Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor

Abstract

The present invention relates to polypeptides derived from single domain heavy chain antibodies directed to Tumor Necrosis Factor-alpha. It further relates to single domain antibodies that are Camelidae VHHs. It further relates to methods of administering said polypeptides. It further relates to protocols for screening for agents that modulate the TNF-alpha receptor, and the agents resulting from said screening.

1 . Anti-TNF-alpha polypeptide comprising one or more single domain antibodies directed against tumor necrosis factor alpha (TNF-alpha) and an Fc domain.  
     
     
         2 . Anti-TNF-alpha polypeptide according to  claim 1 , in which the Fc domain is a human Fc domain.  
     
     
         3 . Anti-TNF-alpha polypeptide according to  claim 1 , in which the one or more single domain antibodies directed against tumor necrosis factor alpha (TNF-alpha) are fused to the Fc domain.  
     
     
         4 . Anti-TNF-alpha polypeptide according to  claim 1 , which inhibits the interaction between TNF alpha and one or more TNF alpha receptors.  
     
     
         5 . Anti-TNF-alpha polypeptide according to  claim 1 , which modifies the biological activity of TNF-alpha after binding to TNF alpha.  
     
     
         6 . Anti-TNF-alpha polypeptide according to  claim 1 , which binds into the receptor-binding groove of TNF-alpha.  
     
     
         7 . Anti-TNF-alpha polypeptide according to  claim 1 , in which said one or more single domain antibodies directed against TNF-alpha bind to TNF-alpha with an affinity of better than 10 −6  M.  
     
     
         8 . Anti-TNF-alpha polypeptide according to  claim 1 , in which said one or more single domain antibodies directed against TNF-alpha have an amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine and glutamine at position 45 and a tryptophan at position 103 according to the Kabat numbering.  
     
     
         9 . Anti-TNF-alpha polypeptide according to  claim 1 , in which said one or more single domain antibodies directed against TNF-alpha have an arginine residue at position 103 according to the Kabat numbering.  
     
     
         10 . Anti-TN F-alpha polypeptide according to  claim 1 , in which said one or more single domain antibodies directed against TNF-alpha are one or more  Camelidae  VHHs.  
     
     
         11 . Anti-TNF-alpha polypeptide according to  claim 10 , in which said one or more single domain antibodies directed against TNF-alpha are one or more humanized  Camelidae  VHHS.  
     
     
         12 . Anti-TN F polypeptide according to  claim 1 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence represented by any of SEQ ID NOs: 1 to 16 or 79 to 84, or comprises: 
 a) a sequence that is more than 70% identical to the sequence of any of SEQ ID NOs: 1 to 16 or 79 to 84;    b) a functional portion of any of SEQ ID NOs: 1 to 16 or 79 to 84 that maintains the interaction with the target with affinity of 1×10 −6  M or better;    c) a functional portion of any of SEQ ID NOs: 1 to 7, 23 to 31, and 62 to 65 that comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target.    
     
     
         13 . Anti-TNF polypeptide according to  claim 12 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 80% identical to the sequence of any of SEQ ID NOs: 1 to 16 or 79 to 84.  
     
     
         14 . Anti-TNF polypeptide according to  claim 13 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 90% identical to the sequence of any of SEQ ID NOs: 1 to 16 or 79 to 84.  
     
     
         15 . Anti-TNF polypeptide according to  claim 12 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence represented by any of SEQ ID NOs: 1 to 16 or 79 to 84.  
     
     
         16 . Anti-TNF polypeptide according to  claim 1 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises the sequence of SEQ ID NO: 4, or comprises: 
 a) a sequence that is more than 70% identical to the sequence of SEQ ID NO: 4;    b) a functional portion of SEQ ID NO: 4 that maintains the interaction with the target with affinity of 1×10 −6  M or better;    c) a functional portion of SEQ ID NO: 4 that comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target.    
     
     
         17 . Anti-TNF polypeptide according to  claim 16 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 80% identical to the sequence of SEQ ID NO:4.  
     
     
         18 . Anti-TNF polypeptide according to  claim 17 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 90% identical to the sequence of SEQ ID NO:4.  
     
     
         19 . Anti-TNF polypeptide according to  claim 16 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises the sequence of SEQ ID NO:4.  
     
     
         20 . Method for treating and/or preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a therapeutically effective amount of an anti-TNF polypeptide according to  claim 1 .  
     
     
         21 . Method for treating and/or preventing and/or alleviating disorders relating to inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, Diabetes Type I, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies, Thyroiditis, or Vasculitis, comprising administering to a subject a therapeutically effective amount of an anti-TNF polypeptide according to  claim 1 .  
     
     
         22 . Composition comprising a polypeptide according to  claim 1  and a suitable pharmaceutical vehicle.  
     
     
         23 . Method for treating and/or preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a therapeutically effective amount of a composition according to  claim 22 .  
     
     
         24 . Method for treating and/or preventing and/or alleviating disorders relating to inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, Diabetes Type I, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies, Thyroiditis, or Vasculitis, comprising administering to a subject a therapeutically effective amount of a composition according to  claim 22 .  
     
     
         25 . Anti-TNF-alpha polypeptide comprising one or more single domain antibodies directed against tumor necrosis factor alpha (TNF-alpha) and an effector group.  
     
     
         26 . Anti-TNF-alpha polypeptide according to  claim 25 , in which the effector group is a human Fc domain.  
     
     
         27 . Anti-TNF-alpha polypeptide according to  claim 26 , in which the one or more single domain antibodies directed against tumor necrosis factor alpha (TNF-alpha) are fused to the Fc domain.  
     
     
         28 . Anti-TNF-alpha polypeptide according to  claim 25 , which inhibits the interaction between TNF alpha and one or more TNF alpha receptors.  
     
     
         29 . Anti-TNF-alpha polypeptide according to  claim 25 , which modifies the biological activity of TNF-alpha after binding to TNF alpha.  
     
     
         30 . Anti-TNF-alpha polypeptide according to  claim 25 , which binds into the receptor-binding groove of TNF-alpha.  
     
     
         31 . Anti-TNF-alpha polypeptide according to  claim 25 , in which said one or more single domain antibodies directed against TNF-alpha bind to TNF-alpha with an affinity of better than 10 −6  M.  
     
     
         32 . Anti-TNF-alpha polypeptide according to  claim 25 , in which said one or more single domain antibodies directed against TNF-alpha have an amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine and glutamine at position 45 and a tryptophan at position 103 according to the Kabat numbering.  
     
     
         33 . Anti-TNF-alpha polypeptide according to  claim 25 , in which said one or more single domain antibodies directed against TNF-alpha have an arginine residue at position 103 according to the Kabat numbering.  
     
     
         34 . Anti-TNF-alpha polypeptide according to  claim 25 , in which said one or more single domain antibodies directed against TNF-alpha are one or more  Camelidae  VHHs.  
     
     
         35 . Anti-TNF-alpha polypeptide according to  claim 34 , in which said one or more single domain antibodies directed against TNF-alpha are one or more humanized  Camelidae  VHHs.  
     
     
         36 . Anti-TNF polypeptide according to  claim 35 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence represented by any of SEQ ID NOs: 1 to 16 or 79 to 84, or comprises: 
 a) a sequence that is more than 70% identical to the sequence of any of SEQ ID NOs: 1 to 16 or 79 to 84;    b) a functional portion of any of SEQ ID NOs: 1 to 16 or 79 to 84 that maintains the interaction with the target with affinity of 1×10 −6  M or better;    c) a functional portion of any of SEQ ID NOs: 1 to 7, 23 to 31, and 62 to 65 that comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target.    
     
     
         37 . Anti-TNF polypeptide according to  claim 36 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 80% identical to the sequence of any of SEQ ID NOs: 1 to 16 or 79 to 84.  
     
