WO2009010539A2 - Recepteur de l'interleukine-6 (il-6) derive de macaca fascicularis - Google Patents

Recepteur de l'interleukine-6 (il-6) derive de macaca fascicularis Download PDF

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Publication number
WO2009010539A2
WO2009010539A2 PCT/EP2008/059325 EP2008059325W WO2009010539A2 WO 2009010539 A2 WO2009010539 A2 WO 2009010539A2 EP 2008059325 W EP2008059325 W EP 2008059325W WO 2009010539 A2 WO2009010539 A2 WO 2009010539A2
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amino acid
seq
acid sequence
protein
binding
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WO2009010539A3 (fr
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Els Anna Alice Beirnaert
Sophie Germaine Amanda De Keersmaeker
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Ablynx NV
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Ablynx NV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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 a protein from cynomolgus monkey (Macaca fascicularis) that functions as a receptor for interleukin-6 (“IL-6”), and to a nucleic acid encoding the same.
  • IL-6 interleukin-6
  • the present invention further relates to compounds that interact with (as defined herein) said IL-6 receptor protein.
  • Said compounds maybe small chemical entities or biological molecules, and may in particular be proteins or polypeptides. More in particular, said compounds may be immunoglobulins or fragments thereof, such as antibodies, antibody fragments (such as Fab' fragments, F(ab') 2 fragments and Fv fragments), constructs derived from antibody fragments (such as ScFv fragments and diabodies), or domain antibodies, single domain antibodies, dAb's or Nanobodies.
  • the compounds described herein are preferably cross-reactive with the human IL-6 receptor.
  • the present inventors have now isolated a nucleic acid (cDNA) from cynomolgus monkey that encodes a receptor for IL-6.
  • cDNA nucleic acid
  • the nucleotide sequence of said cDNA is given in SEQ ID NO:3 and Figure IB and the corresponding amino acid sequence is given in SEQ ID NO: 4 and Figure 3B.
  • the published DNA sequence of the human IL-6 receptor is given in SEQ ID NO:5 and Figure 1C and the corresponding amino acid sequence is given in SEQ ID NO: 6 and Figure 3C.
  • Figure 2 shows an alignment of the cDNA sequence of Imazeki et al. and of the cDNA sequence of the present invention, respectively.
  • Figure 4 shows an alignment of the amino acid sequence of Imazeki et al., of the amino acid sequence of the present invention, and of the amino acid sequence of the human IL-6 receptor, respectively.
  • the amino acid sequence of SEQ ID NO: 4 differs at 5 amino acid positions (positions 4, 5, 48, 85 and 327) from the amino acid sequence disclosed by Imazeki et al. At 4 of these 5 positions, the amino acid sequence of Imazeki et al. is identical to the amino acid sequence of the human IL-6 receptor. As a result, it is believed that compounds that can interact with the human LL-6R receptor and with the sequence disclosed by Imazeki et al. may not necessarily also be capable of interacting with the amino acid sequence of SEQ ID NO: 4.
  • proteins or polypeptides that can bind specifically (as defined herein) to human IL-6 receptor may not necessarily also be capable of specific binding to the amino acid sequence of SEQ ID NO: 4.
  • proteins according to the present invention that function as a receptor for IL-6 in cynomolgus monkeys include in addition to the amino acid sequence of SEQ ID NO: 4, any proteins or polypeptides derived thereof that are still capable of specifically binding to IL-6, and in particular to cynomolgus IL-6.
  • modified proteins or polypeptides of SEQ ID NO: 4 wherein one or more than one amino acid residue in the above-mentioned amino acid sequence is replaced by a different amino acid residue and/or wherein one or more than one amino acid residue is deleted and/or wherein one or more than one amino acid residue is added to the amino acid sequence of SEQ ID NO: 4, while maintaining the biological activity of the native cynomolgus IL-6R.
  • such proteins may be proteins wherein an amino acid sequence and/or an amino acid residue, apart from a region in the amino acid sequence of SEQ ID NO: 4 that is essential for binding to IL-6, are deleted or replaced with another amino acid sequence and/or amino acid residue and/or may be proteins wherein an amino acid sequence and/or an amino acid residue are added to the amino acid sequence of SEQ ID NO: 4, such as added at the N-terminal or C-terminal end thereof.
  • modified proteins or polypeptides at least one of the amino acid residue positions 4, 5, 48, 85 and 327 may be occupied by an amino acid residue that occurs at at least one of the corresponding amino acid residue positions 4, 5, 48, 85 and 327 of SEQ ID NO: 4; more preferably, modified proteins or polypeptides according to the invention have at two or three of the amino acid residue positions 4, 5, 48, 85 and 327 an amino acid residue that occurs at two or three of the corresponding amino acid residue positions 4, 5, 48, 85 and 327 of SEQ ID NO: 4; yet more preferably, have at four or five of the amino acid residue positions 4, 5, 48, 85 and 327 an amino acid residue that occurs at four or five of the corresponding amino acid residue positions 4, 5, 48, 85 and 327 of SEQ ID NO: 4.
  • antibodies, binders and/or compounds that bind to SEQ ID NO: 4, while not binding to SEQ ID NO: 6, should also be capable of binding to the above described modified proteins or polypeptides of the invention, while not binding to SEQ ID NO: 6; alternatively, it is encompassed in the present invention that antibodies, binders and/or compounds that bind to SEQ ID NO: 4 and to SEQ ID NO: 6, should also be capable of binding to the above described modified proteins or polypeptides of the invention and to SEQ ID NO: 6.
  • fusion proteins comprising the amino acid sequence of SEQ ID NO: 4 or a modified protein or polypeptide as described above.
  • Such fusion proteins may for instance comprise the amino acid sequence of SEQ ID NO: 4 or a modified protein or polypeptide as described above and further one or more detectable labels or other signal- generating groups or moieties, depending on the intended use of the labelled protein of the invention. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person and for example described on pages 109- 110 of WO 08/020079.
  • fluorescent labels may include, but are not limited to fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labels, radio-isotopes, metals, metal chelates, metallic cations, chromophores and enzymes.
  • suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.
  • antigenic determinants based on SEQ ID NO: 4 or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. Any part, fragment, region domain, or other epitope based on SEQ ID NO: 4 or derived therefrom that could be used for eliciting an immune response in a subject, such as a mammal, is also encompassed in the present invention.
  • said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope.
  • the proteins, polypeptides or antigenic determinants according to the present invention may be formulated as a preparation or composition comprising at least one protein, polypeptide or antigenic determinant of the invention and at least one diluent or excipient and/or adjuvant, and optionally one or more further polypeptides and/or compounds.
  • the proteins of the present invention may be prepared with a suitable diluent, excipient and/or adjuvant for immunization of a mammal with said proteins, polypeptides or antigenic determinants.
