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 ALGGSEAELAAFEKEIAAFESELQA 
                 
                     
                 
                   YKGKGNPEVEX 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 LQ AYRHN (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 .- 37 . (canceled) 
     
     
         38 . A method for purifying a target of interest comprising, contacting a sample comprising a target of interest with a composition comprising:
 a polypeptide agent attached to a solid support, wherein the polypeptide agent has an amino acid sequence comprising: MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAE LAAFEKFIA FESELQAYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:4),   wherein the polypeptide has an amino acid sequence that differs from SEQ ID NO: 1,   wherein the polypeptide specifically binds the target of interest,   wherein the polypeptide's specific binding to 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 agent is coupled to the solid support through non-covalent association or a covalent bond and the solid support comprises a bead, a glass slide, a chip, a gelatin, or an agarose;   the contacting is performed under conditions that permit binding of the composition to the target of interest; and   removing a portion of the sample that is not bound to the composition; optionally further comprising dissociating the composition from the target of interest and recovering the target of interest.   
     
     
         39 .- 44 . (canceled) 
     
     
         45 . The method of  claim 38 , wherein the solid support comprises a bead, and the composition is suitable for use in affinity chromatography to purify the target of interest. 
     
     
         46 . A method of  claim 38 , wherein the target of interest can be eluted from the composition. 
     
     
         47 . The method of  claim 38 , wherein a nucleic acid molecule encoding the polypeptide is packaged in an expression vector, that is used to transduce a cell line to cause the cell line to express the polypeptide. 
     
     
         48 . A method for purifying a target of interest comprising,
 contacting a sample comprising a target of interest with a composition comprising:   a virus-like particle coupled to a solid support, wherein the virus-like particle expresses a polypeptide as a membrane protein, the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,   wherein no cysteine or proline residues are substituted into any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,   wherein the polypeptide has an amino acid sequence that differs from SEQ ID NO: 1,   wherein the polypeptide specifically binds the target of interest,   wherein the polypeptide's specific binding to the target of interest is greater than binding of a polypeptide according to SEQ ID NO: 1 to the target of interest; and   the contacting is performed under conditions that permit binding of the composition to the target of interest, optionally wherein the solid support comprises a bead, a glass slide, a chip, a gelatin, or an agarose; and   removing a portion of the sample that is not bound to the composition, and optionally further comprising contacting a portion of the sample that is not bound to the composition with an antibody directed against the polypeptide of the composition, the antibody being generated from membrane bound virus-like particles (VLP) expressing the polypeptide released from a mammalian cell is engineered to express a fusion protein comprising the polypeptide fused to a chimeric antigen receptor (CAR), the fusion protein being expressed on the generated VLPs, wherein the antibodies are suitable for use in an assay to detect residual polypeptides detached from the solid support.   
     
     
         49 . (canceled) 
     
     
         50 . The method of  claim 48 , wherein the polypeptide of the composition further comprises a peptide tag, wherein the peptide tag comprises a hexahistidine moiety or a FLAG tag. 
     
     
         51 . The method of  claim 48 , wherein the portion of the sample that is not bound to the composition is discarded. 
     
     
         52 . (canceled) 
     
     
         53 . A method for removing one or more contaminants from a sample comprising a target of interest comprising,
 contacting a sample comprising a target of interest with a composition comprising:   a virus-like particle coupled to a solid support, wherein the virus-like particle expresses a polypeptide as a membrane protein, the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,   wherein no cysteine or proline residues are substituted into any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,   wherein the polypeptide has an amino acid sequence that differs from SEQ ID NO: 1,   wherein the polypeptide specifically binds one or more contaminants to be removed from a sample comprising the target of interest,   wherein the polypeptide's specific binding to one or more contaminants is greater than binding of a polypeptide according to SEQ ID NO: 1 to the one or more contaminants; and   the contacting is performed under conditions that permit binding of the composition to the one or more contaminants, optionally, wherein the solid support comprises a bead, a glass slide, a chip, a gelatin, or an agarose and the virus-like particles is coupled to the solid support through non-covalent association; and   collecting a portion of the sample that is not bound to the composition.   
     
     
         54 . The method of  claim 53 , wherein the polypeptide of the composition further comprises a peptide tag, wherein the peptide tag comprises a hexahistidine moiety or a FLAG tag. 
     
     
         55 .- 56 . (canceled) 
     
     
         57 . An affinity resin comprising a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of:
 MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 LQX 44 YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);   MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 A YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:4);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:5);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 L QX 44 YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 LQ X 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);   MGSWAEFKQRLAAIKTRLEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPE V EALX 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:8);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAAFEKFIAAFESELQAY Z 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:9);   MG SWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:10); and   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 LQX 42 YZ 2  X 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:11); and combinations thereof; wherein Xn is a natural or non-natural amino acid; wherein each Xn is the same or different natural or non-natural amino acid; and/or   wherein Z 1  and/or Z 2  is 2 to 30 natural or non-natural amino acids; and wherein the amino acid sequence is not SEQ ID NO:1.   
     
     
         58 . The affinity resin of  claim 57 ,
 wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO:1 by an amino acid substitution at one or more residues;   wherein the amino acid substitution at one or more residues comprises a conservative substitution;   wherein the amino acid substitution at one or more residues comprises a non-conservative substitution;   wherein the amino acid substitution at one or more residues comprises a substitution at a solvent accessible residue;   wherein the amino acid substitution at one or more residues comprises a substitution at a solvent inaccessible residue; or   wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO:1 by an amino acid deletion at one or more residues.   
     
     
         59 .- 63 . (canceled) 
     
     
         64 . A method of making an affinity resin comprising a step of:
 attaching to a solid support a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of:   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 LQX 44 YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);   MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 A YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:4);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:5);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 L QX 44 YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 LQ X 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);   MGSWAEFKQRLAAIKTRLEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPE V EALX 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:8);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAAFEKEIAAFESELQAY Z 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:9);   MG SWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:10); and   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 L QX 42 YZ 2 X 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:11); and combinations thereof;   wherein Xn is a natural or non-natural amino acid; wherein each Xn is the same or different natural or non-natural amino acid; and/or   wherein Z 1  and/or Z 2  is 2 to 30 natural or non-natural amino acids; and wherein the amino acid sequence is not SEQ ID NO: 1, optionally wherein the polypeptide agent is attached to the solid support by covalent bonding or non-covalent association and wherein the solid support comprises a bead, glass slide, chip, gelatin, or agarose.   
     
     
         65 .- 67 . (canceled) 
     
     
         68 . A composition comprising a solid support coupled to a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of:
 MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 LQX44YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);   MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 AYKG KGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:4);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 EL X 43 AYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:5);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 L QX 44 YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 LQ X 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);   MGSWAEFKQRLAAIKTRLEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPE VEALX 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:8);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAAFEKEIAAFESELQAY Z 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:9);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:10); and   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 L QX 42 YZ 2 X 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:11); and combinations thereof; wherein X n  is a natural or non-natural amino acid; wherein each X n  is the same or different natural or non-natural amino acid; and/or   wherein Z 1  and/or Z 2  is 2 to 30 natural or non-natural amino acids; and   wherein the amino acid sequence is not SEQ ID NO:1, optionally wherein the solid support comprises a bead, glass slide, chip, gelatin, or agarose.   
     
     
         69 . The composition of  claim 68 ,
 wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO:1 by an amino acid substitution at one or more residues;   wherein the amino acid substitution at one or more residues comprises a conservative substitution;   wherein the amino acid substitution at one or more residues comprises a non-conservative substitution;   wherein the amino acid substitution at one or more residues comprises a substitution at a solvent accessible residue;   wherein the amino acid substitution at one or more residues comprises a substitution at a solvent inaccessible residue; or   wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO:1 by an amino acid deletion at one or more residues.   
     
     
         70 .- 75 . (canceled) 
     
     
         76 . A composition comprising a polypeptide agent conjugated to a detectable agent and/or tag,
 wherein the polypeptide agent has an amino acid sequence comprising a sequence selected from the group consisting of:   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 LQX 44 YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);   MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 A YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYKGK GNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:4);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYRHN (SEQ ID NO:5);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAX 30 FEX 33 X 34 IAX 37 FEX 40 X 41 L QX 44 YKGKGNPEVEALX 57 X 58 EAX 61 AIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 LQ X 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);   MGSWAEFKQRLAAIKTRLEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPE V EALX 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:8);   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAAFEKEIAAFESELQAY Z 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:9);   MGSWX 5 X 6 FKX 9 X 10 LAX 13 IKX 16 X 17 LEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO:10); and   MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAX 28 FEX 31 X 32 IAX 35 FEX 38 X 39 L QX 42 YZ 2  X 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO:11); and combinations thereof; wherein X n  is a natural or non-natural amino acid; wherein each X n  is the same or different natural or non-natural amino acid; and/or   wherein Z 1  and/or Z 2  is 2 to 30 natural or non-natural amino acids; and wherein the amino acid sequence is not SEQ ID NO: 1; and optionally wherein the detectable agent is quantifiable.   
     
     
         77 . The composition of  claim 76 , wherein the detectable agent comprises a chromagen, a fluorescent dye, or radionuclide or wherein the tag comprises a polyhistidyl tag, a myc tag, or a FLAG tag. 
     
     
         78 .- 80 . (canceled) 
     
     
         81 . The composition of  claim 76 , wherein the polypeptide agent is conjugated to a chromatography bead, resin, glass slide, chip, gelatin, or agarose. 
     
     
         82 .- 84 . (canceled) 
     
     
         85 . A composition of  claim 76 , wherein the polypeptide agent is a protein or is multimeric.