Single chain analogs of the TGF-beta superfamily (morphons)

Abstract

Disclosed are a family of single-chain polypeptide constructs designed to agonize or mimic members of the TGF-β superfamily by binding to a cell surface receptor complementary to the superfamily member. The single-chain constructs of the invention called “morphons” contain in a single biologically active subunit interacting finger and heel regions which together define a tertiary protein structure complimentary to the ligand binding surface of a receptor that binds a TGF-β superfamily member. Also disclosed are truncated versions of the morphon constructs. Methods are disclosed for making and using single-chain morphons that have binding affinity for predetermined receptors of the TGF-β superfamily.

What is claimed is:  
     
         1 . A single-chain synthetic amino acid biopolymer construct comprising an amino acid sequence 
 defining finger 1, finger 2, and heel regions together complementary to the ligand binding interactive surface of a TGF-β superfamily member receptor structure and    having an amino acid sequence which mimics the structure of a member of the TGF-β super-family such that said-construct preferentially binds said receptor.    
     
     
         2 . The construct of  claim 1  wherein, upon binding with said receptor structure, said construct mimics the bioactivity of a TGF-β superfamily member.  
     
     
         3 . The construct of  claim 2  wherein, upon binding with said receptor structure, said construct initiates a morphogenic cascade.  
     
     
         4 . The construct of  claim 1  wherein: 
 the amino acid sequence of the finger 1 region is folded to define a secondary structure comprising, in order from the N to C direction, a first beta sheet, a helical turn, a loop, and a second beta sheet disposed substantially anti-parallel to said first beta sheet;  
 the amino acid sequence of the heel region is folded to define a secondary structure comprising an alpha helical coil; and  
 the amino acid sequence of the finger 2 region is folded to define a secondary structure comprising, in order from the N to C direction, a beta sheet, a loop, and a second beta sheet disposed substantially anti-parallel to said first beta sheet.  
 
     
     
         5 . The construct of  claim 1  or  4 , wherein said finger 1 and finger 2 regions comprise anti-parallel β pleated sheets which extend along adjacent, substantially parallel axes, and said heel region comprises a helical coil disposed about an axes transverse to said parallel axes.  
     
     
         6 . The construct of  claim 4 , wherein the finger 1 sequence is selected from the group of finger 1 sequences consisting of 
 TGF-β1 SEQ. ID. No.1, residues 2 through 29,    TGF-β2 SEQ. ID. No.2, residues 2 through 29,    TGF-β3 SEQ. ID. No.3, residues 2 through 29,    TGF-β4 SEQ. ID. No.4, residues 2 through 29,    TGF-β5 SEQ. ID. No.5, residues 2 through 29,    dpp SEQ. ID. No.6, residues 2 through 29,    Vg-1 SEQ. ID. No.7, residues 2 through 29,    Vgr-1 SEQ. ID. No.8, residues 2 through 29,    60A SEQ. ID. No.9, residues 2 through 29,    BMP-2A SEQ. ID.No.10, residues 2 through 29,    BMP3 SEQ. ID. No.11, residues 2 through 29,    BMP-4 SEQ. ID. No.12, residues 2 through 29,    BMP-5 SEQ. ID. No.13, residues 2 through 29,    BMP-6 SEQ. ID. No.14, residues 2 through 29,    Dorsalin SEQ. ID. No.15, residues 2 through 29,    OP-1 SEQ. ID. No.16, residues 2 through 29,    OP-2 SEQ. ID. No.17, residues 2 through 29,    OP-3 SEQ. ID. No.18, residues 2 through 29,    GDF-1 SEQ. ID. No.19, residues 2 through 29,    GDF-3 SEQ. ID. No.20, residues 2 through 29,    GDF-9 SEQ. ID. No.21, residues 2 through 29,    Inhibin α SEQ. ID. No.22, residues 2 through 29,    Inhibin βA SEQ. ID. No.23, residues 2 through 29, and    Inhibin βB SEQ. ID. No.24, residues 2 through 29.    
     
