Stabilized single domain antibodies

Abstract

The present invention relates to heterospecific polypeptide constructs comprising at least one single domain antibody directed against a therapeutic and/or diagnostic target and at least one single domain antibody directed against a serum protein, said construct having a prolonged lifetime in biological circulatory systems. The invention further relates to methods for stabilising VHHs in biological circulatory systems.

1 . A polypeptide construct comprising: 
 at least one single domain antibody directed against a therapeutic and/or diagnostic target, and    at least one single domain antibody directed against a serum protein.    
     
     
         2 . A polypeptide construct according to  claim 1  wherein: 
 the number of anti-target single domain antibodies is at least two, and    at least two anti-target single domain antibodies do not share the same sequence, or all the anti-target single domain antibodies share the same sequence.    
     
     
         3 . A polypeptide construct according to  claim 1  wherein: 
 the number of anti-serum protein single domain antibodies is at least two, and    at least two anti-serum-protein single domain antibodies do not share the same sequence, or all the anti-serum-protein single domain antibodies share the same sequence.    
     
     
         4 . A polypeptide construct according to  claim 1  wherein the at least one single domain antibody is a  Camelidae  VHHs antibody.  
     
     
         5 . A polypeptide construct according to  claim 1  wherein the at least one single domain antibody is a humanised  Camelidae  VHHs antibody.  
     
     
         6 . A polypeptide construct according to  claim 1  wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen or a fragment thereof.  
     
     
         7 . A polypeptide construct according to  claim 1  wherein the at least one single domain anti-serum protein antibody corresponds to a sequence represented by any of SEQ ID NOS: 1 to 4, and 28 to 40.  
     
     
         8 . A polypeptide construct according to  claim 1  wherein the target is TNF-alpha.  
     
     
         9 . A polypeptide construct according to  claim 7  corresponding to the sequence represented by any of SEQ ID NO: 5 to 18.  
     
     
         10 . (canceled)  
     
     
         11 . A nucleic acid encoding a polypeptide construct according to  claim 9 .  
     
     
         12 . (canceled)  
     
     
         13 . A method for treatment, prevention and/or alleviation of disorders relating to inflammatory processes comprising administering to a subject in need of such treatment a polypeptide construct according to  claim 8 .  
     
     
         14 . (canceled)  
     
     
         15 . The method according to  claim 13  wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.  
     
     
         16 . A polypeptide construct according to  claim 1  wherein a the target is vWF  
     
     
         17 . A polypeptide construct according to  claim 1  wherein the target is collagen.  
     
     
         18 . A polypeptide construct according to  claim 16  wherein at least one anti-target single domain antibody is anti-vWF VHHs.  
     
     
         19 . A polypeptide construct according to  claim 18  corresponding to the sequence represented by any of SEQ ID NOS: 19 to 21.  
     
     
         20 . (canceled)  
     
     
         21 . A nucleic acid encoding a polypeptide construct according to  claim 19 .  
     
     
         22 . A method for treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof comprising administering to a subject in need of such treatment A polypeptide construct according to  claim 16 .  
     
     
         23 . (canceled)  
     
     
         24 . A method according to  claim 22  wherein said disorders are any of cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis, and said conditions are those arising from coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.  
     
     
         25 . A method according to  claim 22  wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.  
     
     
         26 . A polypeptide construct according to  claim 1  wherein the target is IgE.  
     
     
         27 . A polypeptide construct according to  claim 26  wherein the at least one anti-target single domain antibody is anti-IgE VHHs.  
     
     
         28 . A polypeptide construct according to  claim 26  corresponding to the sequence represented by any of SEQ ID NOS: 22 to 24.  
     
     
         29 . (canceled)  
     
     
         30 . A nucleic acid encoding a polypeptide construct according to  claim 28 .  
     
     
         31 . (canceled)  
     
     
         32 . A method for the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions comprising administering to a subject in need of such treatment a polypeptide construct according to  claim 26 .  
     
     
         33 . A method according to  claim 32  wherein said disorders or conditions are any of hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.  
     
     
         34 . A method according to  claim 32  wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.  
     
     
         35 . A polypeptide construct according to  claim 1  wherein the target is IFN-gamma.  
     
     
         36 . A polypeptide construct according to  claim 35  wherein at least one anti-target single domain antibody is anti-IFN-gamma VHHs.  
     
     
         37 . A polypeptide construct according to  claim 35  corresponding to a sequence represented by SEQ ID NOs: 25 to 27.  
     
     
         38 . (canceled)  
     
     
         39 . A nucleic acid encoding a polypeptide construct according to  claim 37 .  
     
     
         40 . (canceled)  
     
     
         41 . A method for the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active comprising administering to a subject in need of such treatment a polypeptide construct according to  claim 35 .  
     
     
         42 . A method according to  claim 41  wherein said disorders or conditions are any of Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.  
     
     
         43 . A method according to  claim 41  wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.  
     
     
         44 . A composition comprising a polypeptide construct according to  claim 8  or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.  
     
     
         45 . A composition comprising a polypeptide construct according to  claim 16  or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.  
     
     
         46 . A composition comprising a polypeptide construct according to  claim 26  or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.  
     
     
         47 . A polypeptide construct according to  claim 1  directed against a single target wherein said target is involved in a disease process.  
     
     
         48 . (canceled)  
     
     
         49 . A nucleic acid encoding a polypeptide construct according to  claim 47 .  
     
     
         50 .- 52 . (canceled)  
     
     
         53 . A method for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation comprising administering to a subject in need of such treatment the polypeptide construct according to  claim 47 .  
     
     
         54 . (canceled)  
     
     
         55 . A method for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time comprising administering to a subject in need of such treatment the polypeptide construct according to  claim 47 .  
     
     
         56 . A according to any of claims  53  and  55 , wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.  
     
     
         57 . (canceled)  
     
     
         58 . A method of producing a polypeptide according to  claim 1  comprising 
 (a) culturing host cells comprising nucleic acid capable of encoding a polypeptide according to  claim 1 , under conditions allowing the expression of the polypeptide, and,    (b) recovering the produced polypeptide from the culture.    
     
     
         59 . A method according to  claim 58 , wherein said host cells are bacterial or yeast.  
     
     
         60 . A method for prolonging the half-life of a single domain antibody in the blood stream of a subject, said antibody directed against a therapeutic and/or diagnostic target, comprising joining thereto one or more single domain antibodies directed against a serum protein.  
     
     
         61 . A method according to  claim 60  wherein said anti-target single domain antibodies do not share the same sequence.  
     
     
         62 . A method according to  claim 60  wherein said anti-serum protein single domain antibodies do not share the same sequence.  
     
     
         63 . A method according to  claim 60  wherein said single domain antibodies are  Camelidae  VHH antibodies.  
     
     
         64 . A method according to  claim 60  wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen or a fragment thereof.  
     
     
         65 . A method according to  claim 60  wherein said serum protein comprises a sequence corresponding to any of SEQ ID NOs: 1 to 4, a homologous sequence, a functional portion thereof, or a homologous sequence of a functional portion thereof.  
     
     
         66 . A composition comprising a polypeptide according to  claim 1  or a nucleic acid capable of encoding said polypeptide and a pharmaceutically acceptable vehicle.