, but are not limited to, matrix metalloprotease (MMP) cleaved collagen type 1 (C1 M), collagen type 2 (C2M), collagen type 3 (C3M), C reactive protein (CRPM), RANKL, and OPG.
[0006] The present invention also includes an interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits an elevated level of at least one RA-associated biomarker prior to or at the time of treatment. In one aspect of the invention, the interleukin-6 receptor (IL-6R) antagonist is used at a dose of about 75 mg to about 300 mg. In one aspect of the invention, the interleukin-6 receptor (IL-6R) antagonist is used once every other week.
[0007] The present invention also provides methods for decreasing the level of one or more RA-associated biomarker(s) in a subject, or improving one or more RA-associated parameter(s) in a subject, wherein the methods comprise sequentially administering to a subject in need thereof a single initial dose of a pharmaceutical composition comprising an IL-6R antagonist, followed by one or more secondary doses of the pharmaceutical composition comprising the IL- 6R antagonist.
[0008] According to certain embodiments, the present invention provides methods for decreasing the level of one or more RA-associated biomarker(s) in a subject, or improving one or more RA-associated parameter(s) in a subject, wherein the methods comprise administering to the subject about 75 mg to about 300 mg of a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds IL-6R. According to this aspect of the invention, the pharmaceutical composition may be administered to the subject at a dosing frequency of, e.g., once every other week.
[0009] The present invention also includes an IL-6R antagonist as disclosed herein for use in treating or preventing RA, for improving an RA-associated parameter, for decreasing the level of at least one RA-associated biomarker, and/or for treating any of the other indications or conditions disclosed herein.
[0010] According to certain embodiments, the present invention provides methods for treating RA in a subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein the subject has been diagnosed with RA and has also been selected for treatment on the basis of the subject exhibiting a reduced level of a RA-associated biomarker before treatment, as compared to a reference level of the biomarker (e.g., expression of the biomarker in a subset of subjects diagosed with RA and/or expression of the biomarker in healthy subjects). The present invention also provides methods for treating RA by administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein the subject has been diagnosed with rheumatoid arthritis, has already been treated with the anti-IL6R antibody for a defined period of time, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker (e.g., OPG) after treatment for the defined period of time (e.g., 24 weeks), wherein the reduced expression of the biomarker is determined based on a comparison to the level of expression of the respective biomarker in the subject prior to treatment with the anti-IL-6R antibody (e.g., a reduction of OPG expression that is equal to or greater than about a 9% reduction in expression as compared to pre-treatment expression levels).
[0011] The present invention also includes an interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits a lower level of at least one RA-associated biomarker prior to or at the time of treatment (e.g., as compared to a population of RA patients or a subset of the RA patient population), as compared to a reference level of the biomarker. The present invention also includes an interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits a lower level of at least one RA-associated biomarker after treatment with the IL-6R antagonist for a defined period of time (e.g., 24 weeks) as compared to the level of the biomarker prior to treatment. In one aspect of the invention, the lower level of at least one RA-associated biomarker after treatment with the IL-6R antagonist for a defined period of time is equal to or greater than about a 9% reduction in the exhibition of the biomarker as compared to pre-treatment levels. In one aspect of the invention, the interleukin-6 receptor (IL- 6R) antagonist is used at a dose of about 75 mg to about 300 mg. In one aspect of the invention, the interleukin-6 receptor (IL-6R) antagonist is used once every other week.
[0012] Other embodiments of the present invention will become apparent from a review of the ensuing detailed description.
DETAILED DESCRIPTION
[0013] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1 %. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1 , 99.2, 99.3, 99.4, etc.). As used herein, the terms "treat", "treating", or the like, mean to alleviate symptoms, eliminate the causation of symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.
[0015] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described.
Rheumatoid arthritis-Associated Biomarkers
[0016] The present invention also includes methods involving the use, quantification, and analysis of RA-associated biomarkers. As used herein, the term "RA-associated biomarker" means any biological response, cell type, parameter, protein, polypeptide, enzyme, enzyme activity, metabolite, nucleic acid, carbohydrate, or other biomolecule which is present or detectable in an RA patient at a level or amount that is different from (e.g., greater than or less than) the level or amount of the marker present or detectable in a non-RA patient. Exemplary RA-associated biomarkers include, but are not limited to, e.g., C1 M (matrix metalloprotease (MMP) cleaved collagen type 1 ), C2M (MMP cleaved collagen type 2), C3M (MMP cleaved collagen type 3), CRPM (MMP cleaved C reactive protein), MMP-3 (matrix metalloproteinase-3), CTX-1 (carboxy-terminal collagen crosslinks/C-terminal telopeptide I), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), and OC (osteocalcin).
Methods for detecting and/or quantifying such RA-associated biomarkers are known in the art; kits for measuring such RA-associated biomarkers are available from various commercial sources; and various commercial diagnostic laboratories offer services which provide
measurements of such biomarkers as well.
[0017] According to certain aspects of the invention, methods for treating RA are provided which comprise: (a) selecting a subject who exhibits a level of at least one RA-associated biomarker prior to or at the time of treatment which signifies the disease state, and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist. In certain embodiments of this aspect of the invention, the subject is selected on the basis of an elevated level of C1 M, C2M, C3M, or CRPM.
[0018] According to other aspects of the invention, methods for treating RA are provided which comprise administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-6R antagonist, wherein administration of the pharmaceutical composition to the subject results in a decrease in at least one RA-associated biomarker (e.g., C1 M, C2M, C3M, CRPM, etc.) at a time after administration of the pharmaceutical composition, as compared to the level of the biomarker in the subject prior to the administration. [0019] As will be appreciated by a person of ordinary skill in the art, an increase or decrease in an RA-associated biomarker can be determined by comparing (i) the level of the biomarker measured in a subject at a defined time point after administration of the pharmaceutical composition comprising an IL-6R antagonist to (ii) the level of the biomarker measured in the patient prior to the administration of the pharmaceutical composition comprising an IL-6R antagonist (i.e., the "baseline measurement"). The defined time point at which the biomarker is measured can be, e.g., at about 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 15 days, 20 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 84 days, 85 days, or more after administration of the of the pharmaceutical composition comprising an IL-6R antagonist.
[0020] According to certain particular embodiments of the present invention, a subject may exhibit a decrease in the level of one or more of C1 M, C2M, C3M, and/or CRPM following administration of a pharmaceutical composition comprising an IL-6R antagonist (e.g., an anti-IL- 6R antibody). For example, at about day 4, day 8, day 14, day 15, day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day 71 , day 84, or day 85, following administration of a first, second, third or fourth dose of a pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl ), the subject, according to the present invention, may exhibit a decrease in C1 M of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline" is defined as the level of C1 M in the subject just prior to the first administration). Similarly, at about day 4, day 8, day 14, day 15, day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day 71 , day 84 or day 85, following administration of a first, second, third or fourth dose of a pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl ), the subject, according to the present invention, may exhibit a decrease in C2M of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline" is defined as the level of C2M in the subject just prior to the first administration). Similarly, at about day 4, day 8, day 14, day 15, day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day 71 , day 84 or day 85, following administration of a first, second, third or fourth dose of a pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl ), the subject, according to the present invention, may exhibit a decrease in C3M of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline" is defined as the level of C3M in the subject just prior to the first administration). Similarly, at about day 4, day 8, day 14, day 15, day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day 71 , day 84 or day 85, following administration of a first, second, third or fourth dose of a
pharmaceutical composition comprising about 75, 150, 200 or 300 mg of an anti-hlL-6R antibody (e.g., mAbl ), the subject, according to the present invention, may exhibit a decrease in CRPM of about 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline (wherein "baseline" is defined as the level of CRPM in the subject just prior to the first administration).