  • such a preparation or composition may be in a form suitable for any kind of administration to a mammal, and preferably for oral and/or parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion).
  • suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.
  • the present invention also relates to nucleic acid sequences that encode the proteins, and modified proteins, polypeptides, fusion proteins or antigenic determinants as described above.
  • a compound in order to have (significant) biological activity in cynomolgus monkey (e.g. in one of the disease models mentioned herein), a compound should preferably be able to bind specifically (as defined herein) to the amino acid sequence of SEQ ID NO: 4.
  • the invention also provides compounds that can interact with the amino acid sequence of SEQ ID ISIO: 4; and also provides methods and means for generating such compounds.
  • interact with is generally meant that the such compounds can either (i) interact with (as defined herein) the IL- ⁇ receptor protein described herein (i.e.
  • the amino acid sequence of SEQ ID NO:4) and/or (ii) modulate the IL- ⁇ receptor protein described herein; and/or (iii) modulate one or more of the interactions, pathways, mechanisms, signalling, biological effects and/or biological responses in which the IL-6 receptor protein described herein is involved (such as IL-6 or IL-6R mediated signalling); and/or (iv) otherwise effect a significant biological change through interaction of the compound with the IL-6 receptor protein described herein.
  • the invention provides compounds that can interact with the amino acid sequence of SEQ ID NO: 4 and that can also interact with the amino acid sequence of the human IL-6 receptor; and also provides methods and means for generating such compounds.
  • the invention provides proteins and polypeptides that can bind specifically (as defined herein) to the amino acid sequence of SEQ ID NO: 4; and also provides methods and means for generating such proteins and polypeptides, hi particular, the invention provides crossreactive proteins and polypeptides that can bind specifically to both the amino acid sequence of SEQ ID NO: 4 as well as the amino acid sequence of the human IL-6 receptor; and also provides methods and means for generating such proteins and polypeptides.
  • the invention provides proteins and polypeptides that can bind (and in particular, specifically bind) to the amino acid sequence of SEQ ID NO: 4 in at least one (such as any two, any three or all) of the ELISA-based assays described in Examples 1, 2, 3 and/or 4A/4B described below, and or in a standard BlACORE assay (using standard conditions); and also provides methods and means for generating such proteins and polypeptides.
  • the invention provides crossreactive proteins and polypeptides that can bind (and in particular, specifically bind) to both the amino acid sequence of SEQ ID NO: 4 as well as the amino acid sequence of the human IL-6 receptor in at least one (such as any two, any three or all) of the ELISA-based assays described in Examples 1 , 2, 3 and/or 4A/4B described below, and or in a standard BIACORE assay (using standard conditions); and also provides methods and means for generating such proteins and polypeptides.
  • the invention relates to a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:4, preferably the nucleic acid sequence of SEQ ID NO:3, but also including degenerated nucleic acid sequences.
  • Said nucleic acid is preferably in essentially isolated form, and may be in the form of a construct or vector containing one or more further nucleic acid sequences, such as one or more regulatory elements.
  • the invention also relates to a protein or polypeptide that comprises or essentially consists of the amino acid sequence of SEQ ID NO: 4.
  • Said protein or polypeptide is preferably in essentially isolated form.
  • SEQ ID NO: 4 differs at 5 amino acid positions (positions 4, 5, 48, 85 and 327) from the amino acid sequence disclosed by Imazeki et al. (SEQ ID NO:2).
  • the invention also relates to a chimeric protein or polypeptide that comprises or essentially consists of the amino acid sequence of SEQ ID NO:2 having one to four substitutions from among the 5 substituted amino acid positions (positions 4, 5, 48, 85 and 327) of SEQ ID NO:4.
  • a first chimeric protein or polypeptide with one amino acid substitution has the amino acid sequence of SEQ ID NO:2 except for the amino acid of SEQ ID NO:4 at position 4.
  • Another chimeric protein or polypeptide with one amino acid substitution has the amino acid sequence of SEQ ID NO: 2 except for the amino acid of SEQ ID NO:4 at position 5. Additional chimeric proteins or polypeptides with one amino acid substitution having the amino acid sequence of SEQ ID NO: 2 except for the amino acid of SEQ ID NO: 4 at position 48 or 85 or 327 also are provided. Chimeric proteins or polypeptides with two amino acid substitutions include the amino acid sequence of SEQ ID NO: 2 except for the amino acids of SEQ ID NO: 4 at positions 4 and 5; 4 and 48; 4 and 85; 4 and 327; 5 and 48; 5 and 85; 5 and 327; 48 and 85; 48 and 327; or 85 and 327.
  • Chimeric proteins or polypeptides with three amino acid substitutions include the amino acid sequence of SEQ ID NO: 2 except for the amino acids of SEQ ID NO: 4 at positions 4, 5 and 48; 4, 5 and 85; 4, 5 and 327; 4, 48 and 85; 4, 48 and 327; 4, 85 and 327; 5, 48 and 85; 5, 48 and 327; 5, 85 and 327; or 48, 85 and 327.
  • Chimeric proteins or polypeptides with four amino acid substitutions include the amino acid sequence of SEQ ID NO: 2 except for the amino acids of SEQ ID NO: 4 at positions 4, 5, 48 and 85; 4, 5, 48 and 327; 4, 5, 85 and 327; 4, 48, 85 and 327;or 5, 48, 85 and 327.
  • Said chimeric protein or polypeptide is preferably in essentially isolated form.
  • Nucleic acids encoding the chimeric protein or polypeptide including preferably the nucleotide sequence of SEQ ID NO: 1 substituted as appropriate with nucleotides from SEQ ID NO: 3 that encode the substituted amino acids.
  • Nucleic acids containing degenerate nucleotide sequences also are provided.
  • Expression vectors comprising the foregoing nucleic acids also are provided.
  • the invention relates to a compound that can interact with the amino acid sequence of SEQ ID NO: 4 (or the chimeric protein or polypeptide).
  • Said compound may in particular be a protein or polypeptide or a nucleic acid.
  • Such proteins or polypeptides may for example be based on and/or comprise a suitable protein scaffold, such as binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as AffibodiesTM), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, VoI 23: 1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):6
  • Such a protein or polypeptide may in particular comprise or essentially consist of an immunoglobulin sequence (e.g. an antibody), a suitable fragment thereof, or a construct based thereon (such as Fab' fragments, F(ab') 2 fragments, ScFv constructs, "diabodies” and other multispecif ⁇ c constructs, see for example the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep;23(9):l 126-36), and may more in particular comprise or essentially consist of an immunoglobulin variable domain sequence or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g.
