De novo binding domain containing polypeptides and uses thereof

Abstract

Provided herein are de novo binding domain containing polypeptides (DBDpp) that specifically bind a target of interest. Nucleic acids encoding the DBDpp, and vectors and host cells containing the nucleic acids are also provided. Libraries of DBDpp, methods of producing and screening such libraries and the DBDpp identified from such libraries and screens are also encompassed. Methods of making and using the DBDpp are additionally provided. Such uses include, without limitation, affinity purification, and diagnostic and therapeutic applications.

1 . A polypeptide for binding a target of interest, the polypeptide comprising;
 an amino acid sequence comprising;   
       
         
           
                 
               
                   (SEQ ID NO: 4) 
                 
                   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFE 
                 
                     
                 
                   SELQAYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN, 
                 
             
                
                
                
                
               
            
           
         
         wherein the polypeptide is derived from modifications to the amino acid sequence of SEQ ID NO:1, 
         wherein the polypeptide specifically binds a target of interest, and 
         wherein the polypeptide the specific binding the target of interest is greater than binding of a polypeptide according to SEQ ID NO:1 to the target of interest; 
         optionally wherein the polypeptide does not contain SEQ ID NO:50, and wherein the modifications to SEQ ID NO:1 comprise conservative or non-conservative substitutions and do not include a substitution with a cysteine or a proline. 
       
     
     
         2 - 7 . (canceled) 
     
     
         8 . The polypeptide of  claim 1 , wherein the target of interest specifically bound by the polypeptide is a cancer antigen, optionally wherein the cancer antigen is PD-L1, CD137, or CD123. 
     
     
         9 . The polypeptide of  claim 1 ,
 (a) wherein the polypeptide specifically binds PD-L1 and wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO: 43, and SEQ ID NO:44;   (b) wherein the polypeptide specifically binds CD137 and wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19;   (c) wherein the polypeptide specifically binds CD123 and wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS; 92-126 and SEQ ID NO:127; or   (d) wherein the polypeptide competes with the polypeptide of (c) for binding to CD123.   
     
     
         10 - 11 . (canceled) 
     
     
         12 . A method for transforming a reference polypeptide into a polypeptide having specific binding for a target of interest, the method comprising;
 modifying a plurality of amino acid residues from a reference polypeptide to generate a plurality of candidate binding polypeptides;   wherein the reference polypeptide comprises a variant of a non-naturally occurring polypeptide and comprises three anti-parallel alpha helices joined by linker peptides,   wherein the amino acid residues to be modified are solvent accessible or solvent inaccessible and,   wherein the modification comprises one or more conservative or non-conservative amino acid substitutions and does not include a substitution with a cysteine or a proline;   packaging the plurality of candidate binding polypeptides in a plurality of vectors to generate a candidate library; and   screening the candidate library for candidate binding polypeptides that exhibit specific binding to the target of interest;   optionally further comprising identifying potentially immunogenic amino acid residues in a candidate binding polypeptide and modifying at least one of the potentially immunogenic amino acid residues in the candidate polypeptide, wherein the modification comprises an amino acid substitution.   
     
     
         13 - 17 . (canceled) 
     
     
         18 . A de novo binding domain polypeptide (DBDpp), wherein the DBDpp comprises three anti-parallel alpha helices and is a variant of a synthetic polypeptide, wherein the DBDpp immunospecifically binds amino acids of SEQ ID NO: 59, or a protein that is at least 95% identical to CD137, optionally wherein the DBDpp has a dissociation constant (KD) between about 10 −4 M and about 10 −12 M,
 optionally wherein, the amino acid sequence comprises MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQA YRHN (SEQ ID NO:4), and wherein Xn is a natural or non-natural amino acid, and optionally, wherein Xn is not cysteine or proline,   optionally wherein the DBDpp comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NO:12 SEQ ID NO: 19, and   optionally wherein the DBDpp binds to a tumor.   
     
     
         19 - 22 . (canceled) 
     
     
         23 . A fusion protein comprising the de novo binding domain polypeptide (DBDpp) of  claim 18  and further comprising one or more additional DBDpp exhibiting binding specificity for a tumor target. 
     
     
         24 . A de novo binding domain polypeptide (DBDpp) of  claim 18 , wherein the DBDpp is labeled, optionally, wherein the label is a biotin moiety or is selected from the group consisting of an enzymatic label, a fluorescent label, a luminescent label, and a bioluminescent label. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . The de novo binding domain polypeptide (DBDpp) of  claim 18 , wherein the DBDpp is conjugated to a therapeutic or cytotoxic agent. 
     
     
         28 . The de novo binding domain polypeptide (DBDpp) of  claim 18 , further comprising a pharmaceutically acceptable carrier. 
     
     
         29 . A kit comprising the de novo binding domain polypeptide (DBDpp) of  claim 18 . 
     
     
         30 . An isolated nucleic acid molecule encoding the de novo binding domain polypeptide (DBDpp) of  claim 18 . 
     
     
         31 . A vector comprising the isolated nucleic acid molecule of  claim 30 , optionally further comprising a nucleotide sequence which regulates the expression of the de novo binding domain polypeptide (DBDpp) encoded by the nucleic acid molecule. 
     
     
         32 . (canceled) 
     
     
         33 . A host cell comprising the nucleic acid molecule of  claim 30 . 
     
     
         34 . A cell line engineered to express the de novo binding domain polypeptide (DBDpp) of  claim 18 . 
     
     
         35 - 85 . (canceled)