[0021] The present invention also includes methods for determining whether a subject is a suitable subject for whom administration of a pharmaceutical composition comprising an IL-6R antagonist would be beneficial. For example, if an individual, prior to receiving a pharmaceutical composition comprising an IL-6R antagonist, exhibits a level of an RA-associated biomarker which signifies the disease state, the individual is therefore identified as a suitable patient for whom administration of a pharmaceutical composition of the invention (a composition
comprising an anti-IL-6R antibody) would be beneficial. According to certain exemplary embodiments, an individual may be identified as a good candidate for anti-IL-6R therapy if the individual exhibits one or more of the following: (i) a C1 M level greater than about 180 ng/mL, greater than about 190 ng/mL, greater than about 200 ng/mL, greater than about 210 ng/mL, greater than about 220 ng/mL, greater than about 230 ng/mL, greater than about 240 ng/mL, greater than about 250 ng/mL, greater than about 300 ng/mL, greater than about 350 ng/mL, greater than about 400 ng/mL, greater than about 450 ng/mL, or greater than about 500 ng/mL; (ii) a C2M level greater than about 0.25 ng/mL, greater than about 0.26 ng/mL, greater than about 0.26 ng/mL, greater than about 0.27 ng/mL, greater than about 0.28 ng/mL, greater than about 0.29 ng/mL, greater than about 0.30 ng/mL, greater than about 0.31 ng/mL, greater than about 0.32 ng/mL, greater than about 0.33 ng/mL, greater than about 0.34 ng/mL, greater than about 0.35 ng/mL, or greater than about 0.40 ng/mL; (iii) a C3M level greater than about 45 ng/mL, greater than about 46 ng/mL, greater than about 47 ng/mL, greater than about 48 ng/mL, greater than about 49 ng/mL, greater than about 50 ng/mL, greater than about 51 ng/mL, greater than about 52 ng/mL, greater than about 53 ng/mL, greater than about 54 ng/mL, greater than about 55 ng/mL, or greater than about 60 ng/mL; and/or (iv) a CRPM level greater than about 16 ng/mL, greater than about 17 ng/mL, greater than about 18 ng/mL, greater than about 19 ng/mL, greater than about 20 ng/mL, greater than about 21 ng/mL, greater than about 22 ng/mL, greater than about 23 ng/mL, greater than about 24 ng/mL, greater than about 25 ng/mL, greater than about 30 ng/mL, or greater than about 35 ng/mL. Additional criteria, such as other clinical indicators of RA (e.g., an elevated level of high-sensitivity C-reactive protein (hsCRP)), serum amyloid A (SAA), erythrocyte sedimentation rate (ESR) and/or serum hepcidin) may be used in combination with any of the foregoing RA-associated biomarkers to identify an individual as a suitable candidate for anti-IL-6R therapy as described elsewhere herein.
[0022] In other aspects of the invention, biomarker levels and/or changes in biomarker levels with treatment may have predictive value for efficacy of anti-IL6R therapy with particular anti- IL6R agents. For example, RA patients having higher baseline levels of OPG before therapy and/or smaller reductions in OPG levels after 24 weeks of treatment of RA using a particular anti-IL6R therapeutic (i.e., sarilumab) were found to have worse treatment outcomes in terms of radiographic progression (e.g., modified Total Sharp Score and erosion score) at 52 weeks of treatment (see Example 9 below). Without being bound to any particular theory, it is believed that higher levels of OPG are associated with higher levels of gp130 and soluble IL-6R, which serve as alternative targets for IL-6R antibody and interfere with the therapeutic blockade of membrane-associated IL-6R signaling via binding of anti-IL6R antibody to membrane-bound IL- 6R. Thus, additional aspects of the invention are methods of treating rheumatoid arthritis in a subject comprising administration of an anti-IL6R antibody (e.g., sarilumab), wherein the subject has been diagnosed with rheumatoid arthritis and has been selected for treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker (e.g., OPG) pre- treatment, wherein the reduced expression of the biomarker is determined based on a comparison to a reference level of expression of the respective biomarker. An additional aspect of the invention features methods of treating rheumatoid arthritis in a subject comprising administration of an anti-IL6R antibody (e.g., sarilumab), wherein the subject has been diagnosed with rheumatoid arthritis, has been treated with the anti-IL6R antibody, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker (e.g., OPG), wherein the reduced expression of the biomarker is determined based on a comparison to the level of expression of the respective biomarker in the subject prior to treatment with the anti-IL-6R antibody. A particular aspect of the invention features methods methods of treating rheumatoid arthritis in a subject comprising administration of an anti-IL6R antibody (e.g., sarilumab), wherein the subject has been diagnosed with rheumatoid arthritis, has been treated with the anti-IL6R antibody, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of the OPG biomarker, wherein the reduced expression of the OPG biomarker in comparison to pre- treatment expression levels is equal to or greater than a 9% reduction in OPG expression. lnterleukin-6 Receptor Antagonists
[0023] As disclosed in detail above, the present invention includes methods which comprise administering to a subject in need thereof a therapeutic composition comprising an interleukin-6 receptor (IL-6R) antagonist. As used herein, an "IL-6R antagonist" is any agent which binds to or interacts with IL-6R and inhibits the normal biological signaling function of IL-6R when IL-6R is expressed on a cell in vitro or in vivo. Non-limiting examples of categories of IL-6R antagonists include small molecule IL-6R antagonists, anti-IL-6R aptamers, peptide-based IL-6R antagonists (e.g., "peptibody" molecules), and antibodies or antigen-binding fragments of antibodies that specifically bind human IL-6R.
[0024] The present invention includes methods that comprise administering to a patient a human antibody, or an antigen-binding fragment thereof, that binds specifically to hlL-6R. As used herein, the term "hlL-6R" means a human cytokine receptor that specifically binds human interleukin-6 (IL-6). In certain embodiments, the antibody that is administered to the patient binds specifically to the extracellular domain of hlL-6R. The extracellular domain of hlL-6R is shown in the amino acid sequence of SEQ ID NO:1
[0025] The term "antibody," as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1 ). The VH and VL regions can be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-IL- 6R antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
[0026] The term "antibody," as used herein, also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
[0027] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
[0028] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
[0029] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen- binding fragment of an antibody of the present invention include: (i) VH-CH1 ; (ii) VH-CH2; (iii) VH- CH3; (iv) VH-CH1 -Ch2; (V) VH-CH1 -Ch2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1 ; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1 -CH2; (xii) VL-Ch1 -Ch2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
[0030] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, may be adapted for use in the context of an antigen- binding fragment of an antibody of the present invention using routine techniques available in the art.
[0031 ] The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.
[0032] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
[0033] The term "recombinant human antibody," as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
[0034] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of
approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
[0035] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular
Immunology 30:105) to levels typically observed using a human lgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
[0036] An "isolated antibody," as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
[0037] The term "specifically binds," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" IL-6R, as used in the context of the present invention, includes antibodies that bind IL-6R or portion thereof with a KD of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less than about 0.5 nM, as measured in a surface plasmon resonance assay. An isolated antibody that specifically binds human IL-6R may, however, have cross-reactivity to other antigens, such as IL-6R molecules from other (non-human) species.
[0038] The anti-IL-6R antibodies useful for the methods of the present invention may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes methods involving the use of antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the
corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
[0039] The present invention also includes methods involving the use of anti-IL-6R antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes the use of anti-IL-6R antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
[0040] The term "surface plasmon resonance," as used herein, refers to an optical
phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
[0041] The term "KD," as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
[0042] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen. Preparation of Human Antibodies
[0043] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to human IL-6R.
[0044] Using VELOCIMMUNE™ technology (see, for example, US 6,596,541 , Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to IL-6R are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.
[0045] Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific
lymphocytes.