  • an immunoglobulin sequence e.g. an antibody
  • a suitable fragment thereof e.g. an antibody
  • a construct based thereon such as Fab' fragments, F(ab') 2 fragments, ScFv constructs, "diabodies”
  • the protein or polypeptide when it is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a Vn sequence that is derived from a human antibody) or be a so-called V RH sequence (as defined herein) that is derived from a so-called “heavy chain antibody” (as defined herein).
  • a conventional four-chain antibody such as, without limitation, a Vn sequence that is derived from a human antibody
  • V RH sequence as defined herein
  • such a protein or polypeptide may comprise or essentially consist of a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® (as defined herein, and including but not limited to a V HH sequence); other single variable domains, or any suitable fragment of any one thereof.
  • a domain antibody or an amino acid sequence that is suitable for use as a domain antibody
  • a single domain antibody or an amino acid sequence that is suitable for use as a single domain antibody
  • a “dAb” or an amino acid sequence that is suitable for use as a dAb
  • Nanobody® as defined herein, and including but not limited to a V HH sequence
  • such a protein or polypeptide may comprise or essentially consist of a Nanobody® (as defined herein) or a suitable fragment thereof.
  • Nanobody® as defined herein
  • Nanoclone® are registered trademarks of ⁇ blynx N.
  • Such a protein or polypeptide may also be a humanized Nanobody, or may be a multivalent or multispeciflc construct comprising one or more Nanobodies (at least one of which interacts with the amino acid sequence of SEQ ID NO:4) and optionally one or more further amino acid sequences (which may for example be one or more other Nanobodies).
  • WO 08/020079 by Ablynx N. V. entitled "Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with IL-6-mediated signalling", which describe Nanobodies against the human IL-6R, uses thereof (i.e.
  • Nanobodies that interact with the human IL-6 receptor can be formatted as multivalent or multispeciflc constructs (i.e. in particular on page 7, 60 and 118 to 129 of WO 08/020079), for example as constructs with increased half-life in serum (i.e. in particular on page 13, 38 to 40, 57 and 113 to 126of WO 08/020079).
  • proteins or polypeptides described herein are preferably such that they are capable of specific binding to the amino acid sequence of SEQ ID NO: 4, by which is generally meant that such proteins or polypeptides will either (i) bind to the amino acid sequence of SEQ ID NO: 4 with a dissociation constant (K D ) of lO "5 to 10 "12 moles/liter or less, and preferably 10 "7 to 10 "12 moles/liter or less and more preferably 10 "8 to 10 "12 moles/liter (i.e.
  • K D dissociation constant
  • K A association constant of 10 5 to 10 12 liter/ moles or more, and preferably 10 to 10 liter/moles or more and more preferably 10 to 10 liter/moles); and/or (ii) bind to the amino acid sequence of SEQ ID NO: 4 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, even more preferably less than 1 nM, such as less than 500 pM.
  • Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • proteins or polypeptides described herein are cross- reactive with the amino acid sequence of the human IL-6 receptor given in SEQ ID NO: 6, by which is meant that said proteins and polypeptides can bind specifically (as defined herein) to both the amino acid sequence of SEQ ID NO: 4 as well as the amino acid sequence of SEQ ⁇ D NO: 6.
  • proteins or polypeptides described herein can bind to the amino acid sequence of SEQ ID NO: 4 with an affinity of less than 10 nM, even more preferably less than 1 nM.
  • proteins or polypeptides described herein can bind to the amino acid sequence of SEQ ID NO: 4 with an affinity of less than 10 nM, even more preferably less than 1 nM; and are also capable of binding to the amino acid sequence of SEQ ID NO: 6 with an affinity of less than 10 nM, even more preferably less than 1 nM.
  • the proteins and polypeptides of the invention may bind (i) with higher affinity to the amino acid sequence of SEQ ID NO:4 than to the amino acid sequence of SEQ ID NO:6; (ii) with higher affinity to the amino acid sequence of SEQ ID NO: ⁇ than to the amino acid sequence of SEQ ID NO:4; or with about the same affinity to the amino acid sequence of SEQ ID NO:4 than to the amino acid sequence of SEQ ID NO: 4.
  • the affinity with which a protein or polypeptide of the invention bind to the amino acid sequence of SEQ ID NO:4 is no more than 10.000 times, preferably no more than 1000 times, more preferably no more than 100 times higher (or lower, respectively) than the affinity with which the protein or polypeptide of the invention binds to the amino acid sequence of SEQ ID NO:6.
  • amino acid sequence of SEQ ID NO:4 differs from the amino acid sequence of SEQ ID NO:6 at three positions (amino acid residues 4, 5 and 183, which are R, R and F, respectively, in the human sequence; and G, G and L, respectively, in the amino acid sequence of the invention) which together form part of the same epitope on the folded receptor (see the co-pending US provisional application US 61/063,208 by Ablynx N.V, entitled "Improved amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment ofIl ⁇ 6R related diseases and disorders".
  • amino acid sequence of the invention differs at two of these three positions from the amino acid sequence that was published by Imazeki et al. (amino acid residues 4 and 5, which are R and R, respectively, in the Imazeki sequence; and G and G, respectively, in the amino acid sequence of the invention).
  • the differences in amino acid sequence between the amino acid sequence of the invention on the one hand, and the human amino acid sequence as well as the Imazeki sequence on the other hand mean that these amino acid sequences differ in respect of the amino acid residues that form this epitope. This is further confirmed by the fact that the amino acid sequence of SEQ ID NO: 618 in the International patent application WO08/020079 by Ablynx N. V.
  • a protein or polypeptide described herein is such that:
  • amino acid sequence that corresponds to an IL6 receptor protein wherein the amino acid residues at the positions that correspond to positions 4, 5 and 183 in SEQ ID NO: 4 (which are GGL in SEQ ID NO: 4) are GGL, GRL, RGL, GGF, GRF, RGF 5 RRL or RRF, in particular GRF, RGF, RRL or RRF, and more in particular RRF, with an affinity of less than 10 nM, even more preferably less than 1 nM.
  • a protein or polypeptide described herein is such that: it can bind to the amino acid sequence of SEQ ID NO: 4 with an affinity of less than 10 nM, even more preferably less than 1 nM; and further such that: it is also capable of binding to at least one of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8 with an affinity of less than 10 nM, even more preferably less than 1 nM.
  • a protein or polypeptide described herein is such that: it can bind to the amino acid sequence of SEQ ID NO: 4 with an affinity of less than 10 nM, even more preferably less than 1 nM; and further such that: it is also capable of binding to any two of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8 with an affinity of less than 10 nM, even more preferably less than 1 nM.
  • a protein or polypeptide described herein is such that:
  • proteins and polypeptides described herein, and in particular the proteins and polypeptides that are described in the preceding paragraphs, may further be such that they bind (and/or are capable of binding) to the epitope that, among other amino acid residues, is formed by the amino acid residues at positions 4, 5 and 183 (i.e. of which the amino acid residues at positions 4, 5 and 183 form part), i.e. on the amino acid sequence of SEQ ID NO: 4 and/or on one or more of the amino acid sequences with which the proteins or polypeptides are cross-reactive.
  • proteins and polypeptides described herein, and in particular the proteins and polypeptides that are described in the preceding paragraphs may further be such that they bind (and/or are capable of binding) to another epitope on the amino acid sequence of SEQ ID NO: 4 (and/or on one or more of the amino acid sequences with which the proteins or polypeptides are cross-reactive) than the epitope of which the amino acid residues at positions 4, 5 and 183 form part.
  • proteins and polypeptides described herein, and in particular the proteins and polypeptides that are described in the preceding paragraphs, may farther be such that they are capable of modulating (as defined herein) the binding of IL-6 to its receptor; the signalling that is mediated by the binding of IL-6 to its receptor and/or more generally the signalling that is associated with IL-6, its receptor or the mechanism or biological pathways in which IL-6 and/or its receptor are involved; and/or of modulating the biological mechanisms, responses and effects associated with such signalling or these pathways.
  • proteins and polypeptides capable of modulating (as defined herein) the binding of IL-6 to its receptor reference is again made to the International patent application WO 08/020079 of Ablynx N.V. (i.e. in particular to pages 2 to 5, 40 to 42 and 147).
  • proteins and polypeptides described herein, and in particular the proteins and polypeptides that are described in the preceding paragraphs, may also (and/or further) be such that they are capable of competing with one or more of the following amino acid sequences: IL6R03 (SEQ ID NO: 13), IL6R10 (SEQ ID NO: 19), IL6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/or IL6R201 (SEQ ID NO: 31) for binding to the human amino acid sequence of SEQ ID NO: 6, and/or such that they can cross-block (as defined herein) the binding of IL6R03 (SEQ ID NO: 13), IL6R10 (SEQ ID NO: 1.9), IL6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/or IL6R201 (SEQ ID NO:
  • proteins and polypeptides described herein, and m particular the proteins and polypeptides thai are described in the preceding paragraphs, may also (and/or further) be such that they are not capable of competing with with one or more of the following amino acid sequences: IL6R03 (SEQ ID NO: 13), IL6R10 (SEQ ID NO: 19), IL6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/or IL6R201 (SEQ ID NO: 31) for binding to the human amino acid sequence of SEQ ID NO: 6, and/or such that they are not capable of cross-blocking (as defined herein) the binding of IL6R03 (SEQ ID NO: 13), IL6R10 (SEQ ID NO: 19), 1L6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/
  • an amino acid sequence such as a Nanobody, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof
  • an amino acid sequence that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against” or “directed against” said antigenic determinant, epitope, antigen or protein.
  • the term "specificity" has the meaning given to it in paragraph n) on pages 53-56 of WO 08/020079; and as mentioned therein refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen -binding protein (such as a Nanobody or a polypeptide of the invention) molecule can bind.
  • the specificity of an antigen-binding protein can be determined based on affinity and/or avidity, as described on pages 53-56 of WO 08/020079 (incorporated herein by reference), which also describes some preferred techniques for measuring binding between an antigen-binding molecule (such as a Nanobody or polypeptide of the invention) and the pertinent antigen.
  • antigen-binding proteins such as the amino acid sequences, Nanobodies and/or polypeptides of the invention
  • K D dissociation constant
  • K A association constant
  • any K R value greater than 10 4 mol/liter (or any K A value lower than 10 4 M "1 ) liters/mol is generally considered to indicate non-specific binding.
  • a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 iiM, preferably less than 200 oM, more preferably less than 10 nM, such as less than 500 pM.
  • Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • dissociation constant may be the actual or apparent dissociation constant.
  • Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned on pages 53-56 of WO 08/020079.
  • modulating or “to modulate” generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay.
  • modulating or “to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.
  • moduleating may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention.
  • this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved.
  • Modulating may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved.
  • a change i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect
  • a change i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect
  • a change i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect
  • an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in vivo assay) assay known per se, depending on the target or antigen involved, hi particular, art action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention.
  • Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions).
  • Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.
  • cross-block means the ability of an amino acid sequence or other binding agents (such as a polypeptide of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target.
  • the extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to a target, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays.
  • One particularly suitable quantitative cross-blocking assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology.
  • Another suitable quantitative cross- blocking assay uses an ELISA-basecl approach to measure competition between amino acid sequence or another binding agents in terms of their binding to the target.
  • the Biacore machine for example the Biacore 3000
  • the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target.
  • 200- 800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used).
  • test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture.
  • concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence.
  • concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip.
  • the amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis).
  • A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix.
  • the test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded.
  • the chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds.
  • the solution of A* alone is then passed over the target-coated surface and the amount of binding recorded.
  • the chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target.
  • the solution of B* alone is then passed over the target-coated surface and the amount of binding recorded.
  • a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such that during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g.
  • the Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version.
  • an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence.
  • the cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface.
  • C-terminal His-tagged target could alternatively be used.
  • various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).
  • the general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules.
  • the plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target.
  • the amount of bound target is then measured using a reagent that is appropriate to detect the target.
  • An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence.
  • the first amino acid sequence e.g.
  • an Ab-X is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added.
  • An excess amount of the second amino acid sequence, i.e. Ab-Y is then added to the ELISA plate such that the moles of Ab-Y target binding sites per well are at least 10 fold higher than the moles of Ab-X target binding sites that were used, per well, during the coating of the ELISA plate, target is then added such that the moles of target added per well are at least 25-fold lower than the moles of Ab-X target binding sites that were used for coating each well.
  • the background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y), target buffer only (i.e. without target) and target detection reagents.
  • the positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence buffer only (i.e. without second solution phase amino acid sequence), target and target detection reagents.
  • the ELISA assay may be ran in such a manner so as to have the positive control signal be at least 6 times the background signal
  • the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution
  • Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal (i.e
  • the proteins and polypeptides described herein are such that they are capable of competing with at least one of the Nanobodies IL6R03 (SEQ ID NO: 13), IL6R04 (SEQ ID NO: 14), IL6R10 (SEQ ID NO: 19), IL6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/or IL6R201 (SEQ ID NO: 31) described in the International patent application WO 08/020079 by Ablynx N. V.
