Multivalent fibronectin based scaffold domain proteins

Abstract

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 nucleic acid encoding 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; (iii) comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 1; and (iv) binds to a first target molecule with a K D  of less than 500 nM;   (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; (iii) comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1; and (iv) binds to a second target molecule 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, or the amino acid sequence of SEQ ID NO: 20.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the first  10 Fn3 domain binds to a first target molecule with a K D  of less than 100 nM and the second  10 Fn3 domain binds to a second target molecule with a K D  of less than 100 nM. 
     
     
         3 . The nucleic acid 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 nucleic acid 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 nucleic acid 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 nucleic acid of  claim 1 , wherein the polypeptide is selected from:
 (i) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 8-15 and 29-31; and   (ii) a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 8-15 and 29-31.   
     
     
         7 . The nucleic acid of  claim 1 , wherein the polypeptide further comprises 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 . (canceled) 
     
     
         9 . The nucleic acid of  claim 7 , wherein the PK moiety and the polypeptide are linked via at least one disulfide bond, a peptide bond, or a polypeptide. 
     
     
         10 . The nucleic acid of  claim 1 , wherein the first and second target molecules are the same. 
     
     
         11 . The nucleic acid of  claim 1 , wherein the first and second target molecules are different. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The nucleic acid of  claim 1 , wherein said polypeptide has been deimmunized to remove one or more T-cell epitopes. 
     
     
         15 . The nucleic acid of  claim 1 , wherein the first and second  10 Fn3 domains are linked via a glycine-serine based linker. 
     
     
         16 . The nucleic acid of  claim 15 , wherein the glycine-serine based linker is selected from the amino acid sequence of SEQ ID NOs: 21 and 22. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . The nucleic acid of  claim 1 , wherein the first and second  10 Fn3 domains are linked via a glycine-proline based linker. 
     
     
         20 . The nucleic acid of  claim 19 , wherein the glycine-proline based linker is selected from the amino acid sequence of SEQ ID NOs: 32, 33, and 34. 
     
     
         21 . A nucleic acid encoding 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; (iii) comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 1; and (iv) binds to a first target molecule with a K D  of less than 500 nM;   (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 (iii) comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1; and (iv) binds to a second target molecule with a K D  of less than 500 nM;   wherein the first and second  10 Fn3 domains are linked via a proline-alanine based polypeptide linker.   
     
     
         22 . The nucleic acid of  claim 20 , selected from:
 (i) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 63-70; and   (ii) a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 63-70.   
     
     
         23 . The nucleic acid of  claim 21 , wherein the first target molecule is IGF-IR and the second target molecule is VEGFR2. 
     
     
         24 . The nucleic acid of  claim 21 , wherein the first target molecule is VEGFR2 and the second target molecule is IGF-IR. 
     
     
         25 . A method of detecting insulin-like growth factor-I receptor (IGF-IR) and/or vascular endothelial growth factor receptor 2 (VEGFR2) in a sample, the method comprising contacting the sample with a polypeptide which binds to IGF-IR and VEGFR2 under conditions that allow formation of a complex between the polypeptide and IGF-IR and/or VEGFR2, and detecting the complex, wherein the polypeptide comprises:
 (A)(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 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 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; or   (B)(a) an N-terminal domain comprising a first  10 Fn3 domain, 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 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  10 Fn3 domain, 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 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; or   (C)(a) an N-terminal domain comprising a first  10 Fn3 domain, 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 IGF-IR with a K D  of less than 500 nM; and (iv) is at least 85% identical to SEQ ID NO: 35; and   (b) a C-terminal domain comprising a second  10 Fn3 domain, 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 VEGFR2 with a K D  of less than 500 nM; and (iv) is at least 85% identical to SEQ ID NO: 40;   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.