Cystine knot scaffold platform

Abstract

Provided are non-naturally occurring cystine knot peptides (CKPs) that bind to VEGF-A. Additionally, provided are methods of using non-naturally occurring CKPs that bind to VEGF-A, Including diagnostic and therapeutic compositions and methods. Non-naturally CKPs that bind low density lipoprotein receptor-related protein 6 (LRP6) are also provided.

1 . A non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A), wherein the CKP comprises the cystine scaffold structure:
   Z 1 C 1 L1C 2 L2C 3 L3C 4 L4C 5 L5C 6 Z 2      
       wherein:
 Z 1  and Z 2  are any amino acid or an unnatural amino acid; 
 L1 is Loop 1 and has a structure selected from the group consisting of: X 1 X 2 X 3 X 4 X 5 X 6  (SEQ ID NO: 2), X 1 X 2 X 3 X 4 X 5 X 6 X 7  (SEQ ID NO: 3), X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8  (SEQ ID NO: 4), X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9  (SEQ ID NO: 5), and X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10  (SEQ ID NO: 6), wherein each of X 1 -X 10  is any amino acid or an unnatural amino acid; 
 L2 is Loop 2 and has the structure: X 1 X 2 X 3 X 4 X 5  (SEQ ID NO: 7), wherein each of X 1 -X 5  is any amino acid or an unnatural amino acid; 
 L3 is Loop 3 and has the structure: X 1 X 2 X 3  wherein each of X 1 -X 3  is any amino acid or an unnatural amino acid; 
 L4 is Loop 4 and has the structure: X 1 , wherein X 1  is any amino acid or an unnatural amino acid; 
 L5 is Loop 5 and has the structure: X 1 X 2 X 3 X 4 X 5  (SEQ ID NO: 7), wherein each of X 1 -X 5  is any amino acid or an unnatural amino acid; 
 wherein the unnatural amino acid is selected from the group consisting of L-propargylglycine-PEG 6 -, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NH meta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethyl indole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4′-biphenylalanine, 3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine (also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine (also known as L-tert-Leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine, L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester of glycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, and glycine morpholine amide; 
 wherein the CKP binds to VEGF-A with an affinity of 500 pM or better. 
 
     
     
         2 . The CKP of  claim 1 , wherein the CKP has an altered disulfide bond connectivity—with reference to a wild-type  Ecballium elaterium  trypsin inhibitor EETI-II protein having the amino acid sequence set forth in SEQ ID NO: 1; wherein the altered disulfide bond connectivity is C1-C4, C2-C3 and C5-C6. 
     
     
         3 . The CKP of  claim 1  or  claim 2 , wherein Z 1  and/or Z 2  is G. 
     
     
         4 . A non-naturally occurring cystine knot peptide (CKP) comprising an amino acid selected from the group consisting of: GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113), GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114), GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139), GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140), GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142), GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine (Nle), GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X is norleucine (Nle), GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X is norleucine (Nle), and GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein X is norleucine. 
     
     
         5 . The CKP of  claim 4 , wherein the CKP binds VEGF-A. 
     
     
         6 . A non-naturally occurring cystine knot peptide (CKP) comprising an amino acid selected from the group consisting of: GCDVX 1 QPYWGCGPDI-D/E-CLS-N/K/X 2 -CICHWYNSCG (SEQ ID NO: 534), GCDVX 1 QPYWGCGPDI-N/K/X 2 -CLS-D/E-CICHWYNSCG (SEQ ID NO: 535), GCNIX 1 LPYWGCGRDF-D/E-CME-N/K/X 2 -CICQYYQSCG (SEQ ID NO: 538), GCNIX 1 LPYWGCGRDF-N/K/X 2 -CME-D/E_CICQYYQSCG (SEQ ID NO: 539), GCNIX 1 LPFWGCGRDF-D/E-CVS-N/K/X 2 -CICQYYQSCG (SEQ ID NO: 540), and GCNIX 1 LPFWGCGRDF-N/K/X 2 -CVS-D/E-CICQYYQSCG (SEQ ID NO: 541), wherein X 1  is norleucine and X 2  is ornithine. 
     
     
         7 . The CKP of  claim 6 , wherein the CKP binds VEGF-A. 
     
     
         8 . A non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A, wherein the CKP comprises the cystine scaffold structure:
   Z 1 C 1 L1C 2 L2C 3 L3C 4 L4C 5 L5C 6 Z 2      
       wherein:
 Z 1  and Z 2  are any amino acid, more than one amino acid, or an unnatural amino acid; 
 L1 is Loop 1 and has a structure selected from the group consisting of: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 , X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , and X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein each of X 1 -X 10  is any amino acid or an unnatural amino acid; 
 L2 is Loop 2 and has the structure: X 1 X 2 X 3 X 4 X 5 , wherein each of X 1 -X 5  is any amino acid or an unnatural amino acid; 
 L3 is Loop 3 and has the structure: X 1 X 2 X 3  wherein each of X 1 -X 3  is any amino acid or an unnatural amino acid; 
 L4 is Loop 4 and has the structure: X 1 , wherein X 1  is any amino acid or an unnatural amino acid; L5 is Loop 5 and has the structure: X 1 X 2 X 3 X 4 X 5 , wherein each of X 1 -X 5  is any amino acid or an unnatural amino acid; 
 wherein the unnatural amino acid selected from the group consisting of L-propargylglycine-PEG 6 -, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NH meta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethyl indole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4′-biphenylalanine, 3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine (also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine (also known as L-tert-Leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine, L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester of glycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, and glycine morpholine amide, and 
 wherein the CKP has an altered disulfide bond connectivity with reference to a wild-type  Ecballium elaterium  trypsin inhibitor EETI-II protein having the amino acid sequence set forth in SEQ ID NO: 1; wherein the altered disulfide bond connectivity is C1-C4, C2-C3 and C5-C6; and wherein the CKP has a percent alpha helix content of at least 20%. 
 
     
     
         9 . The CKP of any one of  claims 1 - 8 , wherein:
 (a) the C-terminal carboxyl group of the peptide is capped;   (b) the N-terminal amine of the peptide (CKP) is capped; or   (c) the C-terminal carboxyl group and the N-terminal amine of the peptide (CKP) is capped.   
     
     
         10 . A non-naturally occurring CKP that binds to an epitope on VEGF-A comprising at least one of the amino acid residues selected from the group consisting of: V14, V15, F17, D19, Y21, Q22, Y25, I46, K48, N62, D63, L66, M81, I83, K84, P85, H86, G88, Q89, I91, C104, R105, and P106. 
     
     
         11 . The CKP of any one of  claims 1 - 10  conjugated to a therapeutic agent. 
     
     
         12 . The CKP of any one of  claims 1 - 10  conjugated to a label. 
     
     
         13 . An isolated nucleic acid encoding the CKP of any one of  claims 1 - 10 . 
     
     
         14 . An expression vector encoding the nucleic acid molecule of  claim 13 . 
     
     
         15 . A cell comprising the expression vector of  claim 14 . 
     
     
         16 . A method of producing the CKP of any one of  claims 1 - 10  comprising culturing the cell of  claim 15  and recovering the CKP from the cell culture. 
     
     
         17 . A method of producing the CKP of any one of  claims 1 - 10 , comprising chemically synthesizing the CKP. 
     
     
         18 . A composition comprising the CKP of any one of  claims 1 - 11  and a pharmaceutically acceptable carrier. 
     
     
         19 . A method of treating an ocular disease characterized by angiogenesis and/or vascular permeability or leakage in a subject, comprising administering an effective amount of the CKP of any one of  claims 1 - 11  or the composition of  claim 18  to the subject. 
     
     
         20 . The method of  claim 19 , wherein the ocular disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathies, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, retinal vein occlusion (RVO), including Central Retinal Vein Occlusion (CRVO) and branched retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP). 
     
     
         21 . The method of any one of  claims 19  or  20 , wherein the CKP or the composition is administered to the subject via an implantable device. 
     
     
         22 . The CKP of any one of  claims 1 - 11 , wherein the CKP is formulated for long acting delivery. 
     
     
         23 . A formulation comprising the CKP of any of  claims 1 - 11  and PLGA. 
     
     
         24 . A non-naturally occurring cystine knot peptide (CKP) that binds to human low density lipoprotein receptor-related protein 6 (LRP6), wherein the CKP comprises the cystine scaffold structure:
   Z 1 C 1 L1C 2 L2C 3 L3C 4 L4C 5 L5C 6 Z 2 ;   
       wherein:
 Z 1  and Z 2  are any amino acid; 
 L 1  is Loop 1 and has a structure selected from the group consisting of: X 1 X 2 X 3 X 4 X 5 X 6 , X 1 X 2 X 3 X 4 X 5 X 6 X 7 , X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 , X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , and X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein each of X 1 -X 10  is any amino acid; 
 L2 is Loop 2 and has the structure: X 1 X 2 X 3 X 4 X 5 , wherein each of X 1 -X 5  is any amino acid; 
 L3 is Loop 3 and has the structure: X 1 X 2 X 3  wherein each of X 1 -X 3  is any amino acid; 
 L4 is Loop 4 and has the structure: X 1 , wherein X 1  is any amino acid; and 
 L5 is Loop 5 and has the structure: X 1 X 2 X 3 X 4 X 5 , wherein each of X 1 -X 5  is any amino acid.