[0046] Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc, using standard procedures known to those skilled in the art. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified lgG1 or lgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
[0047] In general, the antibodies that can be used in the methods of the present invention possess high affinities, as described above, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
[0048] Specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind IL-6R which can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment which comprises the three heavy chain CDRs (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 227, 19, 231 , 35, 51 , 67, 83, 99, 1 15, 131 , 147, 239, 241 , 163, 179, 235, 195 and 21 1. The antibody or antigen- binding fragment may comprise the three light chain CDRs (LCVR1 , LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 1 , 229, 27, 233, 43, 59, 75, 91 , 107, 123, 139, 155, 171 , 187, 203 and 219. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991 ); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
[0049] In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises the six CDRs (HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3) from the heavy and light chain variable region amino acid sequence pairs
(HCVR/LCVR) selected from the group consisting of SEQ ID NOs: 3/1 1 ; 227/229; 19/27;
231/233; 35/43; 51/59; 67/75; 83/91 ; 99/107; 1 15/123; 131/139; 147/155; 239/155; 241 ;155; 163/171 ; 179/187; 235/237; 195/203; and 21 1/219.
[0050] In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 3/1 1 ; 227/229; 19/27; 231/233; 35/43; 51/59; 67/75; 83/91 ; 99/107; 1 15/123; 131/139; 147/155; 239/155; 241 ;155; 163/171 ; 179/187; 235/237; 195/203; and 21 1/219.
[0051] The study summarized in Example 8 below utilized an anti-hlL-6R antibody referred to as "mAb1 ." This antibody is also referred to herein as VQ8F1 1 -21. mAbl (VQ8F1 1-21 ) comprises an HCVR/LCVR amino acid sequence pair having SEQ ID NOs:19/27, and HCDR1 - HCDR2-HCDR3 / LCDR1 -LCDR2-LCDR3 domains represented by SEQ ID NOs:21 - 23 - 25 / SEQ ID NOs:29 - 31 - 33. However, the methods of the present invention can be practiced using any anti-IL-6R antibody disclosed herein, as well as variants and antigen-binding fragments of such antibody. Pharmaceutical Compositions
[0052] The present invention includes methods which comprise administering an IL-6R antagonist to a patient, wherein the IL-6R antagonist is contained within a pharmaceutical composition. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-31 1.
[0053] The dose of antibody administered to a patient according to the methods of the present invention may vary depending upon the age and the size of the patient, symptoms, conditions, route of administration, and the like. The dose is typically calculated according to body weight or body surface area. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering pharmaceutical compositions comprising anti-IL-6R antibodies may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Pharmaceut. Res. 8:1351 ). Specific exemplary doses of anti-IL6R antibodies, and administration regimens involving the same, that can be used in the context of the present invention are disclosed elsewhere herein.
[0054] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
[0055] A pharmaceutical composition of the present invention can be delivered
subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a
pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
[0056] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),
DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ),
OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.
[0057] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science
249:1527-1533.
[0058] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled in an appropriate ampoule.
[0059] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active
ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
[0060] Exemplary pharmaceutical compositions comprising an anti-IL-6R antibody that can be used in the context of the present invention are disclosed, e.g., in US Patent Application
Publication No. 201 1/0171241 ntirety.
Dosage
[0061] The amount of IL-6R antagonist (e.g., anti-IL-6R antibody) administered to a subject according to the methods of the present invention is, generally, a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount of IL- 6R antagonist that results in a detectable improvement in one or more symptoms or indicia of rheumatoid arthritis. A "therapeutically effective amount" also includes an amount of IL-6R antagonist that inhibits, prevents, lessens, or delays the progression of RA in a subject.
[0062] In the case of an anti-IL-6R antibody, a therapeutically effective amount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg, about 1 .0 mg, about 1 .5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 1 10 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg, of the anti-IL-6R antibody. In certain embodiments, 75 mg, 150 mg, 200 mg, or 300 mg of an anti-IL-6R antibody is administered to a subject.
[0063] The amount of IL-6R antagonist contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg/kg). For example, the IL-6R antagonist may be administered to a patient at a dose of about 0.0001 to about 10 mg/kg of patient body weight. Combination Therapies
[0064] The methods of the present invention, according to certain embodiments, comprise administering to the subject one or more additional therapeutic agents in combination with the IL-6R antagonist. As used herein, the expression "in combination with" means that the additional therapeutic agents are administered before, after, or concurrent with the
pharmaceutical composition comprising the IL-6R antagonist.
[0065] For example, when administered "before" the pharmaceutical composition comprising the IL-6R antagonist, the additional therapeutic agent may be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes or about 10 minutes prior to the administration of the pharmaceutical composition comprising the IL-6R antagonist. When administered "after" the pharmaceutical composition comprising the IL-6R antagonist, the additional therapeutic agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours after the
administration of the pharmaceutical composition comprising the IL-6R antagonist.
Administration "concurrent" with the pharmaceutical composition comprising the IL-6R antagonist means that the additional therapeutic agent is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of
Administration of the pharmaceutical composition comprising the IL-6R antagonist, or administered to the subject as a single combined dosage formulation comprising both the additional therapeutic agent and the IL-6R antagonist.
[0066] The present invention includes methods of treating rheumatoid arthritis which comprise administering to a patient in need of such treatment an anti-hlL-6R antibody in combination with at least one additional therapeutic agent. Examples of additional therapeutic agents which can be administered in combination with an anti-hlL-6R antibody in the practice of the methods of the present invention include, but are not limited to NSAIDs, DMARDs, TNFa antagonists, T-cell blockers, CD-20 antagonists (e.g., anti-CD-20 antibodies), IL-1 antagonists, JAK antagonists, IL- 17 antagonists, and any other compound known to treat, prevent, or ameliorate rheumatoid arthritis in a human subject. Specific, non-limiting examples of additional therapeutic agents that may be administered in combination with an anti-hlL-6R antibody in the context of a method of the present invention include, but are not limited to methotrexate, sulfasalazine,
hydroxychloroquine, leflunomide, etanercept, infliximab, adalimumab, golimumab, rilonacept, anakinra, abatacept, certolizumab and rituximab. In the present methods, the additional therapeutic agent(s) can be administered concurrently or sequentially with the anti-hlL-6R antibody. For example, for concurrent administration, a pharmaceutical formulation can be made which contains both an anti-hlL-6R antibody and at least one additional therapeutic agent. The amount of the additional therapeutic agent that is administered in combination with the anti- hlL-6R antibody in the practice of the methods of the present invention can be easily determined using routine methods known and readily available in the art.
Administration Regimens
[0067] The present invention includes methods comprising administering to a subject a pharmaceutical composition comprising an IL-6R antagonist at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved. In certain embodiments involving the administration of a pharmaceutical composition comprising an anti- IL-6R antibody, once a week dosing at an amount of about 75 mg, 150 mg, 200 mg, or 300 mg, can be employed.
[0068] According to certain embodiments of the present invention, multiple doses of an IL-6R antagonist may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an IL-6R antagonist. As used herein, "sequentially administering" means that each dose of IL-6R antagonist is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an IL-6R antagonist, followed by one or more secondary doses of the IL-6R antagonist, and optionally followed by one or more tertiary doses of the IL-6R antagonist.
[0069] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the IL-6R antagonist. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of IL-6R antagonist, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of IL-6R antagonist contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses area at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").
[0070] In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 14 (e.g., 1 , 1 ½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 1 1 , 1 1 ½, 12, 12½, 13, 13½, 14, 14½, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of IL-6R antagonist which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
[0071] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an IL-6R antagonist. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
[0072] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are
administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
[0073] The present invention includes administration regimens comprising an up-titration option (also referred to herein as "dose modification"). As used herein, an "up-titration option" means that, after receiving a particular number of doses of an IL-6R inhibitor, if an RA patient has not achieved a specified improvement in one or more defined therapeutic parameters (e.g., at least a 20% improvement in swollen joint count [SJC] and/or tender joint count [TJC]), or otherwise exhibits a clear lack of efficacy in the opinion of a physician or health care provider, the dose of the IL-6R inhibitor is thereafter increased. For example, in the case of a therapeutic regimen comprising administration of 150 mg doses of an anti-IL-6R antibody (e.g., sarilumab, tocilizumab, etc.) to a patient at a frequency of once every two weeks, if after 8, 10, 12, 14, 16 or more weeks, the patient has not achieved at least a 20% improvement in SJC and/or TJC, or if the patient exhibits a clear lack of efficacy in the opinion of a physician or other health care provider, then the dose of anti-IL-6R antibody is increased to e.g., 200 mg, 300 mg, or more, administered once every two weeks thereafter (e.g., starting at week 10, 12, 14, 16, 18, or later).