  • the proteins and polypeptides described herein are such that they are a) capable of competing with the Nanobodies IL6R03 (SEQ ID NO: 13), IL6R04 (SEQ ID NO: 14), IL6R10 (SEQ ID NO: 19), IL6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/or IL6R201 (SEQ ID NO: 31) for binding to the amino acid sequence of SEQ ID NO: 4; and/or such that they are capable of cross- blocking the binding of the Nanobodies IL6R03 (SEQ ID NO: 13), IL6R04 (SEQ ID NO: 14), ILoRlO (SEQ ID NO: 19), IL6R13 (SEQ ID NO: 22), IL6R65 (SEQ ID NO: 24), IL6R88 (SEQ ID NO: 25) and/or IL6R201 (SEQ ID NO: 31) for binding to the amino
  • the proteins and polypeptides described herein do not comprise any of the amino acid sequences that are listed in the sequence listing of the International patent application WO08/020079 by Ablynx N. V.
  • the skilled person will be able to provide other proteins and polypeptides that can. bind (and in particular, specifically bind) to the amino acid sequence o ⁇ SEQ ID NO: 4 (and that preferably also have some of the other properties mentioned herein).
  • These may also be, for example, humanized and/or fo ⁇ natted versions of the monovalent amino acid sequences listed in the sequence listing of the International patent application WO08/020079, as well as for example variants that have been obtained by affinity maturation or by changing one or more specific amino acid residues.
  • the proteins and polypeptides described herein can generally be used, in a cynomolgus monkey, to modulate, and in particular inhibit and/or prevent, binding of IL-6R to IL-6 and/or binding of the IL-6/IL-6R complex to gpl30; and thus to modulate, and in particular inhibit and/or prevent, the signalling that is mediated by IL-6R, IL-6, and/or IL6/IL-6R complex; to modulate the biological pathways in which EL-6R, IL-6, and/or IL6/IL-6R complex are involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways.
  • modulating the interaction between IL-6/IL- 6R complex and gpl30 means either binding to IL-6R (i.e. as such or as present in the IL-6/IL-6R complex) in such a way that the formation of the IL-6/IL-6R complex is disrupted and therefore the binding of said complex to gpl30 is modulated (e.g. inhibited) or - binding to ⁇ L-6R (i.e. as such or as present in the IL-6/IL-6R complex) in such a way that the formation of the IL-6/IL-6R complex stays unaffected but the binding of said complex to g ⁇ !30 is modulated (e.g. inhibited).
  • proteins and polypeptides described herein that are cross-reactive with the human IL-6 receptor will be capable of effecting the above activity or activities in both cynomoigus monkeys as well as in human subjects.
  • the proteins and polypeptides described herein that are cross-reactive with the human IL-6 receptor can be used for the prevention and treatment in human subjects of diseases and disorders associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex, and/or with the signaling pathway(s) and/or the biological functions and responses in which IL-6 and/or the IL-6/IL-6R complex are involved, and in particular for the prevention and treatment of diseases and disorders associated with IL-6R, IL-6 and/or with the ⁇ L-6/IL-6R complex, and/or with the signaling pathway(s) and/or the biological functions and responses in which IL-6R, IL-6 and/or the IL-6/IL-6R complex are involved, which are characterized by excessive and/or unwanted signalling mediated by IL-6, IL-6R and/or the IL-6/IL-6R complex or by the pathway(s) in which IL-6, IL-6R and/or the IL-6/IL-6R complex are involved is involved.
  • diseases and disorders associated with IL-6R, IL-6 and/or with the IL-6/IL-6R complex, and/or with the signaling pathway(s) and/or the biological functions and responses in which IL-6, IL-6R and/or the IL- 6/IL-6R complex are involved will be clear to the skilled person based on the disclosure herein, and for example include the following diseases and disorders: sepsis (Starnes et al., 1999) and various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia (Klein et al, 1991), lymphoma, B- lymphoproliferative disorder (BLPD) and prostate cancer.
  • MM multiple myeloma disease
  • RCC renal cell carcinoma
  • plasma cell leukaemia Klein et al, 1991
  • lymphoma lymphoma
  • B- lymphoproliferative disorder BLPD
  • Non-limiting examples of other diseases caused by excessive IL-6 production or signalling include bone resorption (osteoporosis) (Roodman et al., 1992; Jilka et al., 1992), cachexia (Strassman et al., 1992), psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie et al., 1994), inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia (Grau et al., 1990); Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus (Campbell el al.
  • IL-6R, IL-6 and/or IL-6/IL-6R complex related disorders will be clear to the skilled person.
  • proteins and polypeptides described herein can be tested for biological and/or pharmacological activity in animal models that involve the use of cynomolgus monkeys, such as the model described in Shmkura et al, Anticancer Research 18: 1217-1222 (1998).
  • cynomolgus monkeys such as the model described in Shmkura et al, Anticancer Research 18: 1217-1222 (1998).
  • WO 08/020079 i.e. in particular to page 8 of WO 08/020079
  • Ablynx N.V. entitled "Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with IL-6-mediated signalling"
  • the invention also relates to a pharmaceutical composition that comprises at least one compound as described herein and at least one pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition that comprises at least one protein or polypeptide as described herein that is cross-reactive with the human IL-6 receptor, and at least one pharmaceutically acceptable carrier.
  • WO08/020079 i.e. in particular to pages 4 to 9 and 142 to 151 of WO 08/020079
  • Ablynx N.V. entitled "Amino acid sequences directed against IL- 6R and polypeptides comprising the same for the treatment of diseases and disorders associated with IL-6-mediated signalling".
  • the invention also relates to methods for producing, generating and/or identifying amino acid sequences (for example, proteins and polypeptides as described herein) that can bind to and/or have affinity for the amino acid sequence of SEQ ID NO: 4.
  • one particularly useful method for producing an amino acid sequence (for example, proteins and polypeptides as described herein) that can bind to and/or has affinity for the amino acid sequence of SEQ ID NO: 4 generally comprises the steps of: the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said amino acid sequence, protein polypeptide or compound (also referred to herein as a "nucleic acid of the invention”), optionally followed by: isolating and/or purifying the amino acid sequence, protein polypeptide or compound, thus obtained.
  • such a method may comprise the steps of: cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence, protein polypeptide or compound of the invention; optionally followed by: isolating and/or purifying the amino acid sequence, protein polypeptide or compound of the invention thus obtained.
  • methods for generating amino acid sequences, proteins, polypeptides or compounds according to the invention may comprise the steps of: a) providing a set, collection or library of amino acid sequences; and b) screening said set, collection or library of amino acid sequences for amino acid sequences that can bind to and/or have affinity for the amino acid sequence of SEQ ID NO: 4; and c) isolating the amino acid sequence(s) that can bind to and/or have affinity for the amino acid sequence of SEQ ID NO: 4.
  • the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences.