     
         7 . The construct of  claim 4  or  6 , wherein the heel sequence is selected from the group of heel sequences consisting of 
 TGF-β1 SEQ. ID. No.1, residues 35 through 62,  
 TGF-β2 SEQ. ID. No.2, residues 35 through 62,  
 TGF-β3 SEQ. ID. No.3, residues 35 through 62,  
 TGF-β4 SEQ. ID. No.4, residues 35 through 62,  
 TGF-β5 SEQ. ID. No.5, residues 35 through 62,  
 dpp SEQ. ID. No.6, residues 35 through 65,  
 Vg-1 SEQ. ID. No.7, residues 35 through 65,  
 Vgr-1 SEQ. ID. No.8, residues 35 through 65,  
 60A SEQ. ID. No.9, residues 35 through 65,  
 BMP-2A SEQ. ID. No.10, residues 35 through 64,  
 BMP3 SEQ. ID. No.11, residues 35 through 66,  
 BMP-4 SEQ. ID. No.12, residues 35 through 64,  
 BMP-5 SEQ. ID. No.13, residues 35 through 65,  
 BMP-6 SEQ. ID. No.14, residues 35 through 65,  
 Dorsalin SEQ. ID. No.15, residues 35 through 65,  
 OP-1 SEQ. ID. No.16, residues 35 through 65,  
 OP-2 SEQ. ID. No.17, residues 35 through 65,  
 OP-3 SEQ. ID. No.18, residues 35 through 65,  
 GDF-1 SEQ. ID. No.19, residues 35 through 70,  
 GDF-3 SEQ. ID. No.20, residues 35 through 64,  
 GDF-9 SEQ. ID. No.21, residues 35 through 65,  
 Inhibin α SEQ. ID. No.22, residues 35 through 65,  
 Inhibin βA SEQ. ID. No.23, residues 35 through 69, and  
 Inhibin βB SEQ. ID. No.24, residues 35 through 68.  
 
     
     
         8 . The construct of  claim 4  or  6 , wherein the finger 2 sequence is selected from the group of finger 2 sequences consisting of 
 TGF-β1 SEQ. ID. No.1, residues 65 through 94,  
 TGF-β2 SEQ. ID. No.2, residues 65 through 94,  
 TGF-β3 SEQ. ID. No.3, residues 65 through 94,  
 TGF-β4 SEQ. ID. No.4, residues 65 through 94,  
 TGF-β5 SEQ. ID. No.5, residues 65 through 94,  
 dpp SEQ. ID. No.6, residues 68 through 98,  
 Vg-1 SEQ. ID. No.7, residues 68 through 98,  
 Vgr-1 SEQ. ID. No.8, residues 68 through 98,  
 60A SEQ. ID. No.9, residues 68 through 98,  
 BMP-2A SEQ. ID. No.10, residues 67 through 97,  
 BMP3 SEQ. ID. No.11, residues 69 through 99,  
 BMP-4 SEQ. ID. No.12, residues 67 through 97,  
 BMP-5 SEQ. ID. No.13, residues 68 through 98,  
 BMP-6 SEQ. ID. No.14, residues 68 through 98,  
 Dorsalin SEQ. ID. No.15, residues 68 through 99,  
 OP-1 SEQ. ID. No.16, residues 68 through 98,  
 OP-2 SEQ. ID. No.17, residues 68 through 98,  
 OP-3 SEQ. ID. No.18, residues 68 through 98,  
 GDF-1 SEQ. ID. No.19, residues 73 through 103,  
 GDF-3 SEQ. ID. No.20, residues 67 through 97,  
 GDF-9 SEQ. ID. No.21, residues 68 through 98,  
 Inhibin α SEQ. ID. No.22, residues 68 through 101,  
 Inhibin βA SEQ. ID. No.23, residues 72 through 102, and  
 Inhibin βB SEQ. ID. No.24, residues 71 through 101.  
 