     
         38 . Anti-TNF polypeptide according to  claim 37 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 90% identical to the sequence of any of SEQ ID NOs: 1 to 16 or 79 to 84.  
     
     
         39 . Anti-TNF polypeptide according to  claim 36 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence represented by any of SEQ ID NOs: 1 to 16 or79 to 84.  
     
     
         40 . Anti-TNF polypeptide according to  claim 25 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises the sequence of SEQ ID NO: 4, or comprises: 
 a) a sequence that is more than 70% identical to the sequence of SEQ ID NO: 4;    b) a functional portion of SEQ ID NO: 4 that maintains the interaction with the target with affinity of 1×10 −6  M or better;    c) a functional portion of SEQ ID NO: 4 that comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target.    
     
     
         41 . Anti-TNF polypeptide according to  claim 40 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 80% identical to the sequence of SEQ ID NO:4.  
     
     
         42 . Anti-TNF polypeptide according to  claim 41 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises a sequence that is more than 90% identical to the sequence of SEQ ID NO:4.  
     
     
         43 . Anti-TNF polypeptide according to  claim 40 , wherein the one or more one single domain antibodies directed against TNF-alpha comprises the sequence of SEQ ID NO:4.  
     
     
         44 . Method for treating and/or preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a therapeutically effective amount of an anti-TNF polypeptide according to  claim 25 .  
     
     
         45 . Method for treating and/or preventing and/or alleviating disorders relating to inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, Diabetes Type I, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies, Thyroiditis, or Vasculitis, comprising administering to a subject a therapeutically effective amount of an anti-TNF polypeptide according to  claim 25 .  
     
     
         46 . Composition comprising a polypeptide according to  claim 25  and a suitable pharmaceutical vehicle.  
     
     
         47 . Method for treating and/or preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a therapeutically effective amount of a composition according to  claim 46 .  
     
     
         48 . Method for treating and/or preventing and/or alleviating disorders relating to inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, Diabetes Type I, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies, Thyroiditis, or Vasculitis, comprising administering to a subject a therapeutically effective amount of a composition according to  claim 46 .  
     
     
         49 . A method for synthesizing a single-domain-effector group (dAb-effector group) suitable for in vivo use comprising the steps of: (a) selecting an antibody single variable domain having an epitope binding specificity; and (b) attaching the single domain of step (a) to an effector group.  
     
     
         50 . A method according to  claim 49  wherein the antibody single variable domain is a heavy chain variable domain.  
     
     
         51 . A method according to  claim 49  wherein the effector group comprises any one or more of those groups selected from the group consisting of: an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, an Fc region of an antibody and a hinge region of an antibody molecule.  
     
     
         52 . A method according to  claim 49 , wherein the effector group constitutes an Fc region of an antibody.  
     
     
         53 . A method according to  claim 49 , wherein the effector group consists of a CH2 and CH3 domain.  
     
     
         54 . A method according to  claim 51 , wherein the effector group consists of a CH2 domain, a CH3 domain and the hinge region of an antibody molecule.  
     
     
         55 . A method according to  claim 49 , wherein the antibody single variable domain is a non- Camelid  variable domain.  
     
     
         56 . A method according to  claim 55 , wherein the antibody single variable domain is a human variable domain.  
     
     
         57 . A method according to  claim 49 , wherein the antibody single variable domain comprises one or more human framework regions.  
     
     
         58 . A method according to  claim 49 , wherein the antibody single variable domain comprises four framework regions as defined by Kabat, which are derived from a human.  
     
     
         59 . A method according to  claim 58 , wherein one or more of the human framework regions as defined by Kabat are identical on the amino acid level to those encoded by human germline antibody genes.  
     
     
         60 . A method according to  claim 49 , wherein the antibody single variable domain is isolated, in part, by human immunization.  
     
     
         61 . A method according to  claim 49 , wherein the antibody single variable domain is not isolated by animal immunization.  
     
     
         62 . A method according to  claim 49 , wherein the effector group is of  Camelid  or human origin.  
     