Treatment Populations
[0074] The present invention includes methods which comprise administering to a subject in need thereof a therapeutic composition comprising an IL-6R antagonist. As used herein, the expression "a subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or indicia of rheumatoid arthritis (e.g., chronic disease anemia, fever, depression, fatigue, rheumatoid nodules, vasculitis, neuropathy, scleritis, pericarditis, Felty's syndrome and/or joint destruction) and/or who has been diagnosed with rheumatoid arthritis.
Evaluation of RA severity and RA treatment efficacy
[0075] A variety of clinical indices, patient-reported outcomes, and/or other techniques for evaluating RA patients pre- or post-treatment may be used with the present invention.
Exemplary evaluation techniques for use with the present invention include, but are not limited to, modified Total Sharp Score (mTSS), erosion score (ES), and joint space narrowing (JSN).
EXAMPLES
[0076] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1. Generation of Human Antibodies to Human IL-6 Receptor.
[0077] Immunization of rodents can be done by any methods known in the art (see, for example, Harlow and Lane (1988) supra; Malik and Lillehoj, Antibody techniques: Academic Press, 1994, CA). In a preferred embodiment, hlL-6R antigen is administered directly to mice which comprise DNA loci encoding both human Ig heavy chain variable region and Kappa light chain variable region (Veloclmmune™, Regeneron Pharmaceuticals, Inc.; US 6,596,541 ), with an adjuvant to stimulate the immune response. Such an adjuvant includes complete and incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptides) (see O'Hagan, Vaccine Adjuvant, by Human Press, 2000, NJ). Such an adjuvant can prevent rapid dispersal of polypeptide by sequestering the antigen in a local depot, and may contain factors that can stimulate host immune response. In one embodiment, hlL-6R is administered indirectly as DNA plasmid that contains hlL-6R gene and expresses hlL-6R using the host cellular protein expression machinery to produce antigen polypeptide in vivo. In both approaches, the immunization schedule requires several administrations spaced by a few weeks. The antibody immune response is monitored by standard antigen-specific
immunoassay. When animals reached their maximum immune response, the antibody expressing B cells were harvested and fused with mouse myeloma cells to preserve their viability, forming hybridoma cells. To select functionally desirable monoclonal antibodies, conditioned media of the hybridoma cells or transfected cells were screened for specificity, antigen-binding affinity, and potency in blocking hlL-6 binding to hlL-6R (described below).
Example 2. Anti-hlL6R antibodies generated via direct isolation of splenocytes
[0078] DNA encoding VH and VL domains may be isolated directly from a single antigen positive B cell. Briefly, the hlL-6Ra immunized transgenic mouse was terminated and splenocytes were harvested. Red blood cells were removed by lysis followed by pelleting the harvested splenocytes. Resuspended splenocytes were first incubated with a cocktail of human IgG, FITC-anti-mFc, and biotin-IL6Ra for 1 hour. The stained cells were washed twice with PBS, then stained with a cocktail of human and rat IgG, APC-anti-mlgM, and SA-PE for one hour. The stained cells were washed once with PBS and were analyzed by flow cytometry on a MoFlo (Cytomation). Each IgG positive, IgM negative, and antigen positive B cell was sorted and plated into a separate well on a 96-well plate. RT-PCR of antibody genes from these B cells was performed according to a method described by Wang et al. (2000) (J Immunol Methods
244:217-225). Briefly, cDNAs for each single B cell were synthesized via RT-PCR. Each resulting RT product was then split and transferred into two corresponding wells on two 96-well plates. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human IgG heavy chain variable region leader sequence and a 3' primer specific for mouse heavy chain constant region, to form an amplicon. The amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human IgG heavy chain variable region sequence and a nested 3' primer specific for mouse heavy chain constant region. The other set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human kappa light chain variable region leader sequence and a 3' primer specific for mouse kappa light chain constant region to form an amplicon. The amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human kappa light chain variable region sequence and a nested 3' primer specific for mouse kappa light chain constant region. The heavy chain and light chain PCR products were cloned into Sap l-linearized antibody vectors containing lgG1 heavy chain constant region and kappa light chain constant region, respectively. The heavy chain plasmid has a lox2272 site and a lox51 1 site flanking the heavy chain expression cassettes. In addition, immediately downstream of the lox2272 in the heavy chain plasmid there is a hygromycin- resistance gene that lacks a promoter and an initiating ATG. The hygromycin-resistance gene is also transcriptionally linked to a downstream eGFP gene via an IRES sequence. The light chain plasmid has a loxP site and lox2272 site flanking the light chain expression cassette. In addition, The light chain plasmid has a SV40 promoter immediately before an ATG at the lox2272 site, such that upon integration into an appropriate host cell the lox2272-proximal SV40 promoter and initiating ATG from the light chain plasmid is brought adjacent to the hygromycin- resistance gene in the heavy chain plasmid in the proper reading frame to allow transcription and translation of the hygromycin-resistance and eGFP genes. Purified recombinant plasmids having a heavy chain variable region sequence and plasmids having a light chain variable region sequence from the same B cell were then combined and transfected, together with a plasmid that expresses the Cre recombinase, into a modified CHO host cell line. The modified CHO host cell line contains, from 5' to 3', a loxP site, an eCFP, a lox2272 site, DsRed, and a lox51 1 site at a transcriptionally active locus. Consequently, the host CHO cell can be isolated by flow cytometry as a blue-positive, red-positive, and green-negative cell. When recombinant plasmids expressing heavy chain and light chain genes are transfected together with a plasmid expressing the Cre recombinase, site-specific recombination mediated by the Cre recombinase results in the integration of the antibody plasmids at the chromosomal locus containing the lox sites and replacement of the eCFP and DsRed genes. Recombinants can then be isolated as blue-negative, red-negative, and green-positive cells by flow cytometry. Accordingly, CHO cells transfected with recombinant plasmids having a heavy chain variable region sequence and plasmids having a light chain variable region sequence from the same B cell were sorted by flow cytometry, and proper recombinants that show the blue-negative, red-negative, and green- positive phenotype were isolated, and stable recombinant antibody-expressing CHO cell lines were established from isolated clones.
Example 3. Antigen Binding Affinity Determination
[0079] KD of the antigen binding to the selected antibodies described above were determined by surface kinetics on a real-time biosensor surface plasmon resonance assay (BIAcore™). More specifically, the affinity of the antibodies for human IL-6R was measured using a BIAcore® 2000 or BIAcore® 3000. The antibody was captured on an anti-mouse IgG surface and exposed to various concentrations of recombinant hlL-6R protein either in monomeric or dimeric form. Kinetic analysis using BIAevaluation™ software was performed to obtain the association and dissociation rate constants.
[0080] Binding affinities of the antibodies to hlL-6R was also measured for either hybridoma- conditioned media or purified proteins by plate-based competition immunoassay. The antibody proteins were purified using Protein G affinity chromatography from hybridoma cell conditioning medium that was bovine IgG-depleted (Invitrogen). For the competition ELISA, briefly, constant amounts of antibody at different levels were premixed with serial dilutions of antigen protein, hlL- 6R-hFc, ranging from 0 to 10 μg ml, and incubated for two hours at room temperature to reach pseudo-binding equilibrium between the antibody and antigen. These solutions were then transferred to 96-well hlL-6R-hFc pre-coated plates to allow the free-antibody in the mixtures to bind to plate-coated hlL-6R-hFc. The plates were typically coated with 1 to 2 μg ml hlL-6R-hFc protein in PBS solution overnight at 4°C followed by BSA nonspecific blocking. After washing off excess antibody in solution, plate-bound antibodies were detected with an HRP-conjugated goat anti-mouse IgG or IgA polyclonal antibody reagent and developed using either colorimetric or chemiluminescence substrates. The dependency of the signals on the concentrations of antigen in solution was analyzed with a 4-parameter fit analysis using Prism™ software (Graph Pad) and reported as IC50. Competition immunoassay were also carried out using steady state solution phase Kinexa™ instrument (Sapidyne Inc.).
