Artificial antibody polypeptides

Abstract

The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

1 .- 50 . (canceled) 
     
     
         51 . A fibronectin type Ill (Fn3) polypeptide monobody comprising a plurality of Fn3 β-strand domain sequences that are linked to a plurality of loop region sequences,
 wherein the Fn3 β-strand domain sequences of the mono body have at least 50% total amino acid sequence homology to the corresponding amino acid sequence of wild-type Fn3's β-strand domain sequences, wherein the wild-type Fn3 has the amino acid sequence of SEQ ID NO: 110, 
 wherein one or more of the monobody loop region sequences vary by deletion, insertion or replacement of at least two amino acids from the corresponding loop region sequences in said wild-type Fn3, 
 wherein the Fn3 polypeptide monobody comprises a stabilizing mutation of at least one amino acid residue as compared to said wild-type Fn3 molecule, wherein the stabilizing mutation is a substitution of at least one amino acid residue that is involved in an unfavourable electrostatic interaction, wherein the stabilizing mutation is a substitution of at least one Asp 7, Asp 23 or Glu 9 with another amino acid residue, and wherein said stabilizing mutation increases the melting point of the Fn3 polypeptide monobody by more than 0.1° C. at neutral pH as compared to a Fn3 polypeptide monobody that is identical except for the change. 
 
     
     
         52 . The monobody of  claim 51 , wherein corresponding Asp 7, Glu 9 or Asp 23 residue has been substituted with an asparagine (Asn) or lysine (Lys) residue. 
     
     
         53 . The monobody of  claim 51 , wherein Asp 7 or Asp 23, or both have been substituted with an asparagine (Asn) or lysine (Lys) residue. 
     
     
         54 . The monobody of  claim 51 , wherein Glu 9 has been substituted with Asn or Lys residue. 
     
     
         55 . The monobody of  claim 51 , wherein at least one loop region is capable of binding to a specific binding partner (SBP) to form a polypeptide:SBP complex having a dissociation constant of less than 10 −6  moles/liter. 
     
     
         56 . The monobody of  claim 51 , wherein the corresponding loop regions in wild-type Fn3 comprise amino acid residues:
 i) from 15 to 16 inclusive in an AB loop;   ii) from 22 to 30 inclusive in a BC loop;   iii) from 39 to 45 inclusive in a CD loop;   iv) from 51 to 55 inclusive in a DE loop;   v) from 60 to 66 inclusive in an EF loop; and   vi) from 76 to 87 inclusive in an FG loop   
     
     
         57 . The monobody of  claim 56 , wherein the monobody loop region sequences vary from the wild-type Fn3 loop region sequences by the deletion or replacement of at least 2 amino acids. 
     
     
         58 . The monobody of  claim 56 , wherein the monobody loop region sequences vary from the wild-type Fn3 loop region sequences by the insertion of from 3 to 25 amino acids. 
     
     
         59 . The monobody of  claim 51 , wherein Asp 7, Asp 23 and Glu 9 have been substituted with a neutral or positively charged amino acid residue. 
     
     
         60 . The monobody of  claim 51 , wherein the stabilizing mutation is a substitution of at least one of Asp 7, Asp 23 or Glu 9 with a neutral amino acid residue. 
     
     
         61 . The monobody of  claim 51 , wherein the stabilizing mutation is a substitution of at least one of Asp 7, Asp 23 or Glu 9 with a positively charged amino acid residue. 
     
     
         62 . The monobody of  claim 60 , wherein Asp 7 is substituted with a neutral amino acid. 
     
     
         63 . The monobody of  claim 61 , wherein Asp 7 is substituted with a positive amino acid. 
     
     
         64 . The monobody of  claim 60 , wherein Glu 9 is substituted with a neutral amino acid. 
     
     
         65 . The monobody of  claim 61 , wherein Glu 9 is substituted with a positive amino acid. 
     
     
         66 . The monobody of  claim 60 , wherein Asp 23 is substituted with a neutral amino acid. 
     
     
         67 . The monobody of  claim 61 , wherein Asp 23 is substituted with a positive amino acid. 
     
     
         68 . An isolated nucleic acid molecule encoding the Fn3 monobody according to  claim 51 . 
     