  • the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a na ⁇ ve set, collection or library of immunoglobulin sequences; a synthetic or semi-synlhelic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.
  • the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as Vn domains or V ⁇ domains) or of light chain variable domains.
  • the set, collection or library of amino acid sequences may be a set, collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.
  • the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with the amino acid sequence of SEQ ID NO: 4 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof.
  • said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
  • the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening.
  • suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1 105-1116 (2005).
  • the method for generating amino acid sequences that can bind to and/or have affinity for the amino acid sequence of SEQ TD NO: 4 comprises at least the steps of: a) providing a collection or sample of cells expressing amino acid sequences; b) screening said collection or sample of cells for cells that express an amino acid sequence that can bind to and/or have affinity for the amino acid sequence of SEQ ID NO: 4; and c) either (i) isolating said amino acid sequence; or (ii) isolating from said cell a nucleic acid sequence that encodes said amino acid sequence, followed by expressing said amino acid sequence.
  • the collection or sample of cells may for example be a collection or sample of B-cells.
  • the sample of cells may be derived from a mammal that has been suitably immunized with the amino acid sequence of SEQ ID NO: 4 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof.
  • said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
  • step b) is preferably performed using a flow cytometry technique such as FACS.
  • FACS flow cytometry technique
  • the method for generating an amino acid sequence that can bind to and/or have affinity for the amino acid sequence of SEQ ID NO: 4 may comprise at least the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for the amino acid sequence of SEQ ID NO: 4; and c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.
  • the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a na ⁇ ve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.
  • the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V H domains or V HH domains) or of light chain variable domains.
  • the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.
  • the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with the amino acid sequence of SEQ ID NO: 4 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof.
  • said antigenic determinant maybe an extracellular part, region, domain, loop or other extracellular epitope(s).
  • the set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains.
  • the set, collection or library of nucleotide sequences may encode a set, collection or library OfV 1-1 H sequences.
  • the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening.
  • suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
  • the invention also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.
  • one or more amino acid sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the invention.
  • nucleic acid sequence encoding an the amino acid sequence of SEQ ID NO: 4 may be suitably humanized (or alternatively camelized) and suitably expressed; and/or one or more nucleic acid sequences encoding an the amino acid sequence of SEQ ID NO: 4 may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the invention.
  • the methods described above may also comprise a further screening or selection step for specific binding to the human IL-6 receptor protein of SEQ ID NO: 6.
  • individual proteins or polypeptides that are capable of specific binding to the amino acid sequence of SEQ ID NO: 4 may be screened or tested for specific binding to the amino acid sequence of SEQ ID NO: 6 (or visa versa), in order to provide proteins or polypeptides that are cross-reactive with the human IL-6 receptor.
  • the invention is illustrated by the following non-limiting experimental part.
  • Figure IA Published DNA sequence of scynoIL-6R (published by Imazeki et al. - SEQ ID NO: 1
  • Figure IB DNA sequence of scynoIL-6R obtained from cDNA as described in Example 6
  • Figure 1C DNA sequence of shIL-6R (human IL-6R - SEQ ID NO: 5)
  • Figure 2 Alignment of cynomolgus IL-6R cDNA sequence of the present invention with the cynomolgus IL-6R cDNA sequence as published by Imazeki et al. (1998) Int. J.
  • Figure 3 A Amino acid sequence of scynoIL- ⁇ R (published by Imazeki et al - SEQ ID NO:
  • Figure 3B Amino acid sequence of scynoIL-6R obtained from cDNA as described in
  • Example 6 (invention - SEQ ID NO: 4).
  • FIG. 3C Amino acid sequence of shIL-6R (SEQ ID NO: 6).
  • Figure 3D Amino acid sequence of baboon IL-6R. (SEQ ID NO: 7).
  • Figure 3E AA sequence of marmoset IL-6R (SEQ ID NO: 8).
  • Figure 3F AA sequence of rat IL-6R (SEQ ID NO: 9).
  • Figure 3G AA sequence of pig IL-6R (SEQ ID NO: 10).
  • Figure 3H AA sequence of mouse IL-6R (SEQ ID NO: I I).
  • Figure 4 Alignment of cynomolgus IL-6R amino acid sequence of the present invention, cynomolgus IL-6R amino acid sequence as published by Imazeki et al. (1998) Int. J.
  • Figure 5 Alignment of IL-6R amino acid sequences of human, marmoset, baboon, cynomolgus monkey, rat, pig and mouse.
  • Figure 6A Human or cynomolgus plasma sIL-6R is captured on plates coated with
  • Nanobodies to be tested or with anti-IL-6R MAbs are tested or with anti-IL-6R MAbs. Subsequently, bound sIL-6R is detected with biotinylated anti-IL-6R and strep-HRP.
  • FIG. 6B Plasma IL-6R is captured on BNl 2 coated plates. After washing, Nanobodies to be tested are allowed to bind to captured IL-6R and detected with an in-house purified rabbit anti-Nanobody polyclonal antibody.
  • Nanobodies to be tested are pre-incubated in human or primate plasma.
  • the plasma sIL-6R is captured on BNl 2 coated plates and bound Nanobody is detected.
  • Figure 6D Nanobodies to be tested are pre-incubated in primate plasma with human IL-6.
  • Nanobodies that bind to human IL-6R as well as to primate IL-6R will compete with human IL-6 for binding to primate IL-6R. Therefore cross-reactive Nanobodies will inhibit the binding of IL-6 to the plasma sIL-6R in this experiment. After pre- incubation, the plasma sIL-6R in complex with IL-6 or Nanobody is captured on BNl 2 coated plates and bound IL-6 is detected.
  • Nanobodies to neutralize human IL6/cyno IL-6R binding in plasma were assessed for neutralize human IL6/cyno IL-6R binding in plasma.
  • FIGS. 8 and 9 Plasma potency assay analyzing capacity of IL-6R Nanobodies to neutralize IL6/IL6R binding in plasma from human ( Figure 8) or cynomolgus monkey
  • Examples 1 to 4A/4B different ELISA formats are explored to determine the cross- reactivity of IL-6R Nanobodies with various primate sIL-6R, including human IL-6R and cynomolgus IL-6R (invention).
  • the ELISA formats used are schematically shown in Figures 6A to 6D.
  • Fig. 6A In the first assay format (Fig. 6A), different Nanobodies to be tested and antibodies are used to capture human or cyno plasma sIL-6R, after which bound sIL-6R is detected with a polyclonal anti-IL-6R.
  • Fig. 6B represents the second assay format, where Nanobodies to be tested are allowed to bind to BN12 captured plasma sIL-6R.
  • Fig. 6C Nanobodies to be tested are pre-incubated in plasma and the sIL-6R/ Nanobody complexes are subsequently captured on BN12 coated plates. Bound Nanobodies to be tested are detected with anti-His.