[0081] Results are shown in Table 1 (control: humanized monoclonal antibody to human IL-6R (U.S. Patent No. 5,817,790 SEQ ID NO:69 and 71 ). Antibody (HCVR and LCVR amino acid sequences): VQ8A9-6 (3, 1 1 ); VQ8F1 1 -21 (19, 27); VV7G4-1 (35, 43); W7G4-10 (51 , 59) VV6C10-1 (67, 75); W6C10-3 (83, 91 ); VV6C10-4 (99, 107); VV6F12-1 1 (1 15, 123); VV9A6-1 1 (131 , 139); W6A9-5 (147, 155), VV3D8-4 (163, 171 ); W1 G4-7 (179, 187); 248982-13-1-E5 (195, 203); 248982-13-2-A9 (21 1 , 219). Monomer and dimer KD determined by BIAcore™; solution KD by Kinexa™; IC50 by ELISA assays (n.d. = not determined).
Table 1. Antigen Binding Affinity
Figure imgf000025_0001
Example 4. Neutralization of hlL-6 Activity
[0082] hlL-6 blocking activities of the anti-hlL-6R antibodies of the invention were screened by hlL-6 blocking immunoassays, in vitro hlL-6 dependent cell growth bioassays, and surface plasmon resonance (BIAcore™). The immunoassay was used to screen ability of the tested antibody to block hlL-6 binding to hlL-6R, and the in vitro bioassay was used to determine the potency of the antibodies in neutralizing hll_-6R-mediated cellular signal transduction.
[0083] For the immunoassay, hlL-6 recombinant protein was coated on a 96-well plate in PBS buffer overnight at 4°C. This plate was used to capture free hlL-6R-hFc from antibody sample solutions, and the amount of captured hlL-6R-hFc was quantified according to the standard curve. The sample solutions were composed of a constant amount of hlL-6R-hFc recombinant protein (100 pM) and varying amounts of antibody, either in crude hybridoma condition medium or as purified antibody protein, ranging from 0 to about 50 nM in serial dilutions. The antibody- antigen mixtures were incubated at room temperature for -2 hours to allow antibody-antigen binding to reach equilibrium. The equilibrated sample solutions were then transferred to the hlL- 6 coated plates for measurement of free hlL-6R-hFc. After 1 hour binding, the plate was washed and bound hlL-6R-hFc was detected using HRP-conjugated goat anti-hFc polyclonal antibodies (Jackson Immuno Research), and developed using TMB substrate (BD Pharmigen). IC50s were determined as the amount of antibody required to reduce 50% of IL-6R-hFc detectable to plate bound hlL-6 ligand. Results are shown in the first column of Table 2.
[0084] Additionally, the ability of the test antibody to block hlL-6 binding to the hlL-6R receptor was determined using surface plasmon resonance. Purified antigen hlL-6R-hFc molecules were captured by goat anti-human IgG polyclonal antibodies immobilized on CM-5 surface through amine coupling to a density of 250 RU. hlL-6 solution (0.25ml, 50 nM) was injected over the receptor surface and bound hlL-6 recorded (first injection of IL-6). Bound hlL-6 was then removed with a pulse of 3 M MgCI2 following by conditioning buffer. Anti-hlL6R antibody in hybridoma conditioned medium was injected over the captured receptor surface followed by second injection of hlL-6. The percent reduction in hL-6 binding resulting from preformed antibody and receptor complex was used as a score to define hlL-6 blockers from non-blockers (second column, Table 2).
Table 2. Neutralization of hlL-6 Binding
Figure imgf000026_0001
[0085] The ability of hlL-6R antibodies to block hlL-6 activity in vitro was measured in the hlL- 6-dependent myeloma line XG-1. XG-1 cells maintained in hlL-6-containing medium were washed twice with hl L-6-free media and cultured for -24 hours in hlL-6-free medium to deplete residual hlL-6. The starved cells were then spun down and re-suspended in the medium at 4 x 105 cells per ml and plated 20,000 cells per well in a 96-well tissue culture plate. The purified antibody proteins were serially diluted in medium and added to the plated cells at concentrations ranging from 0 to 50 nM. Subsequently, recombinant hlL-6 was added to the wells to a final concentration of 8 pM. Cells were allowed to grow for -72 hours at 37°C in a humidified 5% C02 incubator. At the end of growth period, live cells were measured using CCK-8 kit (Dojindo, Japan). IC50s were determined as described above, and reported in the third column of Table 2.
[0086] The ability of hlL-6R antibodies to block hlL-6 activity was also measured in vitro in the hlL-6-responsive human hepatoma cell line, HepG2. HepG2 cells were transfected with a reporter plasmid containing a STAT3 (Signal Transducer and activator of Transcription 3) response element linked to a luciferase gene. The transfected cells were trypsinized, spun down and re-suspended in the medium at approximately 2.5 x 105 cells per ml and plated at 20,000 cells per well in a 96-well tissue culture plate. The purified antibody proteins were serially diluted in medium and added to the plated cells at concentrations ranging from 0 to 100 nM. Subsequently, recombinant hlL-6 was added to the wells to a final concentration of 50 pM. The response was measured after incubating the cells for 6 hours at 37°C in a humidified 5% C02 incubator. Luciferase activity was measured with the Steady-Glo™ luciferase assay system (Promega). IC50s were determined as described above, and reported in the fourth column of Table 2.
Example 5. Binding Epitope Diversity
[0087] An antibody binding competition immunoassay was performed using as a control humanized antibody to human IL-6R. Briefly, a 96-well immunosorbent plate was coated with 20 ng per well hlL-6R recombinant protein overnight at 4°C. After blocking non-specific binding with BSA, the hlL-6R binding sites on one half of the plate were saturated with binding of the control antibody by addition of 500 ng of the control per well, and to the other half of the plate was added binding buffer only. After three hours binding at room temperature, the purified antibodies were spiked in at a final concentration of 50 ng/ml with and without the preexisting control antibody in the well. After one hour of additional binding, the free antibody was washed away and the plate-bound antibody was detected with HRP-conjugated goat anti-mouse IgG or IgA, polyclonal antibody and the plate was developed using chromatic HRP substrates and absorbance at 450 nm was recorded. Percentage deductions of the binding of the anti-hlL6R antibodies by the presence of the control antibody are listed in Table 3 below. A similar experiment was conducted using surface plasmon resonance technology (Table 3). Both methods generated consistent results. Antibodies VQ8F1 1 , W3D8, VV6A9, VV6C10-1 bound epitopes overlapping with the control antibody; while antibodies VQ8A9, W1 G4, VV6F12, VV7G4, W9A6, and W6C10-3 appeared to bind distinct epitopes as antigen binding was not blocked by the control antibody. Partial competition may result from steric hindrance from the first antibody bound, even though epitopes may not be overlapping.
Table 3. Competition of Antigen Binding with Control Antibody
Figure imgf000028_0001
Example 6. Cross-species Binding Property
[0088] Four antibodies were tested for cross-reactivity to monkey IL-6R recombinant protein using BIAcore™ technology. Briefly, a biosensor chip on which goat anti-mouse Fc polyclonal antibody was immobilized was used to present anti-hlL-6R monoclonal antibodies to a density of about 75 RU. Recombinant human or monkey monomeric IL-6R protein (Macaca fascicularis, extracellular domain; SEQ ID NO:251 ), at a concentration range between 1.25 - 40 nM, was injected over the antibody surface. The binding of the receptor to the antibody and the dissociation of the bound complex were monitored in real-time. Both association rate constant (ka) and dissociate rate constant (kd) were obtained, and KD calculated (Table 4).
