Single domain antibodies directed against tumor 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 . An anti-TNF-alpha polypeptide comprising at least one anti-TNF-alpha single domain antibody.  
   
   
       2 . An anti-TNF-alpha polypeptide according to  claim 1  wherein a single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 1 to 16 and 79 to 84.  
   
   
       3 . An anti-TNF-alpha polypeptide according to  claim 1  further comprising at least one single domain antibody directed against a serum protein.  
   
   
       4 . An anti-TNF-alpha polypeptide according to  claim 3  wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin or fibrinogen.  
   
   
       5 . An anti-TNF-alpha polypeptide according to  claim 3  wherein the anti-serum protein single domain antibody correspond to a sequence represented by any of SEQ ID NOs: 26 to 29 and 85 to 97.  
   
   
       6 . An anti-TNF-alpha polypeptide according to  claim 3  corresponding to a sequence represented by any of SEQ ID NOs: 30 to 43.  
   
   
       7 . An anti-TNF-alpha polypeptide according to  claim 1  further comprising at least one single domain antibody selected from the group consisting of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor single domain antibody and anti-IFN-gamma receptor single domain antibody.  
   
   
       8 . An anti-TNF-alpha polypeptide according to  claim 1 , wherein the number of single domain antibodies directed against TNF-alpha is at least two.  
   
   
       9 . An anti-TNF-alpha polypeptide according to  claim 8  corresponding to a sequence represented by any of SEQ ID NOs: 73 to 76.  
   
   
       10 . An anti-TNF-alpha polypeptide according  claim 1 , wherein at least one single domain antibody is a humanized  Camelidae  VHHs.  
   
   
       11 . An anti-TNF-alpha polypeptide according to  claim 10  wherein a humanized  Camelidae  VHH corresponds to a sequence represented by any of SEQ ID NOs: 17 to 19 and 21 to 24.  
   
   
       12 . A composition comprising an anti-TNF-alpha polypeptide according to  claim 1  and at least one single domain antibody from the group consisting of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor single domain antibody and anti-IFN-gamma receptor single domain antibody, for simultaneous, separate or sequential administration to a subject.  
   
   
       13 . An anti-TNF-alpha polypeptide according to  claim 7  wherein the at least one anti-IFN-gamma single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 44 to 72.  
   
   
       14 . (canceled)  
   
   
       15 . An anti-TNF-alpha polypeptide according to  claim 1 , wherein the anti-TNF-alpha polypeptide is an homologous sequence, a functional portion, or a functional portion of an homologous sequence of the full length anti-TNF-alpha polypeptide.  
   
   
       16 . An anti-TNF-alpha polypeptide according to  claim 1  wherein the at least one single domain antibody is a  Camelidae  VHH.  
   
   
       17 . A nucleic acid encoding an anti-TNF-alpha polypeptide according to  claim 1 .  
   
   
       18 . A method of identifying an agent that modulates the binding of an anti-TNF-alpha polypeptide to Tumor Necrosis Factor-alpha comprising the steps of: 
 (a) contacting an anti-TNF-alpha polypeptide of  claim 1  with a target that is Tumor Necrosis Factor alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and    (b) measuring the binding between the polypeptide and target of step (a), wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates the binding of an anti-TNF-alpha polypeptide and Tumor Necrosis Factor-alpha.    
   
   
       19 . A method of identifying an agent that modulates Tumor Necrosis Factor-alpha-mediated disorders through the binding of an anti-TNF-alpha polypeptide to Tumor Necrosis Factor-alpha comprising: 
 (a) contacting an anti-TNF-alpha polypeptide of  claim 1  with a target that is Tumor Necrosis Factor alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and    (b) measuring the binding between the polypeptide and target of step (a), wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates Tumor Necrosis Factor alpha-mediated disorders.    
   
   
       20 . A method of identifying an agent that modulates the binding of Tumor Necrosis Factor alpha to its receptor through the binding of an anti-TNF-alpha polypeptide to Tumor Necrosis Factor-alpha comprising: 
 (a) contacting an anti-TNF-alpha polypeptide of  claim 1  with a target that is Tumor Necrosis Factor-alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and    (b) measuring the binding between the polypeptide and target of step (a), wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator, identifies said candidate modulator as an agent that modulates the binding of Tumor Necrosis Factor-alpha to its receptor.    
   