     
         69 . An expression vector comprising an expression cassette operably linked to the nucleic acid molecule of  claim 68 . 
     
     
         70 . A host cell transformed with the vector of  claim 69 . 
     
     
         71 . A variegated nucleic acid library encoding Fn3 polypeptide monobodies comprising a plurality of nucleic acid species each comprising a plurality of loop regions, wherein the species encode a plurality of Fn3 β-stand domain sequences that are linked to a plurality of loop region sequences,
 wherein the Fn3 β-strand domain sequences of the mono body have at least 50% total amino acid sequence homology to the corresponding amino acid sequence of wild-type Fn3's β-strand domain sequences, wherein the wild-type Fn3 has the amino acid sequence of SEQ ID NO: 110; 
 wherein one or more of the loop region sequences vary by deletion, insertion or replacement of at least two amino acids from corresponding loop region sequences in said wild-type Fn3; 
 wherein the Fn3 polypeptide monobody comprises a stabilizing mutation of at least one amino acid residue as compared to said wild-type Fn3 molecule, wherein the stabilizing mutation is a substitution of at least one amino acid residue that is involved in an unfavourable electrostatic interaction, wherein the stabilizing mutation is a substitution of at least one Asp 7, Asp 23 or Glu 9 with another amino acid residue, and wherein said stabilizing mutation increases the melting point of the Fn3 polypeptide monobody by more than 0.1° C. at neutral pH as compared to a Fn3 polypeptide monobody that is identical except for the change. 
 
     
     
         72 . The monobody of  claim 71 , wherein corresponding Asp 7, Glu 9 or Asp 23 residue has been substituted with an asparagine (Asn) or lysine (Lys) residue. 
     
     
         73 . The monobody of  claim 71 , wherein Asp 7 or Asp 23, or both have been substituted with an asparagine (Asn) or lysine (Lys) residue. 
     
     
         74 . The monobody of  claim 71 , wherein Glu 9 has been substituted with Asn or Lys residue. 
     
     
         75 . The monobody of  claim 71 , wherein at least one loop region is capable of binding to a specific binding partner (SBP) to form a polypeptide:SBP complex having a dissociation constant of less than 10 −6  moles/liter. 
     
     
         76 . The monobody of  claim 71 , wherein the corresponding loop regions in wild-type Fn3 comprise amino acid residues:
 i) from 15 to 16 inclusive in an AB loop;   ii) from 22 to 30 inclusive in a BC loop;   iii) from 39 to 45 inclusive in a CD loop;   iv) from 51 to 55 inclusive in a DE loop;   v) from 60 to 66 inclusive in an EF loop; and   vi) from 76 to 87 inclusive in an FG loop   
     
     
         77 . The monobody of  claim 76 , wherein the monobody loop region sequences vary from the wild-type Fn3 loop region sequences by the deletion or replacement of at least 2 amino acids. 
     
     
         78 . The monobody of  claim 76 , wherein the monobody loop region sequences vary from the wild-type Fn3 loop region sequences by the insertion of from 3 to 25 amino acids. 
     
     
         79 . The monobody of  claim 71 , wherein Asp 7, Asp 23 and Glu 9 have been substituted with a neutral or positively charged amino acid residue. 
     
     
         80 . The monobody of  claim 71 , wherein the stabilizing mutation is a substitution of at least one of Asp 7, Asp 23 or Glu 9 with a neutral amino acid residue. 
     
     
         81 . The monobody of  claim 71 , wherein the stabilizing mutation is a substitution of at least one of Asp 7, Asp 23 or Glu 9 with a positively charged amino acid residue. 
     
     
         82 . The monobody of  claim 80 , wherein Asp 7 is substituted with a neutral amino acid. 
     
     
         83 . The monobody of  claim 81 , wherein Asp 7 is substituted with a positive amino acid. 
     
     
         84 . The monobody of  claim 80 , wherein Glu 9 is substituted with a neutral amino acid. 
     
     
         85 . The monobody of  claim 81 , wherein Glu 9 is substituted with a positive amino acid. 
     
     
         86 . The monobody of  claim 80 , wherein Asp 23 is substituted with a neutral amino acid. 
     
     
         87 . The monobody of  claim 81 , wherein Asp 23 is substituted with a positive amino acid.