Artificial antibody polypeptides
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. A modified human fibronectin type III (Fn3 ) molecule comprising a stabilizing mutation of at least one residue involved in an unfavorable electrostatic interaction as compared to the wild-type human Fn3, wherein the stabilizing mutation is a substitution of at least one of Asp 7, Asp 23 or Glu 9 with a neutral or positively charged amino acid residue, wherein amino acid residue 6 is Arg.
2. The Fn3 of claim 1 , wherein Asp 7 or Asp 23, or both, have been substituted with an asparagine (Asn) or lysine (Lys) residue.
3. The Fn3 of claim 1 , wherein Glu 9 has been substituted with an asparagine (Asn) or lysine (Lys) residue.
4. The Fn3 of claim 1 , wherein Asp 7, Asp 23, and Glu 9 have been substituted with a neutral or positively charged amino acid residue.
5. The Fn3 of claim 1 , 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.
6. The Fn3 of claim 1 , 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.
7. A modified human tenth type III module of fibronectin (FNfn10) molecule comprising a stabilizing mutation of at least one residue involved in an unfavorable electrostatic interaction as compared to the wild-type human FNfn10molecule, wherein the stabilizing mutation is a substitution of at least one of amino acid residues 7, 9 or 23 with a neutral or positively charged amino acid residue, wherein amino acid residue 6 is Arg.
8. The modified FNfn10of claim 7 , wherein the stabilizing mutation is a substitution of at least one of amino acid residues 7, 9 or 23 with a neutral amino acid residue.
9. The modified FNfn10 of claim 7 , wherein the stabilizing mutation is a substitution of at least one of amino acid residues 7, 9 or 23 with a positively charged amino acid residue.
10. The modified FNfn10 of claim 7 , wherein amino acid residues 7 or 23, or both, have been substituted with an asparagine (Asn) or lysine (Lys) residue.
11. The modified FNfn10of claim 7 , wherein amino acid residue 9 has been substituted with an asparagine (Asn) or lysine (Lys) residue.
12. The modified FNfn10of claim 7 , wherein amino acid residues 7, 9 and 23 have been substituted with a neutral or positively charged amino acid residue.
13. The Fn3 of claim 1 , wherein amino acid residue 1 is Val.
14. The Fn3 of claim 5 , wherein Asp 7 is substituted with a neutral amino acid.
15. The Fn3 of claim 6 , wherein Asp 7 is substituted with a positive amino acid.
16. The Fn3 of claim 5 , wherein Glu 9 is substituted with a neutral amino acid.
17. The Fn3 of claim 6 , wherein Glu 9 is substituted with a positive amino acid.
18. The Fn3 of claim 5 , wherein Asp 23 is substituted with a neutral amino acid.
19. The Fn3 of claim 6 , wherein Asp 23 is substituted with a positive amino acid.
20. The Fn3 of claim 2 , wherein Asp 7 is substituted with an Asn residue.
21. The Fn3 of claim 2 , wherein Asp 7 is substituted with a Lys residue.
22. The Fn3 of claim 3 , wherein Glu 9 is substituted with an Asn residue.
23. The Fn3 of claim 3 , wherein Glu 9 is substituted with a Lys residue.
24. The Fn3 of claim 2 , wherein Asp 23 is substituted with an Asn residue.
25. The Fn3 of claim 2 , wherein Asp 23 is substituted with a Lys residue.
26. The Fn3 of claim 7 , wherein amino acid residue 1 is Val.
27. A modified human fibronectin type III (Fn3 ) molecule comprising a stabilizing mutation of at least one residue involved in an unfavorable electrostatic interaction as compared to the wild-type human Fn3 , wherein the stabilizing mutation is a substitution of Asp 7 or Asp 23 with a positively charged amino acid residue.
28. The Fn3 of claim 27 , wherein the stabilizing mutation is a substitution of Asp 7, with a positively charged amino acid residue.
29. The Fn3 of claim 27 , wherein the stabilizing mutation is a substitution of Asp 23, with a positively charged amino acid residue.