Targeted therapeutics based on engineered proteins for tyrosine kinases receptors, including igf-ir

Abstract

The present invention provides innovative proteins that bind to insulin-like growth factor-I receptor (IGF-IR), as well as other important proteins. The invention also provides innovative proteins in pharmaceutical preparations and derivatives of such proteins and the uses of same in diagnostic, research and therapeutic applications. The invention further provides cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and vectors comprising the polynucleotides encoding the innovative proteins.

We claim: 
     
         1 . A polypeptide comprising an altered tenth fibronectin type III ( 10 Fn3) domain, wherein the altered  10 Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein the amino acid sequences of the BC, DE, and FG loops of the  10 Fn3 domain are at least 80% identical to the amino acid sequences of the respective BC, DE, and FG loops of an  10 Fn3 domain with an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2-125, 184-203, or 226, and (ii) binds human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μM or less. 
     
     
         2 . The polypeptide of  claim 1 , wherein the amino acid sequences of the BC, DE, and FG loops of the  10 Fn3 domain are identical to the amino acid sequences of the respective BC, DE and FG loops of an  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-125, 184-203, or 226. 
     
     
         3 . A polypeptide comprising an altered tenth fibronectin type III ( 10 Fn3) domain, wherein the altered  10 Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein at least one loop of the BC, DE, and FG loops of the  10 Fn3 domain has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of an  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-125, 184-203, and 226, and (ii) wherein the  10 Fn3 domain binds to human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μM or less. 
     
     
         4 . The polypeptide of  claim 1 , wherein the altered  10 Fn3 domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 2-125, 184-203, or 226. 
     
     
         5 . The polypeptide of  claim 1 , wherein the altered  10 Fn3 domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 2-125, 184-203, or 226. 
     
     
         6 . The polypeptide of  claim 1 , wherein the altered  10 Fn3 domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-125, 184-203, or 226. 
     
     
         7 . The polypeptide of  claim 1  or  3 , wherein the altered  10 Fn3 domain binds human IGF-IR with a disassociation constant of about 10 nM or less. 
     
     
         8 . The polypeptide of  claim 1  or  3 , further comprising one or more pharmacokinetic (PK) moieties selected from: a polyoxyalkylene moiety, a human serum albumin binding protein, sialic acid, human serum albumin, transferrin, and an Fc fragment. 
     
     
         9 . The polypeptide of  claim 8 , wherein the PK moiety is the polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol. 
     
     
         10 . The polypeptide of  claim 1 , wherein said polypeptide inhibits the binding of insulin-like growth factor-I (IGF-I) or insulin-like growth factor-II (IGF-II) to IGF-IR and does not activate human IGF-IR at sub IC50 concentrations in a cell-based assay. 
     
     
         11 . A pharmaceutically acceptable composition comprising the polypeptide of  claim 1  or  3 , wherein the composition is essentially endotoxin free. 
     
     
         12 . The polypeptide of  claim 1 , further comprising a second altered  10 Fn3 domain. 
     
     
         13 . The polypeptide of  claim 12 , wherein the second altered  10 Fn3 domain binds to a target selected from the group consisting of EGFR, c-Met, c-kit, Her2, FGFR1, VEGFR2, VEGF-A, VEGF-B, VEGF-C, VEGF-D and folate receptor. 
     
     
         14 . The polypeptide of  claim 13 , wherein the second altered  10 Fn3 domain binds to the target with a disassociation constant of about 1 μM or less. 
     
     
         15 . The polypeptide of  claim 14 , wherein the second altered  10 Fn3 domain binds to the target with a disassociation constant of about 10 nM or less. 
     
     
         16 . A nucleic acid encoding the polypeptide of  claim 1  or  3 . 
     
     
         17 . A vector comprising the nucleic acid of  claim 16 . 
     
     
         18 . A method of treating cancer associated with increased insulin-like growth factor (IGF) activity comprising administering to a subject in need thereof an effective amount of a polypeptide comprising an altered tenth fibronectin type III ( 10 Fn3) domain, wherein the altered  10 Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein at least one loop of the BC, DE, and FG loops of the  10 Fn3 domain has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of an  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-125, 184-203, and 226, and (ii) wherein the  10 Fn3 domain binds to human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μM or less. 
     
     
         19 . The method of  claim 18 , wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, synovial sarcoma, osteosarcoma, cervical cancer, prostate cancer, and pancreatic cancer. 
     
     
         20 . A method of inhibiting insulin-like growth factor (IGF)-mediated proliferation of tumor cells comprising contacting the cell with an effective amount of a polypeptide comprising an altered tenth fibronectin type III ( 10 Fn3) domain, wherein the altered  10 Fn3 domain (i) comprises an AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop, wherein at least one loop of the BC, DE, and FG loops of the  10 Fn3 domain has 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of an  10 Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-125, 184-203, and 226, and (ii) wherein the  10 Fn3 domain binds to human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μM or less.