High affinity adaptor molecules for redirecting antibody specifity

Abstract

Disclosed are methods for identifying high affinity adaptor molecules that bind to both a circulating antibody and a target molecule and redirect the specificity of the circulating antibody to the target molecule. Exemplary high affinity adaptor molecules are also provided.

1 . A method for identifying a high affinity adaptor molecule capable of redirecting antibody specificity, the method comprising:
 (a) providing a randomized library encoding a population of candidate targeting peptides;   (b) selecting a targeting peptide from the display library which binds with high affinity and/or selectivity to a target molecule;   (c) linking the targeting peptide to a ligand moiety via a linking moiety to form a candidate adaptor molecule; and   (d) evaluating the ability of the candidate adaptor molecule to redirect the specificity of the circulating antibody to the target molecule;   
       thereby identifying the adaptor molecule. 
     
     
         2 . The method of  claim 1 , wherein steps (a)-(d) are performed consecutively. 
     
     
         3 . The method of  claim 1 , wherein linking step (c) is performed prior to step (b). 
     
     
         4 . The method of  claim 1 , wherein the library is an mRNA display, ribosome display, yeast display, phage display or synthetic peptide library. 
     
     
         5 . The method of  claim 1 , wherein the targeting peptide binds to the target molecule with a binding affinity of 1 nM or lower. 
     
     
         6 . The method of  claim 1 , wherein the ligand moiety comprises a glycan moiety. 
     
     
         7 . The method of  claim 1 , wherein the ligand moiety is a blood group antigen. 
     
     
         8 . The method of  claim 1 , wherein the ligand moiety is a gal antigen or epitope thereof. 
     
     
         9 . The method of  claim 8 , wherein the ligand moiety consists of one or more gal-α-1-3-gal disaccharide units. 
     
     
         10 . The method of  claim 8 , wherein the ligand moiety is a modified gal antigen having modifications which reduce competitive binding by interfering molecules. 
     
     
         11 . The method of  claim 8 , wherein the ligand moiety is a modified gal antigen having modifications which reduce enzymatic or chemical degradation. 
     
     
         12 . The method of  claim 10 , wherein the modified gal antigen comprises a protecting group at a C6′ position of a terminal galactose residue. 
     
     
         13 . The method of  claim 1 , wherein the ligand moiety is a peptidomimetic of a gal antigen. 
     
     
         14 . The method of  claim 1 , wherein the ligand moiety is a peptide ligand moiety. 
     
     
         15 . The method of  claim 14 , wherein the peptide ligand moiety comprises an epitope that is selectively bound by an antigen binding site of the circulating antibody. 
     
     
         16 . The method of  claim 15 , wherein the peptide ligand moiety comprises an idiotope of an antibody, wherein the idiotope is selectively bound by a circulating anti-idiotypic antibody. 
     
     
         17 . The method of  claim 16 , wherein the peptide ligand moiety comprises a binding site portion of an Fc binding protein. 
     
     
         18 . The method of  claim 1 , wherein the peptide ligand moiety is selected by (i) providing a randomized mRNA display library encoding a population of candidate peptide ligand moieties; and (ii) selecting a peptide ligand moiety from the display library of step (i) which binds with high affinity and/or selectivity to a circulating antibody. 
     
     
         19 . The method of  claim 18 , wherein the candidate peptide ligand moieties are fused to targeting peptides prior to selection step (ii). 
     
     
         20 . The method of  claim 18 , wherein the candidate peptide ligand moieties are fused to targeting peptides following selection step (ii). 
     
     
         21 . The method of  claim 1 , wherein the target molecule is a soluble disease-associated molecule. 
     
     
         22 . The method of  claim 21 , wherein the redirected antibody specificity is evaluated by measuring opsonization or neutralization of the soluble molecule. 
     
     
         23 . The method of  claim 1 , wherein the ligand moiety is linked to the targeting moiety with a bifunctional linker moiety. 
     
     
         24 . The method of  claim 23 , wherein the bifunctional linker moiety links the targeting moiety and the ligand moiety via an amino group in the targeting moiety and a thiol moiety in the ligand moiety. 
     
     
         25 . The method of  claim 1 , wherein the target molecule is a present on the surface of an infected or neoplastic cell. 
     
     
         26 . The method of  claim 25 , wherein the redirected antibody specificity is evaluated by measuring ADCC or CDC-dependent killing of the cell. 
     
     
         27 . A high affinity adaptor molecule identified according to the method of any one of the preceding claims, the adaptor molecule comprising (i) a targeting moiety which binds with high affinity or selectivity to a target molecule, (ii) a ligand moiety which specifically binds to a circulating antibody; and (iii) a linker moiety linking the targeting moiety to the ligand moiety, wherein the adaptor molecule facilitates a functional interaction between the antibody and the target molecule. 
     
     
         28 . The high affinity adaptor molecule of  claim 27 , wherein the targeting moiety is a peptide targeting moiety. 
     
     
         29 . The adaptor molecule of  claim 27 , wherein the targeting moiety binds with high affinity or selectivity to VEGF ligand. 
     
     
         30 . The adaptor molecule of any one of  claims 27 , wherein the targeting moiety comprises one or more sequences selected from SEQ ID NOs 1, 2, 3 and 4. 
     
     
         31 . The adaptor molecule of any one of  claims 27 , wherein the targeting moiety is PEGylated. 
     
     
         32 . The adaptor molecule of any one of  claims 27 , wherein the ligand moiety comprises a Gal antigen which specifically binds to a circulating anti-Gal antibody. 
     
     
         33 . A high affinity adaptor molecule selected from the group consisting of: 
       
         
           
                 
               
                   (a) (SEQ ID NO: 1)- X - Y , comprising: 
                 
                   H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D- 
                 
                     
                 
                   Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro- 
                 
                     
                 
                   D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D- 
                 
                     
                 
                   Val-D-Trp-D-Val-D-Thr-PEG2-Cys- X - Y ; 
                 
                     
                 
                   (b) (SEQ ID NO: 2)- X - Y , comprising: 
                 
                   H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala- 
                 
                     
                 
                   D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D- 
                 
                     
                 
                   Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D- 
                 
                     
                 
                   Arg-D-Thr-D-Ile-D-Thr-PEG2-Cys- X - Y ; 
                 
                     
                 
                   (c) (SEQ ID NO: 3)- X - Y , comprising: 
                 
                   H-Gly-D-Val-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala- 
                 
                     
                 
                   D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D- 
                 
                     
                 
                   Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D- 
                 
                     
                 
                   Trp-D-Ser-D-Thr-D-Thr-PEG2-Cys- X - Y ; 
                 
                   and 
                 
                     
                 
                   (d) (SEQ ID NO: 4)- X - Y , comprising: 
                 
                   H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His- 
                 
                     
                 
                   D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D- 
                 
                     
                 
                   Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu- 
                 
                     
                 
                   D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr-PEG2-Cys- 
                 
                     
                 
                     X - Y ; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein, 
         X is bifunctional chemical linker with maleimide functionality; and 
         Y is an amino modified Gal-1-3-Gal disaccharide.