Table 4. Comparison of Binding Affinity to Human and Monkey IL-6R
Figure imgf000028_0002
[0089] Among the four tested antibodies, VQ8F1 1 , VV6A9, and VQ8A9 strongly reacted to monkey receptor with KD values that differed by about 1.5- to about 3-fold from human receptor binding, respectively. W1 G4, which was not blocked by the control antibody (Table 3), showed no binding to monkey receptor despite strong binding to the human receptor with KD of 241 pM.
Example 7. Effect of Constant Region on Binding Affinity
[0090] The binding affinity to monomeric hlL-6R of four antibodies having mouse IgG, human lgG1 or human lgG4 (wild-type and modified) were determined using BIAcore™ as described above except a goat anti-human Fc polyclonal antibody surface was used to capture hlgG antibodies. Monomeric hlL-6R was injected at concentrations of 12.5, 6.25, 3.12, and 1.56 nM. The ability of the antibodies to neutralize hlL-6-dependent HepG2/STAT3 signal transduction was also determined in a luciferase assay (IC50). IC50s for different IgG isotypes were similar, suggesting no effect of isotype on antibody affinity for antigen.
Table 5. Comparison of IgG Isotypes
Figure imgf000029_0001
Example 8: Biomarker Analysis
[0100] Biomarker analysis was conducted on samples taken from subjects who participated in clinical trials of mAbl . In particular, C1 M, C2M, C3M, and CRPM levels were measured in samples from patients at baseline and at different time points following initiation of study treatment(s). Clinical trials of mAbl are further described below and in U.S. Patent Application Publication 2013/0149310. [0101] A dose-ranging phase 2 portion of a clinical trial of subcutaneous mAb1 in patients with active RA treated with concomitant methotrexate (MTX) was conducted. Sera were collected at baseline, 2 weeks, and 12 weeks from patients randomized to treatment with methotrexate (MTX)+placebo (Pbo), MTX+mAb1 150 mg q2w (150mg of mAbl every other week), or MTX+mAb1 200 mg q2w (200 mg of mAbl every other week). C1 M, C2M, C3M and CRPM were analyzed by ELISA.
[0102] Baseline levels of each biomarker were similar among treatment groups. mAbl treatment at both doses resulted in marked reduction in levels of C1 M, C2M, C3M and CRPM from baseline at 2 weeks (see Table 6 below, percent change from baseline ±SE) and suppression of each marker was maintained at 12 weeks (see Table 7 below, percent change from baseline ±SE). mAbl treatment in patients with RA resulted in significant dose-dependent reduction in MMP-generated neo-epitope biomarkers related to joint and tissue turnover, whereas there was no significant decrease in these biomarkers in the MTX+Pbo group at either 2 and 12 weeks.
Table 6. Percent Change in RA Biomarkers from Baseline at 2 Weeks
Figure imgf000030_0001
Table 7. Percent Change in RA Biomarkers from Baseline at 12 Weeks
Figure imgf000030_0002
Example 9: Further Biomarker Analysis
[0103] Additional biomarker analysis was conducted on samples taken from subjects who participated in clinical trials of mAbl . Clinical trials of mAbl are further described below and in U.S. Patent Application Publication 2013/0149310.
[0104] Patients with rheumatoid arthritis (RA) develop bone and joint damage due to chronic inflammation mediated by critical cytokines, such as IL-6. Blockade of IL-6R signaling by sarilumab significantly reduced structural damage in RA patients, as measured by the modified van der Heijde total Sharp Score, including the erosion score and joint space narrowing components in the Phase 3 (part B) portion of the MOBILITY trial. To elucidate the mechanism of clinical reduction of bone and joint damage by sarilumab, and to investigate the role of IL-6 signaling in the regulation of osteoclasts and fibroblast-like synoviocytes (FLS) and their ability to increase levels of bone resorptive molecules like RANKL (receptor activated NF kB ligand) and joint destructive proteins, such as matrix metalloproteinases (MMPs), we evaluated a panel of markers associated with bone resorption and formation (RANKL, OPG, CTX-1 , osteocalcin and C1 M), synovium (MMP3 and C3M) and cartilage degradation (C2M and P2NP) in patients enrolled in the MOBILITY Part B trial (NCT01061736).
[0105] Sera were analyzed from patients randomized to either Placebo (Pbo) + methotrexate (MTX), or subcutaneous administration of 200 mg sarilumab every other week (q2w) + MTX. Levels of serum biomarkers [CTX-1 , C1 M, osteocalcin (OC), C3M, MMP-3, C2M, P2NP, RANKL and OPG] were measured by ELISA. All biomarkers were analyzed at baseline, and post- treatment at week 2 and 24, with the exceptions of CTX-1 and OC, which were analyzed at baseline, week 24 and week 52 post-treatment. Week 52 samples were assessed for patients who completed study; samples collected at or after week 40 were evaluated if patients discontinued treatment prior to week 52.
[0106] Baseline levels of each biomarker were similar between treatment groups. Levels of C1 M, and C3M were significantly lower in the sarilumab 200 mg q2w+MTX treatment group at 2 and 24 weeks post-treatment compared to baseline (Table 8). By week 24, levels of RANKL and MMP-3 were reduced significantly in the sarilumab 200 mg q2w+MTX group but not in the Pbo+MTX group. Levels of CTX-1 , OPG, C2M and P2NP were similar between treatment groups, although C2M showed a slight decrease following sarilumab+MTX treatment. In contrast, OC, a marker of bone formation, increased from baseline in the sarilumab 200 mg q2w+MTX treatment group at weeks 24 and 52. Table 8. Median percent change in RA Biomarkers from Baseline
Figure imgf000032_0001
[0107] The ratios of RANKL to OPG were also calculated for weeks 0, 2, and 24. As shown in table 9 below, a greater decrease in the log RANKL/OPG ratio occurred in subjects receiving of 200 mg sarilumab every other week (q2w) + MTX, as opposed to placebo + MTX.
Table 9. Log RANKL/OPG Ratios
Figure imgf000032_0002
[0108] Sarilumab reduced bone resorption and joint damage markers and increased osteocalcin, a marker of bone formation, in RA patients. Notably, sarilumab treatment lead to RANKL reduction and RANKL/OPG ratio reduction in RA patients. This data demonstrates that increased IL-6 signaling promotes structural damage through osteoclasts and FLS and by reducing osteoblast bone formation, and conversely, by blocking IL-6 signaling, osteoclast- mediated structural damage can be reduced and osteoblast-mediated bond formation can be restored. Example 10: Biomarker Predictive Analysis
[0109] The biomarkers described in this current study were readily measured in serum collected from patients in each treatment group. Sarilumab + MTX significantly reduced levels of markers of joint inflammation and collagen degradation including MMP-3, C1 M, C2M, and C3M in patients with RA compared with placebo + MTX. Sarilumab + MTX significantly reduced levels of RANKL, the major osteoclastogenic factor, and decreased the ratio of RANKL/OPG. This is the first report that inhibition of IL-6 signaling leads to RANKL reduction in patients with RA. These data indicate that inhibition of IL-6R signaling in patients with RA reduces osteoclast-mediated structural damage and increases osteoblast-mediated bone formation.
[0110] Correlation with baseline levels: Baseline levels of RANKL and the RANKL:OPG ratio were not correlated with baseline mTSS, ES, JSN. However, a trend for the association between baseline OPG levels and baseline radiographic scores was observed, but only in the sarilumab+MTX group and not in the placebo arm.
Table 10: Correlation of Baseline Disease Indices with Baseline Biomarker Levels*
Figure imgf000033_0001
Figure imgf000033_0002
Figure imgf000034_0001
*Correlation between baseline level in biomarker data and baseline efficacy data
[0111] Correlation with radiographic progression: For every 1 pmol/L increase in baseline OPG levels (approximately 20% of baseline levels), the odds ratio increased for radiographic progression by the mTSS and ES but not the JSN. This result is unexpected given the association of OPG with anti-bone resorption, yet the increased levels at baseline were associated with increased radiographic progression at week 52 in the sarilumab treatment group (Table 1 1 below).
