Multivalent fibronectin based scaffold domain proteins
View Patent ↗The present invention relates to multivalent polypeptides comprising at least two fibronectin scaffold domains connected via a polypeptide linker. The invention also relates to multivalent polypeptides for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative proteins.
1. A polypeptide comprising:
(a) an N-terminal domain comprising a first fibronectin type III tenth domain ( 10 Fn3), wherein the 10 Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loop FG; (ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human 10 Fn3 domain (SEQ ID NO: 1), and (iii) binds to insulin-like growth factor-I receptor (IGF-IR) with a K D of less than 500 nM; and
(b) a C-terminal domain comprising a second fibronectin type III tenth domain ( 10 Fn3), wherein the 10 Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loop FG; (ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human 10 Fn3 domain (SEQ ID NO: 1), and (iii) binds to vascular endothelial growth factor receptor 2 (VEGFR2) with a K D of less than 500 nM;
wherein the first and second 10 Fn3 domains are linked via a polypeptide selected from a glycine-serine based linker, a glycine-proline based linker, a proline-alanine based linker or the amino acid sequence of SEQ ID NO: 20, wherein the polypeptide comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 8-11, 29-31, and 63-70.
2. The polypeptide of claim 1 , wherein the first 10 Fn3 domain binds to IGF-IR with a K D of less than 100 nM and the second 10 Fn3 domain binds to VEGFR2 with a K D of less than 100 nM.
3. The polypeptide of claim 1 , wherein the loop BC and loop FG of the first and second 10 Fn3 domains have an altered amino acid sequence relative to the sequence of the corresponding loops of the human 10 Fn3 domain.
4. The polypeptide of claim 1 , wherein the first 10 Fn3 domain is linked at its C-terminus to the amino acid sequence of SEQ ID NO: 19.
5. The polypeptide of claim 1 , wherein the second 10 Fn3 domain is linked at its C-terminus to the amino acid sequence of SEQ ID NO: 17 or 18.
6. The polypeptide of claim 1 , selected from a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 8-11, 29-31, and 63-70.
7. The polypeptide of claim 1 , further comprising one or more pharmacokinetic (PK) moieties selected from: a polyoxyalkylene moiety, a human serum albumin binding protein, sialic acid, human serum albumin, IgG, an IgG binding protein, transferrin, and an Fc fragment.
8. The polypeptide of claim 7 , wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.
9. The polypeptide of claim 7 , wherein the PK moiety and the polypeptide are linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety.
10. The polypeptide of claim 1 , wherein said polypeptide has been deimmunized to remove one or more T-cell epitopes.
11. The polypeptide of claim 1 , wherein the first and second 10 Fn3 domains are linked via a glycine-serine based linker.
12. The polypeptide of claim 11 , wherein the glycine-serine based linker is selected from the amino acid sequence of SEQ ID NOs: 21 and 22.
13. A pharmaceutically acceptable composition comprising the polypeptide claim 1 , wherein the composition is essentially endotoxin free.
14. The polypeptide of claim 1 , wherein the first and second 10 Fn3 domains are linked via a glycine-proline based linker.
15. The polypeptide of claim 14 , wherein the glycine-proline based linker is selected from the amino acid sequence of SEQ ID NOs: 32, 33, and 34.
16. The polypeptide of claim 1 , wherein the BC, DE and FG loops of the first 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively.
17. The polypeptide of claim 1 , wherein the BC, DE and FG loops of the second 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 5, 6 and 7, respectively.
18. A polypeptide comprising:
(a) an N-terminal domain comprising a first fibronectin type III tenth domain ( 10 Fn3), wherein the 10 Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loop FG; (ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human 10 Fn3 domain (SEQ ID NO: 1), (iii) binds to vascular endothelial growth factor receptor 2 (VEGFR2) with a K D of less than 500 nM, and (iv) is at least 85% identical to SEQ ID NO: 40; and
(b) a C-terminal domain comprising a second fibronectin type III tenth domain ( 10 Fn3), wherein the 10 Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loop FG; (ii) has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human 10 Fn3 domain (SEQ ID NO: 1), (iii) binds to insulin-like growth factor-I receptor (IGF-IR) with a K D of less than 500 nM, and (iv) is at least 85% identical to SEQ ID NO: 35;
wherein the first and second 10 Fn3 domains are linked via a polypeptide selected from a glycine-serine based linker, a glycine-proline based linker, a proline-alanine based linker or the amino acid sequence of SEQ ID NO: 20.
19. The polypeptide of claim 18 , selected from a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 12-15.
20. The polypeptide of claim 19 , selected from a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 12-15.
21. The polypeptide of claim 18 , wherein the first 10 Fn3 domain binds to VEGFR2 with a K D of less than 100 nM and the second 10 Fn3 domain binds to IGFR-IR with a K D of less than 100 nM.
22. The polypeptide of claim 18 , wherein the loop BC and loop FG of the first and second 10 Fn3 domains have an altered amino acid sequence relative to the sequence of the corresponding loops of the human 10 Fn3 domain.
23. The polypeptide of claim 18 , further comprising one or more pharmacokinetic (PK) moieties selected from: a polyoxyalkylene moiety, a human serum albumin binding protein, sialic acid, human serum albumin, IgG, an IgG binding protein, transferrin, and an Fc fragment.
24. The polypeptide of claim 23 , wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.
25. The polypeptide of claim 23 , wherein the PK moiety and the polypeptide are linked via at least one disulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety.
26. The polypeptide of claim 18 , wherein said polypeptide has been deimmunized to remove one or more T-cell epitopes.
27. A pharmaceutically acceptable composition comprising the polypeptide claim 18 , wherein the composition is essentially endotoxin free.
28. The polypeptide of claim 18 , wherein the BC, DE and FG loops of the first 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 5, 6 and 7, respectively.
29. The polypeptide of claim 18 , wherein the BC, DE and FG loops of the second 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively.