Compounds

Abstract

Binding members, e.g. human antibody molecules, which bind interleukin-6 (IL-6) and neutralise its biological effects. Use of binding members for IL-6 in medical treatment e.g. for treating inflammatory diseases and tumours associated with IL-6.

1 . An isolated binding member for human IL-6, which binds human IL-6 with a K D  of not more than 30 pM as defined by surface plasmon resonance. 
     
     
         2 . A binding member according to  claim 1 , wherein the K D  is not more than 10 pM. 
     
     
         3 . An isolated binding member for human IL-6, which binds human IL-6 at residue Phe102 and/or Ser204, the residue numbering being defined according to the full length human IL-6 amino acid sequence SEQ ID NO: 161. 
     
     
         4 . A binding member according to  claim 3 , wherein the binding member binds human IL-6 at residue Arg207. 
     
     
         5 . An isolated binding member for human IL-6, wherein the binding member has an IC 50  of less than 1 nM in an assay of inhibition of VEGF release from human synovial fibroblasts stimulated with 0.6 pM human IL-1β and 2.4 nM soluble human IL-6Rα. 
     
     
         6 . A binding member according to any of the preceding claims, wherein the binding member binds cynomolgus IL-6 and wherein cynomolgus IL-6 has an IC 50  less than 10-fold different from the IC 50  of unlabelled human IL-6 in a time resolved fluorescence assay for inhibition of labelled human IL-6 binding to the binding member immobilised on a support, wherein the labelled human IL-6 is at a final concentration equal to twice the Kd of its interaction with the binding member. 
     
     
         7 . A binding member according to any of the preceding claims, which has an IC 50  of less than 100 pM in an assay for inhibition of proliferation of TF-1 cells in response to 20 pM human IL-6. 
     
     
         8 . An isolated binding member for human IL-6, comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the set of CDRs has 22 or fewer amino acid alterations from a set of CDRs in which:
 HCDR1 has amino acid sequence SEQ ID NO: 3;   HCDR2 has amino acid sequence SEQ ID NO: 4;   HCDR3 has amino acid sequence SEQ ID NO: 115;   LCDR1 has amino acid sequence SEQ ID NO: 8;   LCDR2 has amino acid sequence SEQ ID NO: 9; and   LCDR3 has amino acid sequence SEQ ID NO: 120, and wherein
 the binding member has an IC 50  of less than 100 pM in an assay for inhibition of proliferation of TF-1 cells in response to 20 pM human IL-6. 
   
     
     
         9 . An isolated binding member for human IL-6 according to any of  claims 1  to  7 , comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the set of CDRs has 22 or fewer amino acid alterations from a set of CDRs in which:
 HCDR1 has amino acid sequence SEQ ID NO: 3; 
 HCDR2 has amino acid sequence SEQ ID NO: 4; 
 HCDR3 has amino acid sequence SEQ ID NO: 115; 
 LCDR1 has amino acid sequence SEQ ID NO: 8; 
 LCDR2 has amino acid sequence SEQ ID NO: 9; and 
 LCDR3 has amino acid sequence SEQ ID NO: 120. 
 
     
     
         10 . A binding member according to  claim 8  or  claim 9 , comprising a set of CDRs having 20 or fewer substitutions from a set of CDRs in which:
 HCDR1 has amino acid sequence SEQ ID NO: 3; 
 HCDR2 has amino acid sequence SEQ ID NO: 4; 
 HCDR3 has amino acid sequence SEQ ID NO: 115; 
 LCDR1 has amino acid sequence SEQ ID NO: 8; 
 LCDR2 has amino acid sequence SEQ ID NO: 9; and 
 LCDR3 has amino acid sequence SEQ ID NO: 120;
 and wherein the binding member optionally has an insertion of one residue to increase the length of the HCDR3 relative to SEQ ID NO: 115, and/or has an insertion of one residue to increase the length of the LCDR3 relative to SEQ ID NO: 120. 
 
 
     
     
         11 . A binding member according to any of  claims 8  to  10 , comprising an HCDR1 wherein Kabat residue 35 is Ile, Thr or Val. 
     
     
         12 . A binding member according to  claim 11 , wherein HCDR1 is SEQ ID NO: 3. 
     
     
         13 . A binding member according to any of  claims 8  to  12 , comprising an HCDR2 wherein Kabat residue 64 is Lys or Arg. 
     
     
         14 . A binding member according to  claim 13 , wherein HCDR2 has amino acid sequence SEQ ID NO: 4. 
     
     
         15 . A binding member according to any of  claims 8  to  14 , wherein: Kabat residue 95 in HCDR3 is Trp and/or wherein Kabat residue 101 in HCDR3 is Asp. 
     
     
         16 . A binding member according to any of the preceding claims, comprising an HCDR3 wherein:
 Kabat residue 96 is Ala or Glu;   Kabat residue 97 is Asp, Glu or Asn;   Kabat residue 98 is Asp, Gly, Glu or His;   Kabat residue 99 is His, Gly or Thr;   Kabat residue 100 is Pro, Tyr, Asn, Arg, Trp or Ala;   Kabat residue 100A is Pro, Tyr, Ala, Arg, Thr, Gly, Asn, Pro or Ser;   Kabat residue 100B is Trp, Tyr, His, Gln, Pro or Thr;   Kabat residue 100C is Ile, Ala, Val, His, Tyr or Leu; and   Kabat residue 102 is Leu, Val, His, Met or Ile.   
     
     
         17 . A binding member according to  claim 16 , wherein HCDR3 has amino acid sequence SEQ ID NO: 115. 
     
     
         18 . A binding member according to any of  claims 8  to  17 , wherein Kabat residue 34 in LCDR1 is Ala or Thr. 
     
     
         19 . A binding member according to  claim 18 , wherein LCDR1 is SEQ ID NO: 8. 
     
     
         20 . A binding member according to any of  claims 8  to  19 , wherein LCDR2 is SEQ ID NO: 9. 
     
     
         21 . A binding member according to any of the preceding claims, comprising an LCDR3 wherein:
 Kabat residue 89 is Gln, Met or Ala;   Kabat residue 90 is Gln, Asn, Ser or Ala;   Kabat residue 91 is Ser, Asn, Gly, Ala or His;   Kabat residue 92 is Trp, Tyr, Ser, Lys or Phe;   Kabat residue 93 is Leu, Ser, Lys, Arg or Ala;   Kabat residue 94 is Gly, Thr, Ala or Pro;   Kabat residue 96 is Gly or Trp; and   Kabat residue 97 is Ser or Thr.   
     
     
         22 . A binding member according to  claim 21 , comprising an LCDR3 having amino acid sequence SEQ ID NO: 120. 
     
     
         23 . An isolated binding member for human IL-6, comprising an HCDR3, an LCDR3 and/or a set of CDRs of any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23. 
     
     
         24 . A binding member according to any of the preceding claims, comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein:
 HCDR1 is SEQ ID NO: 3;   HCDR2 is SEQ ID NO: 4;   HCDR3 is SEQ ID NO: 115;   LCDR1 is SEQ ID NO: 8;   LCDR2 is SEQ ID NO: 9; and   LCDR3 is SEQ ID NO: 80.   
     
     
         25 . A binding member according to any of the preceding claims, wherein the binding member comprises an antibody molecule comprising an antibody VH domain and an antibody VL domain, wherein the antibody molecule comprises the said set of CDRs, wherein the VH domain comprises HCDR1, HCDR2, HCDR3 and a framework and the VL domain comprises LCDR1, LCDR2, LCDR3 and a framework. 
     
     
         26 . A binding member according to  claim 25 , wherein the antibody molecule comprises an antibody constant region. 
     
     
         27 . A binding member according to  claim 26 , wherein the antibody molecule is an IgG1. 
     
     
         28 . A binding member according to any of  claims 25  to  27 , wherein the framework regions of the VH and/or VL domain are germlined to human germline gene segment sequences. 
     