Table 11 : Correlation of Disease Progression (week 52) with Baseline OPG Levels*
Figure imgf000034_0002
"Logistic regression results on radiographic progression; results of fixed effects; NS=not significant
[0112] To determine if the percent change in biomarkers at week 24 was prognostic or predictive of radiographic change at week 52, logistic regression was performed using the upper tertile vs the lower tertile (≥Q3 vs <Q3) as a cut-off for the percent change in OPG. Patients in the sarilumab + MTX treatment group with less than a 9% percent change (< Q3) in OPG from baseline at week 24 were more significantly more likely to have radiographic progression (mTSS and ES) at week 52 compared to the patients that showed greater percent change in OPG from baseline. Since the extent of OPG change from baseline did not affect the radiographic response in the placebo group significantly, these data indicate that the percent change in OPG at week 24 is predictive of sarilumab response rather than reflecting general prognostic value (Table 12 below). Table 12: Progression of Disease Indices at Wk 52 by Percent Change of OPG at Wk 24*
Figure imgf000035_0001
* >Q3 vs <Q3
[0113] Spearman rank correlations also confirmed that the percent change in OPG at week 24 was negatively correlated with radiographic change for mTSS and ES (JSN trend for significance only) at week 52 in the sarilumab treatment group but not in the placebo + MTX group (Table 13 below).
Percentage Change in OPG at W24 Correlation with Disease Progression at
Figure imgf000035_0002
[0114] Regression analysis was performed for every 200 or 1000 pmol increase in baseline RANKL levels and radiographic progression at week 52. Odd ratios were not significantly different from one, indicating that the RANKL levels were not able to predict the patients with radiographic progression versus the non-progressors in either the placebo group or in the sarilumab treatment group. The RANKL/OPG ratio odds ratios were not significantly different than one for either mTSS, ES or JSN. Additionally, the percent change from baseline in RANKL at week 24 was also evaluated by regression analysis and did not distinguish the patients with and without radiographic progression at week 52 (data not shown).
[0115] These results indicate that OPG levels as well as early changes in OPG levels with sarilumab treatment have predictive values for efficacy of sarilumab treatment in RA patients, in contrast to other RA biomarkers. Example 11. Effect Anti-IL-6R Antibody Dose Modification ("Up-Titration") In Rheumatoid Arthritis Patients
Background
[0116] Sarilumab (also referred to herein as "mAbl ") is a human monoclonal antibody directed against IL-6R and has been shown to be effective with subcutaneous (SC) dose regimens of 150 mg and 200 mg once every two weeks (q2w) in rheumatoid arthritis (RA) patients with active disease despite treatment with methotrexate (MTX). The objective of the present Example was to assess the impact on efficacy and safety of increasing sarilumab dosing from 150 mg to 200 mg q2w in patients who showed poor response to the 150 mg dose.
Methods
[0117] Details of the MOBILITY clinical trial are described elsewhere (see, for example, U.S. Patent Application Publication 2013/0149310). Briefly, adults with moderate-to-severe active RA and inadequate response to MTX were randomized to: (1 ) placebo (pbo) + MTX; (2) sarilumab 150 mg q2w + MTX; or (3) sarilumab 200 mg q2w + MTX. At week 16, patients who did not achieve >20% improvement in swollen joint count (SJC) and tender joint count (TJC) or who had a clear lack of efficacy in the opinion of the investigator were offered rescue therapy with open-label sarilumab 200 mg q2w in all 3 dose arms (PBO, 150 mg q2w, 200 mg q2w).
Results
[0118] 1 197 patients were randomized to three treatment groups. A subset of each randomized group were rescued on or after Week 16. Results are summarized in Tables 14A and 14B.
Table 14A
Figure imgf000036_0001
LDAS (%)
Wk 16 1 .9% 0% 0%
Wk 24 15.4% 23.2% 23.3%
Wk 52 50.6% 56.8% 26.7%
mTSS change from baseline (mean)
Wk 52 2.30 ± 4.58 1 .59 ± 4.05
Table 14B
mAb1 , 200mg Q2W
(continue 200mg Q2W)
mAb1 , 150mg Q2W Rescue at or after mAb1 , 200mg Q2W
Efficacy Rescue at week 16 week 16 Rescue at Week 16 Parameters (N=30) (N=46) (N=28)
ACR20 (%)
Wk 16 7% 24% 7%
Wk 24 50% 28% 29%
Wk 52 50% 52% 46%
ACR50 (%)
Wk 16 0% 7% 0%
Wk 24 20% 15% 7%
Wk 52 27% 28% 32%
DAS28-CRP (mean ± SD)
Wk 16 5.63 ±1.09 4.85 ± 1.21 5.13 ±1.23
Wk 24 4.18 ±1.20 4.63 ±1 .66 4.62 ±1.54
Wk 52 3.74 ±1.22 3.79 ±1 .41 3.73 ±1.42
LDAS (%)
Wk 16 0% 8.7% 16.7%
Wk 24 23.3% 28.3% 33.3%
Wk 52 26.7% 30.4% 33.3%
mTSS change from baseline (mean)
Wk 52 -0.38 ± 4.35
[0119] Patients in the rescued placebo cohort responded to the 200 mg sarilumab rescue dose with improved ACR20 and ACR50 responses and decreased DAS28 CRP mean values at Weeks 24 and 52. The sarilumab 150 mg group rescued at Week 16 (n=30) exhibited a more pronounced response at Week 24 than the cohort rescued from the initial 200 mg group (n=28). The percentage of patients achieving LDAS (DAS28- CRP <3.2) at week 24 and week 52 were similar for sarilumab 150 mg and 200 mg rescued cohorts. The 150 mg and 200 mg rescued cohorts demonstrated comparable benefit by Week 52. Little effect on radiographic progression was observed with the placebo rescued cohort, and only modest effect was observed with those rescued from the sarilumab 150 mg group. Patients rescued from the 200 mg regimen demonstrated minimal radiographic progression over 52 weeks. Incidence of treatment emergent adverse events (TEAEs), serious adverse events, or adverse events leading to treatment discontinuation were similar in the open-label rescue and double-blind periods. Infections were the most frequently reported TEAEs in the open-label rescue period .
Conclusions
[0120] This Example demonstrates that increasing anti-IL-6R antibody dose to 200 mg q2w may have therapeutic value for RA patients with a suboptimal response at Week 16 to the 150mg q2w dose. Patients rescued from the 200mg q2w regimen exhibited minimal radiographic progression . The safety profile observed in the post-rescue period was generally consistent with that observed in the double-blind period.
[0121] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

What is claimed is:
1 . A method for treating rheumatoid arthritis (RA) in a subject, the method
comprising: (a) selecting a subject who exhibits an elevated level of at least one RA-associated biomarker prior to or at the time of treatment, and (b) administering to the subject a
pharmaceutical composition comprising a therapeutically effective amount of an interleukin-6 receptor (IL-6R) antagonist.
2. The method of claim 1 , wherein the IL-6R antagonist is an antibody or antigen- binding fragment thereof that specifically binds IL-6R.
3. The method of claim 2, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises heavy and light chain CDR sequences from a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 3/1 1 ; 227/229; 19/27;
231/233; 35/43; 51/59; 67/75; 83/91 ; 99/107; 1 15/123; 131/139; 147/155; 239/155; 241/155; 163/171 ; 179/187; 235/237; 195/203; and 21 1/219.
4. The method of claim 3, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises heavy and light chain CDR sequences from the HCVR/LCVR sequence pair of SEQ ID NOs: 19/27.
5. The method of claim 4, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises three heavy chain complementarity determining region (HCDR) sequences comprising SEQ ID NOs:21 , 23, 25, respectively, and three light chain complementarity determining (LCDR) sequences comprising SEQ ID NOs: 29, 31 , 33, respectively.