     
         63 . A method according to  claim 49 , wherein the single variable domain comprises one or more human framework regions and the immunoglobulin effector group is of human origin.  
     
     
         64 . A method according to  claim 63 , wherein the single variable domain comprises four human framework regions and the immunoglobulin effector group is of human origin.  
     
     
         65 . A method according to  claim 49 , wherein attaching of the single variable domain to the effector group in step (b) is effected by expressing the single-domain-effector group as a fusion polypeptide.  
     
     
         66 . A dAb-effector group comprising: (a) an antibody single variable domain having an epitope binding specificity; and (b) an effector group attached to said antibody single variable domain.  
     
     
         67 . A medicament comprising the dAb-effector group of  claim 66 .  
     
     
         68 . A dAb-effector group according to  claim 66 , wherein the antibody single variable domain is a heavy chain variable domain.  
     
     
         69 . A dAb-effector group according to  claim 66 , wherein the effector group comprises any one or more of those groups selected from the group consisting of: an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, an Fc region of an antibody and a hinge region of an antibody molecule.  
     
     
         70 . A dAb-effector group according to  claim 69  wherein the effector group consists of a CH2 and CH3 domain.  
     
     
         71 . A dAb-effector group according to  claim 69  wherein the effector group consists of a CH2 domain, a CH3 domain and the hinge region of an antibody molecule.  
     
     
         72 . A dAb-effector group according to  claim 69  wherein the effector group constitutes an Fc region of an antibody.  
     
     
         73 . A dAb-effector group according to  claim 66 , wherein the antibody single variable domain is of human origin.  
     
     
         74 . A dAb-effector group according to  claim 66 , wherein the antibody single variable domain comprises human framework regions.  
     
     
         75 . A dAb-effector group according to  claim 66 , wherein the effector group is of  Camelid  or human origin.  
     
     
         76 . A dAb-effector group according to  claim 66 , wherein the single variable domain comprises one or more human framework regions and the immunoglobulin effector group is of human origin.  
     
     
         77 . Two or more dAb-effector groups according to  claim 66  provided as a higher order structure selected from the group consisting of the following: dimers, trimers and multimers.  
     
     
         78 . Two dAb-effector groups according to  claim 77  provided as a heterodimer or a homodimer.  
     
     
         79 . Two dAb-effector groups according to  claim 78  provided as a homodimer.  
     
     
         80 . A nucleic acid molecule encoding a dAb-effector group according to  claim 66 .  
     
     
         81 . A nucleic acid molecule according to  claim 80  further encoding a signal sequence for export of the dAb and effector group from the cytoplasm of a host cell upon expression.  
     
     
         82 . A vector comprising nucleic acid according to  claim 80 .  
     
     
         83 . A host cell transfected with a vector according to  claim 82 .  
     
     
         84 . A composition comprising a dAb-effector group(s) according to  claim 66  and a pharmaceutically acceptable carrier, diluent or excipient.  
     
     
         85 . A method of treating and/or preventing disease in a patient, wherein the method comprises administering to the patient a dAb-effector group(s) according to  claim 66  or a composition according to claims  84 .  
     
     
         86 . A medicament for the treatment and/or prevention of disease, comprising the dAb-effector group of  claim 66  or the composition of  claim 84 .  
     
     
         87 . A method for the treatment and/or prophylaxis of an inflammatory disease in a patient in need of such treatment and/or prophylaxis which comprises the step of administering to that patient a therapeutically effective amount of a dAb-effector group according to  claim 66 .  
     
     
         88 . A method according to  claim 87  wherein the inflammatory disease is mediated by TNF alpha and is selected from the group consisting of the following: rheumatoid arthritis, psoriasis, Crohn's disease, inflammatory bowel disease (IBD), multiple sclerosis, Alzheimer's, and glomerular nephritis.  
     
     
         89 . A method according to  claim 88 , wherein the TNF alpha is human TNF alpha and the patient is a human.