     
         29 . A binding member according to  claim 28 , wherein the antibody VH domain comprises framework regions germlined to human germline framework Vh3_DP-86_(3-66). 
     
     
         30 . A binding member according to  claim 29 , wherein VH domain comprises framework regions FR1, FR2, FR3 and FR4 having amino acid sequences wherein FR1 is SEQ ID NO: 167, FR2 is SEQ ID NO: 168, FR3 is SEQ ID NO: 169 and FR4 is SEQ ID NO: 170. 
     
     
         31 . A binding member according to  claim 30 , wherein the antibody VH domain has the VH domain amino acid sequence shown in SEQ ID NO: 112. 
     
     
         32 . A binding member according to any of  claims 28  to  31 , wherein the antibody VL domain comprises framework regions germlined to human germline framework Vk1_L12. 
     
     
         33 . A binding member according to  claim 32 , wherein the VL domain comprises framework regions FR1, FR2, FR3 and FR4 having amino acid sequences wherein FR1 is SEQ ID NO: 171, FR2 is SEQ ID NO: 172, FR3 is SEQ ID NO: 173 and FR4 is SEQ ID NO: 174. 
     
     
         34 . A binding member according to  claim 33 , wherein the antibody VL domain has the VL domain amino acid sequence shown in SEQ ID NO: 117. 
     
     
         35 . An isolated antibody molecule comprising a heavy chain comprising amino acid sequence SEQ ID NO: 112 and a light chain comprising amino acid sequence SEQ ID NO: 117. 
     
     
         36 . An isolated antibody molecule that binds IL-6, wherein the antibody molecule comprises a VH domain amino acid sequence at least 90% identical to SEQ ID NO: 112 and a VL domain amino acid sequence at least 90% identical to SEQ ID NO: 117. 
     
     
         37 . An antibody molecule according to  claim 35  or  claim 36 , wherein the antibody molecule is an IgG. 
     
     
         38 . An antibody molecule according to  claim 37 , wherein the IgG is IgG1. 
     
     
         39 . An isolated VH domain of an antibody molecule according to any of  claims 25  to  38 . 
     
     
         40 . An isolated VL domain of an antibody molecule according to any of  claims 25  to  38 . 
     
     
         41 . A composition comprising an isolated binding member according to any of  claims 1  to  34 , or an antibody molecule according to any of  claims 35  to  38 , and a pharmaceutically acceptable excipient. 
     
     
         42 . A composition comprising an isolated binding member according to any of  claims 1  to  34 , or an antibody molecule according to any of  claims 35  to  38 , for use in a method of treatment of the human or animal body by therapy. 
     
     
         43 . A composition according to  claim 42  for use in treating a disorder associated with IL-6. 
     
     
         44 . Use of an isolated binding member according to any of  claims 1  to  34 , or an antibody molecule according to any of  claims 35  to  38 , for the manufacture of a medicament for treating a disorder associated with IL-6. 
     
     
         45 . A composition according to  claim 43  or use according to  claim 44 , wherein the disorder is an inflammatory and/or autoimmune disease. 
     
     
         46 . A composition or use according to  claim 45 , wherein the disorder is rheumatoid arthritis, osteoarthritis, cachexia, chronic obstructive pulmonary disease, Juvenile idiopathic arthritis, asthma, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease or atherosclerosis. 
     
     
         47 . A composition or use according to  claim 45 , wherein the disorder is rheumatoid arthritis. 
     
     
         48 . A composition according to  claim 43  or use according to  claim 44 , wherein the disorder is a tumour and/or cancer. 
     
     
         49 . A method of treating a disorder associated with IL-6 in an individual, comprising administering a binding member according to any of  claims 1  to  34 , or an antibody molecule according to any of  claims 35  to  38 , to the individual. 
     
     
         50 . A method according to  claim 49 , wherein the disorder is an inflammatory and/or autoimmune disease. 
     
     
         51 . A method according to  claim 50 , wherein the disorder is rheumatoid arthritis, osteoarthritis, cachexia, chronic obstructive pulmonary disease, Juvenile idiopathic arthritis, asthma, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease or atherosclerosis. 
     
