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 . A polypeptide comprising a first immunoglobulin single variable domain having a first antigen or epitope binding specificity and a second immunoglobulin single variable domain having a second antigen or epitope binding specificity wherein one or both of said first and second variable domains bind to an antigen or epitope which increases the half-life of the ligand in vivo, and either (i) the first and the second immunoglobulin variable domains are heavy chain variable domains; or (ii) the first and the second immunoglobulin variable domains are light chain variable domains. 
     
     
         2 . The polypeptide according to  claim 1 , wherein the polypeptide is provided as an IgG immunoglobulin comprising four heavy chain single variable domains or four light chain single variable domains. 
     
     
         3 . The polypeptide according to  claim 2 , wherein the single variable domains are identical. 
     
     
         4 . The polypeptide of  claim 1 , wherein the heavy chain domains are Camelid V HH  domains. 
     
     
         5 . The polypeptide according to  claim 1 , wherein the first, and second domains bind independently, such that the polypeptide may simultaneously bind both the first and second epitopes or antigens. 
     
     
         6 . The polypeptide according to  claim 1 , wherein said first and second epitopes are present on separate antigens. 
     
     
         7 . The polypeptide according to  claim 1 , wherein said first and second epitopes are present on the same antigen. 
     
     
         8 . The polypeptide according to  claim 1  wherein the variable regions are covalently associated. 
     
     
         9 . The polypeptide according to  claim 8  wherein the covalent association is mediated by disulphide bonds. 
     
     
         10 . A polypeptide comprising an anti-human TNF-alpha single domain antibody (dAb) and an anti-serum albumin (SA) dAb. 
     
     
         11 . The polypeptide according to  claim 10 , wherein the dAbs are Camelid V HH  domains. 
     
     
         12 . The polypeptide according to  claim 1  wherein the polypeptide comprises a variable domain having one or more framework regions comprising an amino acid sequence that is the same as the amino acid sequence of a corresponding framework region encoded by a human germline antibody gene segment, or the amino acid sequences of one or more of said framework regions collectively comprises up to 5 amino acid differences relative to the amino acid sequence of said corresponding framework region encoded by a human germline antibody gene segment. 
     
     
         13 . The polypeptide according to  claim 1 , wherein the polypeptide comprises a variable domain, wherein the amino acid sequences of FW1, FW2, FW3 and FW4 are the same as the amino acid sequences of corresponding framework regions encoded by a human germline antibody gene segment, or the amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding framework regions encoded by said human germline antibody gene segment. 
     
     
         14 . The polypeptide according to  claim 13 , which comprises an antibody variable domain comprising FW1, FW2 and FW3 regions, and the amino acid sequences of said FW1, FW2 and FW3 are the same as the amino acid sequences of corresponding framework regions encoded by human germline antibody gene segments. 
     
     
         15 . The polypeptide according to a  claim 1  which the heavy chain variable domain is not a Camelid immunoglobulin variable domain. 
     
     
         16 . The polypeptide of  claim 15 , wherein the heavy chain variable domain does not contain one or more amino acids that are specific to Camelid immunoglobulin variable domains as compared to human V H  domains. 
     
     
         17 . Nucleic acid encoding at least the polypeptide according to  claim 1 . 
     
     
         18 . A vector comprising the nucleic acid according to  claim 17 . 
     
     
         19 . A vector according to  claim 18 , further comprising components necessary for the expression of the polypeptide. 
     
     
         20 . A host cell comprising a vector according to  claim 19 . 
     
     
         21 . A pharmaceutical composition comprising the polypeptide according to  claim 1 , and a pharmaceutically acceptable excipient, carrier or diluent. 
     
     
         22 . A polypeptide comprising a first immunoglobulin single variable domain having binding specificity for serum albumin (SA), and a second immunoglobulin single variable domain having binding specificity for an antigen selected from the group consisting of IFN-gamma, tumour necrosis factor (TNF), TNF-alpha, and TNF receptor. 
     
     
         23 . A polypeptide comprising a first immunoglobulin single variable domain having binding specificity for serum albumin (SA), and a second immunoglobulin single variable domain having binding specificity for an antigen selected from the group consisting of human or animal proteins, cytokines, and cytokine receptors. 
     
     
         24 . A polypeptide comprising a first immunoglobulin single variable domain having binding specificity for serum albumin (SA), and a second single immunoglobulin variable domain having binding specificity for a receptor for a cytokine listed in  claim 22 . 
     
     
         25 . The polypeptide of  claim 22 ,  23 , or  24 , where each of the first and second domains is (i) a heavy chain variable domain or (ii) a light chain variable domain. 
     
     
         26 . A polypeptide comprising a dimer, trimer or tetrameter of (i) heavy chain single variable domains or (ii) light chain single variable domains, the domains being specific for the same epitope or adjacent epitopes on the same target. 
     
     
         27 . A polypeptide comprising (i) first and second heavy chain single variable domains or (ii) first and second light chain single variable domains, the domains having the same epitope specificity, wherein the epitope is provided as multiple copies by TNF-alpha. 
     
     
         28 . A polypeptide comprising (i) first and second heavy chain single variable domains, or (ii) first and second light chain single variable domains, wherein each domain has binding specificity to an antigen selected from the group consisting of human or animal proteins, cytokines, and cytokine receptors. 
     
     
         29 . The polypeptide according to  claim 28 , wherein the cytokine receptors is TNF receptor. 
     
     
         30 . A polypeptide comprising (i) first and second heavy chain single variable domains, or (ii) first and second light chain single variable domains, wherein each domain has binding specificity to an antigen selected from the group consisting of IFN-gamma, tumour necrosis factor (TNF), TNF-alpha, and TNF receptor. 
     
     
         31 . The polypeptide according to  claim 26 ,  27 ,  28  or  30 , wherein the variable domains are provided by an antibody scFv fragment. 
     
     
         32 . The polypeptide according to  claim 26 ,  27 ,  28  or  30 , wherein the variable domains are provided by an antibody Fab region. 
     
     
         33 . The polypeptide of  claim 22 ,  26 ,  27 ,  28  or  30 , wherein the or each variable domain or dAb is a Camelid V HH  domain.