Method for obtaining polypeptide constructs comprising two or more single domain antibodies

Abstract

The present invention relates to methods for obtaining a polypeptide construct directed against one or more antigens and/or epitopes and having one or more desired characteristics, wherein the polypeptide construct comprises at least two single domain antibodies. The methods of the present invention involve producing a diversity of polypeptide constructs that are structural variants and screening the produced diversity of polypeptide constructs for a polypeptide construct having said one or more desired characteristics. The present invention further relates to polypeptide constructs directed against one or more antigens and/or epitopes having one or more desired characteristics, wherein the polypeptide construct comprises at least two single domain antibodies. The methods and polypeptide constructs according to the present invention are useful for the identification of optimal therapeutic compounds.

1 . A method for obtaining a polypeptide construct directed against to one or more antigens and/or epitopes and having one or more desired characteristics, wherein said polypeptide construct essentially consists or comprises at least two single domain antibodies, said method at least comprising the steps of:
 (i) selecting a template polypeptide construct directed against one or more antigens or epitopes,   (ii) producing a diversity of polypeptide constructs that are structural variants of the selected template polypeptide construct of step (i), wherein said structural variants each comprise at least two single domain antibodies, and   (iii) screening the produced diversity of polypeptide constructs of step (ii) for a polypeptide construct having said one or more desired characteristics, wherein said polypeptide construct comprises at least two single domain antibodies and is directed against one or more antigens and/or epitopes.   
     
     
         2 . The method according to  claim 1 , wherein said diversity of polypeptide constructs is a library of polypeptide constructs. 
     
     
         3 . The method according to  claim 1 , wherein said structural variants of step (ii) are one or more of the following:
 structural variants with regard to the number and/or identity of said single domain antibodies in said polypeptide constructs, and/or,   structural variants with regard to the relative position of said single domain antibodies within the polypeptide constructs, and/or,   structural variants with regard to the amino acid residues of the CDR region(s) of one or more of said single domain antibodies in said polypeptide constructs, and/or,   structural variants with regard to the amino acid residues of the framework region(s) of one or more of said single domain antibodies in said polypeptide constructs, and/or,   structural variants with regard to the codon usage in the nucleic acid sequences encoding the polypeptide constructs.   
     
     
         4 . The method according to  claim 1 , wherein said structural variants of step (ii) comprise at least two single domain antibodies that are linked via one or more peptide linkers. 
     
     
         5 . The method according to  claim 4 , wherein said structural variants of step ii) are one or more of the following
 structural variants with regard to the composition of said one or more peptide linkers, and/or,   structural variants with regard to the number of said one or more linkers, and/or,   structural variants with regard to the relative position of said one or more linkers in said polypeptide constructs.   
     
     
         6 . The method according to  claim 1 , wherein the single domain antibodies present in said diversity of polypeptide constructs and in said polypeptide construct are all heavy chain variable domains or are all light chain variable domains. 
     
     
         7 . The method according to  claim 6 , wherein the single domain antibodies present in said diversity of polypeptide constructs and in said polypeptide construct are all heavy chain variable domains. 
     
     
         8 . The method according to  claim 7 , wherein the heavy chain variable domains are variable domains obtainable from heavy chain antibodies (VHH). 
     
     
         9 . The method according to  claim 1 , wherein in step (iii) the produced diversity of polypeptide constructs is screened for a polypeptide construct having a suitable binding affinity or avidity, for a polypeptide construct having a suitable solubility, for a polypeptide construct having a suitable stability, for a polypeptide construct having a suitable efficacy, and/or for a polypeptide construct having a suitable potency. 
     
     
         10 .- 13 . (canceled) 
     
     
         14 . The method according to  claim 1 , wherein said diversity of polypeptide construct comprises at least two single domain antibodies that are directed against the same epitopes. 
     
     
         15 . The method according to  claim 1 , wherein said diversity of polypeptide construct comprises at least two single domain antibodies that are directed against at least two different epitopes that are present on the same antigen. 
     
     
         16 . The method according to  claim 1 , wherein said diversity of polypeptide construct comprises at least two single domain antibodies that are directed against at least two different epitopes that are present on different antigens. 
     
     
         17 . The method according to  claim 1 , wherein said polypeptide construct comprises at least three single domain antibodies. 
     
     
         18 . The method according to  claim 17 , wherein said three single domain antibodies are directed against the same epitope. 
     
     
         19 . The method according to  claim 17 , wherein said three single domain antibodies are directed against at least three different epitopes. 
     
     
         20 . The method according to  claim 19 , wherein said at least three different epitopes are present on the same antigen. 
     
     
         21 . The method according to  claim 19 , wherein said at least three different epitopes are present on at least two different antigens. 
     
     
         22 . Polypeptide construct obtainable by the method of  claim 1 .