  • Example 5 binding of Nanobodies to human IL-6R and cyno IL-6R (invention) is determined in a standard BIACORE assay.
  • the Nanobodies to be tested need to be able to block the binding of IL-6 to the IL-6R. Therefore, an IL-6 competition ELISA is developed to determine whether the Nanobodies to be tested can block the interaction of the cyno plasma sIL-6R with human IL-6. This competition assay is also used to determine the cross- reactivity of the Nanobodies to be tested for rhesus, baboon and marmoset sIL-6R. Human IL-6 is pre-incubated in primate plasma with different concentrations of Nanobody. After pre-incubation, the plasma sIL-6R in complex with IL-6 or Nanobody is captured on BNl 2 coated plates and bound IL-6 is detected (Fig. 6D).
  • the non-neutralizing anti-IL-6R MAb BN12 (lot no. P20108D1) and the neutralizing anti-IL-6R MAb BR6 (lot no. P931221D7) are from Diaclone; both MAbs are stored at 4°C.
  • Biotinylated goat anti-human IL-6 (lot no. SV1307011) and biotinylated goat anti-human IL- 6R (lot no. VP0307011) are from R&D systems.
  • the lyophilized antibodies are reconstituted in Tris-buffered saline pH 7.3 (20 mM Tris base, 150 mM NaCl) containing 0.1% BSA to a concentration of 50 micro g/ml, aliquoted and stored at -20 0 C.
  • Recombinant human IL-6 is from eBioscience (lot no. E022146). IL-6 is diluted to 10 microg/mL in D-PBS containing 10% BSA, aliquoted and stored at -2O 0 C.
  • Casein is from Calbiochem, (lot no. B70277) and is stored at RT.
  • Biotinylated anti-His MAb (Serotec, MCAl 396B lot 0307) is stored at 4°C.
  • HRP-conjugated streptavidin (lot no. 00032671) and HRP-conjugated anti-rabbit Ig (lot no, 00033131) are from Dako and are stored at 4°C.
  • Slow TMB (lot no. IE114111) is from Pierce and TMB One (lot no. 23172203) is from Promega; both are stored at 4°C.
  • Example 1 Binding of Nanobodies to plasma sIL-6R - capturing of plasma S ⁇ L-6R
  • 96-well microtiter plates are coated ON with 100 microl per well of 5 microg/niL BNl 2, BR ⁇ or the Nanobody to be tested in 50 niM bicarbonate buffer (pH 9.6) at 4°C.
  • the plates are washed and blocked with 300 microl of 1% casein in PBS for 2 hours at RT. Subsequently, the wells are washed with PBS-T (3 x 300 microl) and incubated with 100 microl of different dilutions of human or cyiio plasma in PBS (1 hour at RT, 600 rpm).
  • the wells are incubated with 100 microl of 250 ng/ml biotinylated goat anti-IL-6R in 1% casein in PBS for 30 minutes at RT (600 rpm).
  • the plates are washed and bound anti-IL-6R is detected for 30 minutes (RT, 600 rpm) with HRP-conjugated streptavidin (1/5000 in 1% casein in PBS, 100 microl/well).
  • HRP-conjugated streptavidin (1/5000 in 1% casein in PBS, 100 microl/well.
  • the plates are developed for 20 minutes at RT with 100 microl/well of slow TMB after which the coloring reaction is stopped with 100 microl IN HCl.
  • the absorbance is determined at 450 nm.
  • 96-well microtiter plates are coated ON with 100 microl per well of 5 microg/mL BNl 2 in 50 niM bicarbonate buffer (pH 9.6) at 4°C. The plates are washed and blocked with 300 microl of 1% casein in PBS for 2 hours at RT. After washing, 100 microl of human or cyno plasma is added (1, 1/10, 1/100 or 1/1000 in PBS) and incubated for 1 hour at RT (600 rpm). The plates are washed and 100 microl of the Nanobody to be tested or control Nanobody Alb8 (SEQ ID NO: 26) (5 microg/ml in PBS) is added and the plates are incubated for 1 hour at RT, 600 rpm.
  • SEQ ID NO: 26 Nanobody Alb8
  • the wells are incubated with 100 microl of an in- house purified rabbit anti-Nanobody polyclonal antibody (1/2000 in PBS 1% casein) for 30 minutes at RT (600 rpm).
  • the plates are washed and incubated for 30 minutes (RT, 600 ipm) with HRP-conjugated anti-rabbit Ig (1/2000 in PBS 1% casein, 100 microl/well).
  • HRP-conjugated anti-rabbit Ig 1/2000 in PBS 1% casein, 100 microl/well.
  • the plates are developed for 15 minutes at RT with 100 microl/well of slow TMB after which the coloring reaction is stopped with 100 microl IN HCl.
  • the absorbance is determined at 450 nm.
  • Example 3 Binding to plasma sIL-6R in solution 96-welI microliter plates are coated ON with 100 microl per well of 5 microg/niL BNl 2 in 50 niM bicarbonate buffer (pH 9.6) at 4°C. The plates are washed and blocked with 300 microl of 1% casein in PBS for 2 hours at RT. In the mean time, the Nanobodies to be tested are pre-incubated in human or cyno plasma for 1 hour at RT (600 rpm). 60 microl Nanobody at 2 microg/ml in PBS-T is added to 60 microl of plasma (undiluted or 1/5 in PBS- T).
  • the blocked plates are washed with PBS-T (3 x 300 microl), 100 microl of the pre-incubated samples is added and the plates are incubated for 1 hour at RT, 600 rpm. After washing (see above), the wells are incubated with 100 micro] of 2 microg/ml of biotinylated anti-His MAb in PBS-Tor 30 minutes at RT (600 rpm). The plates are washed and incubated for 30 minutes (RT, 600 rpm) with HRP -conjugated streptavidin (1/1000 in PBS-T, 100 microl/well). After washing, the plates are developed for 20 minutes at RT with 100 microl/well of slow TMB after which the coloring reaction is stopped with 100 microl IN HCl. The absorbance is determined at 450 urn.
  • Example 4A IL-6 competition ELISA - titration of IL-6
  • 96-well microliter plates are coated ON with 100 microl per well of 5 micro g/mL BN12 in 50 niM bicarbonate buffer (pH 9.6) at 4°C. The plates are washed and blocked with 300 microl of 1% casein in PBS for 2 hours at RT. hi the mean time different concentrations of recombinant human IL-6 are pre-incubated in human or primate plasma. 60 microl of a 1 A dilution series of human IL-6 (1000 to 0.5 ng/mL in PBS 0.1% Tween20) is added to 60 microl plasma in low adhesion microtiter plates and incubated for Ih at RT, 600 rpm.