6. The method of claim 5, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises an HCVR having the amino acid sequence of SEQ ID NO: 19 and an LCVR having the amino acid sequence of SEQ ID NO: 27.
7. The method of any one of claims 1 to 6, wherein the RA-associated biomarker is selected from the group consisting of MMP cleaved collagen type 1 (C1 M), MMP cleaved collagen type 2 (C2M), MMP cleaved collagen type 3 (C3M), MMP cleaved C reactive protein (CRPM), RANKL, and OPG.
8. The method of claim 7, wherein the subject is selected on the basis of exhibiting a biomarker expression level selected from the group consisting of a mean serum C1 M level of greater than 180 ng/mL prior to or at the time of treatment ("baseline"), a mean serum C2M level of greater than 0.25 ng/mL prior to or at the time of treatment ("baseline"), a mean serum C3M level of greater than 50 ng/mL prior to or at the time of treatment ("baseline"), and a mean serum CRPM level of greater than 17 ng/mL prior to or at the time of treatment ("baseline").
9. The method of any one of claims 2 to 8, wherein the pharmaceutical composition comprises about 75 mg to about 300 mg of the antibody or antigen-binding fragment that specifically binds IL-6R, and wherein the pharmaceutical composition is administered to the subject subcutaneously.
10. The method of claim 8, wherein the subject exhibits a decrease in biomarker expression selected from the group consisting of at least a 29% decrease in mean serum C1 M level from baseline at day 14 or later following the administration, at least a 8% decrease in mean serum C2M level from baseline at day 14 or later following the administration, at least a 16% decrease in mean serum C3M level from baseline at day 14 or later following the administration, and at least a 17% decrease in mean serum CRPM level from baseline at day 14 or later following the administration.
1 1. The method of claim 10, wherein the subject exhibits a decrease in biomarker expression selected from the group consisting of at least a 47% decrease in mean serum C1 M level from baseline at day 14 or or later following the administration, at least a 12% decrease in mean serum C2M level from baseline at day 14 or later following the administration, at least a 24% decrease in mean serum C3M level from baseline at day 14 or later following the administration, at least a 24% decrease in mean serum C3M level from baseline at day 14 or later following the administration, and at least a 24% decrease in mean serum CRPM level from baseline at day 14 or later following the administration.
12. A method for treating rheumatoid arthritis (RA) in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein administration of the pharmaceutical composition to the subject results in a decrease in at least one RA-associated biomarker in the subject by about day 14, 84, or 168, following administration of the pharmaceutical composition as compared to the level of the biomarker in the subject prior to the administration.
13. The method of claim 12, wherein the RA-associated biomarker is selected from the group consisting of MMP cleaved collagen type 1 (C1 M), MMP cleaved collagen type 2 (C2M), MMP cleaved collagen type 3 (C3M), MMP cleaved C reactive protein (CRPM), RANKL, and OPG.
14. The method of claim 12 or 13, wherein the pharmaceutical composition comprises about 75 mg to about 300 mg of the antibody or antigen-binding fragment that specifically binds IL-6R.
15. The method of claim 14, wherein administration of the pharmaceutical composition to the subject results in a decrease in biomarker expression selected from the group consisting of at least a 29% decrease in mean serum C1 M in the subject at day 14 following administration of the pharmaceutical composition as compared to the mean serum C1 M level in the subject prior to the administration, at least a 8% decrease in mean serum C2M in the subject by day 14 following administration of the pharmaceutical composition as compared to the mean serum C2M level in the subject prior to the administration, at least a 16% decrease in mean serum C3M in the subject by day 14 following administration of the pharmaceutical composition as compared to the mean serum C3M level in the subject prior to the administration, and at least a 17% decrease in mean serum CRPM in the subject by day 14 following administration of the pharmaceutical composition as compared to the mean serum CRPM level in the subject prior to the administration.
16. The method of claim 15, wherein administration of the pharmaceutical composition to the subject results in a decrease in biomarker expression selected from the group consisting of at least a 47% decrease in mean serum C1 M in the subject at day 14 following administration of the pharmaceutical composition as compared to the mean serum C1 M level in the subject prior to the administration, at least a 12% decrease in mean serum C2M in the subject by day 14 following administration of the pharmaceutical composition as compared to the mean serum C2M level in the subject prior to the administration, at least a 24% decrease in mean serum C3M in the subject by day 14 following administration of the pharmaceutical composition as compared to the mean serum C3M level in the subject prior to the administration, and at least a 24% decrease in mean serum CRPM in the subject by day 14 following administration of the pharmaceutical composition as compared to the mean serum CRPM level in the subject prior to the administration..
17. The method of claim 15 or 16, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises heavy and light chain CDR sequences from a
HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs:3/1 1 ; 227/229; 19/27; 231/233; 35/43; 51/59; 67/75; 83/91 ; 99/107; 1 15/123; 131/139; 147/155; 239/155;
241/155; 163/171 ; 179/187; 235/237; 195/203; and 21 1/219.
18. The method of claim 17, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises heavy and light chain CDR sequences from the HCVR/LCVR sequence pair of SEQ I D NOs:19/27.
19. The method of claim 18, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises three heavy chain complementarity determining region (HCDR) sequences comprising SEQ ID NOs:21 , 23, 25, respectively, and three light chain complementarity determining (LCDR) sequences comprising SEQ ID NOs: 29, 31 , 33, respectively.
20. The method of claim 19, wherein the antibody or antigen-binding fragment that specifically binds IL-6R comprises an HCVR having the amino acid sequence of SEQ ID NO:19 and an LCVR having the amino acid sequence of SEQ ID NO:27.
21. The method of any one of claims 12 to 20, wherein the pharmaceutical composition is administered to the subject subcutaneously or intravenously.
22. The method of any one of claims 12 to 21 , wherein a second therapeutic agent is administered to the subject before, after or concurrent with the pharmaceutical composition.
23. The method of claim 22, wherein the second therapeutic agent is selected from the group consisting of a NSAID, DMARD, TNFa antagonist, T-cell blocker, CD-20 antagonist, IL-1 antagonist, JAK antagonist, and IL-17 antagonist.
24. A method for treating rheumatoid arthritis (RA) in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein the subject has been diagnosed with rheumatoid arthritis and has been selected for treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker pre-treatment, wherein the reduced expression of the biomarker is determined based on a comparison to a reference level of expression of the respective biomarker.
25. A method for treating rheumatoid arthritis (RA) in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds interleukin-6 receptor (IL-6R), wherein the subject has been diagnosed with rheumatoid arthritis, has been treated with the anti-IL6R antibody for a defined period of time, and has been selected for further treatment with the IL-6R antibody on the basis of exhibiting reduced expression of a biomarker, wherein the reduced expression of the biomarker is determined based on a comparison to the level of expression of the respective biomarker in the subject prior to treatment with the anti-IL- 6R antibody.
26. The method of claim 24 or 25, wherein the biomarker is selected from the group consisting of
27. The method of claim 25, wherein the defined period of time is selected from the group consisting of about day 14, 84, or 168 after administration of an initial dose of the anti- IL6R antibody.
28. The method of claim 25, wherein the biomarker is OPG and the comparison to the level of expression in the subject prior to treatment is expression of OPG that is equal to or greater than a 9% reduction in OPG expression after the defined period of time.
29. An interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits an elevated level of at least one RA-associated biomarker prior to or at the time of treatment.
30. An interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits a lower level of at least one RA-associated biomarker prior to or at the time of treatment, as compared to a reference level of the biomarker.
31. An interleukin-6 receptor (IL-6R) antagonist for use in a method for treating rheumatoid arthritis (RA) in a subject, wherein the subject exhibits a lower level of at least one RA-associated biomarker after treatment with the IL-6R antagonist for a defined period of time as compared to the level of the biomarker prior to treatment.
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