     
         52 . A method according to  claim 51 , wherein the disorder is rheumatoid arthritis. 
     
     
         53 . A method according to  claim 49 , wherein the disorder is a tumour and/or cancer. 
     
     
         54 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding member according to any of  claims 1  to  34 , a VH domain according to  claim 39 , a VL domain according to  claim 40 , or an antibody molecule according to any of  claims 35  to  38 . 
     
     
         55 . A host cell in vitro transformed with nucleic acid according to  claim 54 . 
     
     
         56 . A method of producing a binding member, an antibody molecule or an antibody VH or VL domain, comprising culturing host cells according to  claim 55  under conditions for production of the binding member, antibody molecule or antibody VH or VL domain. 
     
     
         57 . A method according to  claim 55 , further comprising isolating and/or purifying the binding member, antibody molecule, VH domain or VL domain. 
     
     
         58 . A method according to  claim 56  or  claim 57 , further comprising formulating the binding member, antibody molecule, VH domain or VL domain into a composition comprising at least one additional component. 
     
     
         59 . A method for producing an antibody antigen-binding domain for IL-6, the method comprising
 providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein the parent VH domain HCDR1, HCDR2 and HCDR3 are a set of HCDRs as shown in Table 7, a VH domain which is an amino acid sequence variant of the parent VH domain, and optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and   testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for IL-6.   
     
     
         60 . A method according to  claim 59 , wherein said one or more VL domains is provided by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain comprising LCDR1, LCDR2 and LCDR3, wherein the parent VL domain LCDR1, LCDR2 and LCDR3 are a VL set of CDRs as shown in Table 7, producing one or more VL domains each of which is an amino acid sequence variant of the parent VL domain. 
     
     
         61 . A method according to  claim 59  or  claim 60 , wherein said VH domain which is an amino acid sequence variant of the parent VH domain is provided by CDR mutagenesis. 
     
     
         62 . A method according to any of  claims 59  to  61  further comprising producing the antibody antigen-binding domain as a component of an IgG, scFv or Fab antibody molecule. 
     
     
         63 . A method for producing a binding member that binds IL-6, which method comprises:
 providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region;   combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1, HCDR2, and/or HCDR3 shown in Table 7, such that said donor nucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains;   expressing the nucleic acids of said product repertoire to produce product VH domains;   optionally combining said product VH domains with one or more VL domains;   selecting a binding member for IL-6, which binding member comprises a product VH domain and optionally a VL domain; and   recovering said binding member or nucleic acid encoding it.   
     
     
         64 . A method according to  claim 63  wherein the donor nucleic acids are produced by mutation of said HCDR1 and/or HCDR2. 
     
     
         65 . A method according to  claim 63  wherein the donor nucleic acid is produced by mutation of HCDR3. 
     
     
         66 . A method according to  claim 63 , comprising providing the donor nucleic acid by random mutation of nucleic acid. 
     
     
         67 . A method according to any one of  claims 59  to  66 , further comprising attaching a product VH domain that is comprised within the recovered binding member to an antibody constant region. 
     
     
         68 . A method according to any one of  claims 59  to  67  comprising providing an IgG, scFv or Fab antibody molecule comprising the product VH domain and a VL domain. 
     
     
         69 . A method according to any one of  claims 59  to  68 , further comprising testing the antibody antigen-binding domain or binding member that binds IL-6 for ability to neutralize IL-6. 
     
     
         70 . A method according to  claim 69 , wherein a binding member that comprises an antibody molecule that binds and neutralizes IL-6 is obtained. 
     
     
         71 . A method according to  claim 70 , wherein the antibody molecule is an scFv. 
     
     
         72 . A method according to  claim 70 , wherein the antibody molecule is an IgG. 
     
     
         73 . A method for producing an antibody molecule composition, comprising obtaining an antibody molecule using a method according to any of  claims 56  to  72 , and formulating the antibody molecule into a composition comprising at least one additional component.