  • the blocked BN12 coated plates are washed with PBS-T, 100 microL of the IL- 6/plasma mixtures are transferred to the plates and incubated for 1 hour at RT (600 rpm).
  • the plates are washed and bound IL-6 is detected with 200 ng/mL of biotinylated anti-IL-6 in PBS-T (100 microl/well).
  • the wells are incubated with 100 microl HRP- conjugated streptavidin (1/1000 in PBS-T).
  • the plates are washed and developed for 15 minutes at RT with 100 microl/well of TMB One after which the coloring reaction is stopped with 100 microl IN HCl.
  • the absorbaiice is determined at 450 nm. 4 parameter logistic curves are fitted in GraphPad Prism to determine the EC50 values of IL-6 in the different plasma's.
  • IL-6 competition ELISA Nanobody titration - detection of IL-6 96-well microtiter plates are coated ON with 100 microl per well of 5 microg/mL BNl 2 in 50 raM bicarbonate buffer (pH 9.6) at 4 0 C. The coating solution is aspirated and the plates are blocked with 300 microl of 1% casein in PBS for 2 hours at RT.
  • IL-6R Nanobodies to be tested are incubated in human or primate plasma supplemented with human IL-6 at a concentration corresponding to the EC50 of IL-6 (50 ng/mL in human, rhesus and cyno plasma; 25 ng/mL in baboon plasma).
  • 30 micro L Nanobody diluted in PBS 0.1% Tween20, 30 microL IL-6 (50 or 25 ng/mL in PBS 0.1% Tween20) and 60 microL plasma are sequentially added to the wells of low adhesion microtiter plates and incubated for 1 hour at RT (600 rpm).
  • the blocked BN 12 coated plates are washed with PBS- T, 100 microL of the IL-6/ Nanobody /plasma mixtures are transferred to the plates and incubated for 1 hour at RT (600 rpm).
  • the plates are washed and bound IL-6 is detected with biotinylaled anti-IL-6 and HRP -conjugated streptavidin as described above.
  • the plates are washed and developed with TMB One as above. The absorbance is determined at 450 nra.
  • Nanobodies to human IL-6R and cyno IL-6R were determined in a standard BIACORE assay.
  • the Nanobodies tested were described in the International patent application WO 08/020079.
  • SEQ ID refers to the SEQ ID number according to WO 08/020079.
  • the cynomolgus monkey IL-6R sequence was verified on lymph node cDNA from cynomolgus monkey (see Figure IB; SEQ ID NO: 3 and Figure 3B; SEQ ID NO: 4).
  • the sequence that was retrieved contained 5 amino acid substitutions compared to the cynomolgus sequence that was published by Imazeki et al. ( Figure 4).
  • Example 7 Cross-reactivity of different Nanobodies to cynomolgus IL-6R
  • IL6R202 SEQ ID NO: 32; bivalent humanized Nanobody
  • IL6R203 SEQ ID NO: 33; trivalent humanized Nanobody
  • the mixture was captured by a non-neutralizing anti-IL-6R Mab and detected using a biotinylated anti-IL-6 MAb. Data were presented as nM binding units to account for the difference in valency between compounds.
  • Nanobodies IL6R04 (SEQ ID NO: 14), IL6R65 (SEQ ID NO: 24) and IL6R8S (SEQ ID NO: 25) were analyzed in a human and cynomolgus plasma potency assay and compared to Nanobodies IL6R201 (SEQ ID NO: 31), IL6R202 (SEQ ID NO: 32) and IL6R203 (SEQ ID NO: 33). Data are presented in Figures 9 and 10.
  • IL6R03 SEQ ID NO: 13
  • IL6R65 SEQ ID NO: 24
  • IL6R13 SEQ ID NO: 22
  • IL6R88 SEQ ID NO: 25
  • LAPGGCP AQEVARGVLTSLPGDSVTLTCPGGEPEDNATVHWVLRKP AEGSHL SRW AGVGRRLLLRSVQLHDSGNYSCYRAGRP AATVHLLVD VPPEEPQLSCFRKSPLS NVVCEWGPRSTPSPTTKA VLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDS J SFYIVSMCV ASSVGSKLSKTQTFQGCGILQPDPP ANITVTAV ARNPRWLSVTWQDPH SWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEE FGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPTQAPTTNKDDDNILSGDSANATSL PVQD
  • LAPGGCP AQEV ARGVLTSLPGDSVTLTCPGGEPEDNATVHWVLRKP AAGSYL SRWAGVGRRLLLRSVQLHDSGNYSCYRAGRP AGTVHLLVD VPPEEPQLSCFRKSPLS NVVCEWGPRSTPSPTTKA VLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLA VPEGDS SFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPP ANITVTA V ARNPRWLSVTWQDPH SWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEE FGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPTQAPTTNKDDDNILSRDSANATSL PVQD
  • MLTVGCTLLV ALLAAP A V ALVLGSCRALEVANGTVTSLPGATVTLICPGKEA AGNVTIHWVYSGSQNREWTTTGNTLVLRDVQLSDTGDYLCSLNDHLVGTVPLLVD VPPEEPKLSCFRK-NPLVNAICEWRPSSTPSPTTKAVLFAKKINTTNGKSDFQVPCQYS QQLKSFSCQVEILEGDKVYHIVSLCV ANSVGSKSSHNEAFHSLKMVQPDPP ANLVVS A ⁇ PGRPRWLKVSWQHPETWDPSYYLLQFQLRYRPVWSKEFTVLLLPV AQYQCVIHD

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Abstract

La présente invention concerne une protéine dérivée de singe Cynomolgus (Macaca fascicularis)qui agit en tant que récepteur pour l'interleukine-6 (IL-6) et un acide nucléique codant pour une telle protéine. La présente invention concerne également des composés qui interagissent (tel que défini dans la description) avec ladite protéine réceptrice d'IL-6. Lesdits composés peuvent être des petites entités chimiques ou molécules biologiques, et peuvent notamment être des protéines ou des polypeptides. De manière plus spécifique, lesdits composés peuvent être des immunoglobulines ou des fragments de celles-ci, tels que des anticorps, des fragments d'anticorps (tels que des fragments Fab, des fragments F(ab')2 et des fragments Fv), des constructions dérivées de fragments d'anticorps (tels que des fragments scFv et des molécules diacorps), ou des anticorps de domaine, des anticorps à domaine unique, des d'Ab ou nanocorps. Les composés selon l'invention sont de préférence de réaction croisée avec le récepteur IL-6 humain.
PCT/EP2008/059325 2007-07-19 2008-07-16 Recepteur de l'interleukine-6 (il-6) derive de macaca fascicularis Ceased WO2009010539A2 (fr)

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