   
       21 . A kit for screening for agents that modulate Tumor Necrosis Factor-alpha-mediated disorders comprising an anti-TNF-alpha polypeptide of  claim 1  and Tumor Necrosis Factor-alpha.  
   
   
       22 - 25 . (canceled)  
   
   
       26 . A method for treating and/or preventing and/or alleviating disorders relating to inflammatory reactions comprising administering to a subject in need of such treatment an effective amount of the anti-TNF-alpha polypeptide of  claim 1 .  
   
   
       27 . (canceled)  
   
   
       28 . A method for treating and/or preventing and/or alleviating the symptoms of disorders susceptible to modulation by a TNF-alpha modulating substance, comprising administering to a subject in need of such treatment an anti-TNF-alpha polypeptide of  claim 1  that passes through the gastric environment without the substance being inactivated.  
   
   
       29 . (canceled)  
   
   
       30 . A method for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance delivered to the vaginal and/or rectal tract, or the nose, upper respiratory tract and/or lung, comprising administering to the vaginal and/or rectal tract, or the nose, upper respiratory tract and/or lung, of a subject in need of such treatment an effective amount of an anti-TNF-alpha polypeptide of  claim 1 .  
   
   
       31 - 33 . (canceled)  
   
   
       34 . A method for treating and/or preventing and/or alleviating the symptoms of disorders susceptible to modulation by a TNF-alpha modulating substance delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa, comprising administering to the intestinal mucosa of a subject in need of such treatment an anti-TNF-alpha polypeptide of  claim 1 .  
   
   
       35 . (canceled)  
   
   
       36 . A method for treating and/or preventing and/or alleviating the symptoms of disorders susceptible to modulation by a TNF-alpha modulating substance, comprising administering to a subject in need of such treatment an anti-TNF-alpha polypeptide of  claim 1  that passes through the tissues beneath the tongue effectively.  
   
   
       37 . (canceled)  
   
   
       38 . A method for treating and/or preventing and/or alleviating the symptoms of disorders susceptible to modulation by a TNF-alpha modulating substance, comprising administering to a subject in need of such treatment an anti-TNF-alpha polypeptide of  claim 1  that passes through the skin effectively.  
   
   
       39 . A method according to any of claims  26 ,  28 ,  30 ,  34 ,  36 , and  38  wherein said disorders are any of 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, and Vasculitis.  
   
   
       40 . A composition comprising the anti-TNF-alpha polypeptide of  claim 1 , and a suitable pharmaceutical vehicle.  
   
   
       41 . A method of diagnosing a disorder characterised by the dysfunction of Tumor Necrosis Factor-alpha comprising: 
 (a) contacting a sample with an anti-TNF-alpha polypeptide of  claim 1 ,    (b) detecting binding of said polypeptide to said sample, and    (c) comparing the binding detected in step (b) with a standard, wherein a difference in binding relative to said sample is diagnostic of a disorder characterised by dysfunction of Tumor Necrosis Factor-alpha.    
   
   
       42 - 43 . (canceled)  
   
   
       44 . A method for purification of Tumor Necrosis Factor-alpha comprising contacting a sample with the anti-TNF-alpha polypeptide of  claim 1 .  
   
   
       45 . A method for inhibiting the interaction between Tumor Necrosis Factor-alpha and one or more Tumor Necrosis Factor-alpha receptors, comprising contacting Tumor Necrosis Factor-alpha with the anti-TNF-alpha polypeptide of  claim 1 .  
   
   
       46 . A method for producing an anti-TNF-alpha polypeptide of  claim 1  comprising the steps of: 
 (a) obtaining double stranded DNA encoding a  Camelidae  VHH directed to Tumor Necrosis Factor alpha,    (b) cloning and expressing the DNA selected in step (b).    
   
   
       47 . A method of producing an anti-TNF-alpha polypeptide of  claim 1  comprising: 
 (a) culturing host cells comprising nucleic acid that encode an anti-TNF-alpha polypeptide of  claim 1 , under conditions allowing the expression of the polypeptide, and,    (b) recovering the produced polypeptide from the culture.    
   
   
       48 . A method according to  claim 47 , wherein said host cells are bacterial or yeast.  
   
   
       49 . (canceled)