     
     
         9 . The construct of  claim 7 , wherein the finger 2 sequence is selected from the group of finger 2 sequences consisting of 
 TGF-β1 SEQ. ID. No.1, residues 65 through 94,    TGF-β2 SEQ. ID. No.2, residues 65 through 94,    TGF-β3 SEQ. ID. No.3, residues 65 through 94,    TGF-β4 SEQ. ID. No.4, residues 65 through 94,    TGF-β5 SEQ. ID. No.5, residues 65 through 94,    dpp SEQ. ID. No.6, residues 68 through 98,    Vg-1 SEQ. ID. No.7, residues 68 through 98,    Vgr-1 SEQ. ID. No.8, residues 68 through 98,    60A SEQ. ID. No.9, residues 68 through 98    BMP-2A SEQ. ID. No.10, residues 67 through 97,    BMP3 SEQ. ID. No.11, residues 69 through 99,    BMP-4 SEQ. ID. No.12, residues 67 through 97,    BMP-5 SEQ. ID. No.13, residues 68 through 98,    BMP-6 SEQ. ID. No.14, residues 68 through 98,    Dorsalin SEQ. ID. No.15, residues 68 through 99,    OP-1 SEQ. ID. No.16, residues 68 through 98,    OP-2 SEQ. ID. No.17, residues 68 through 98,    OP-3 SEQ. ID. No.18, residues 68 through 98,    GDF-1 SEQ. ID. No.19, residues 73 through 103,    GDP-3 SEQ. ID. No.20, residues 67 through 97    GDF-9 SEQ. ID. No.21, residues 68 through 98,    Inhibin α SEQ. ID. No.22, residues 68 through 101,    Inhibin βA SEQ. ID. No.23, residues 72 through 102, and    Inhibin βB SEQ. ID. No.24, residues 71 through 101.    
     
     
         10 . The construct of  claim 4 , wherein the finger 1 sequence is selected from the group of sequences consisting of 
 dpp SEQ. ID. No.6, residues 2 through 29,    Vg-1 SEQ. ID. No.7, residues 2 through 29,    Vgr-1 SEQ. ID. No.8, residues 2 through 29,    60A SEQ. ID. No.9, residues 2 through 29,    BMP-2A SEQ. ID. No.10, residues 2 through 29,    BMP3 SEQ. ID. No.11, residues 2 through 29,    BMP-4 SEQ. ID. No.12, residues 2 through 29,    BMP-5 SEQ. ID. No.13, residues 2 through 29,    BMP-6 SEQ. ID. No.14, residues 2 through 29,    Dorsalin SEQ. ID. No.15, residues 2 through 29,    OP-1 SEQ. ID. No.16, residues 2 through 29,    OP-2 SEQ. ID. No.17, residues 2 through 29, and    OP-3 SEQ. ID. No.18, residues 2 through 29.    
     
     
         11 . The construct of  claim 4  or  10 , wherein the heel sequence is selected from the group of sequences consisting of 
 dpp SEQ. ID. No.6, residues 35 through 65,  
 Vg-1 SEQ. ID. No.7, residues 35 through 65,  
 Vgr-1 SEQ. ID. No.8, residues 35 through 65,  
 60A SEQ. ID. No.9, residues 35 through 65,  
 BMP-2A SEQ. ID. No.10, residues 35 through 64,  
 BMP3 SEQ. ID. No.11, residues 35 through 66,  
 BMP-4 SEQ. ID. No.12, residues 35 through 64,  
 BMP-5 SEQ. ID. No.13, residues 35 through 65,  
 BMP-6 SEQ. ID. No.14, residues 35 through 65,  
 Dorsalin SEQ. ID. No.15, residues 35 through 65,  
 OP-1 SEQ. ID. No.16, residues 35 through 65,  
 OP-2 SEQ. ID. No.17, residues 35 through 65, and  
 OP-3 SEQ. ID. No.18, residues 35 through 65.  
 
     
     
         12 . The construct of  claim 4  or  10 , wherein the finger 2 sequence is selected from the group of sequences consisting of 
 dpp SEQ. ID. No.6, residues 68 through 98,  
 Vg-1 SEQ. ID. No.7, residues 68 through 98,  
 Vgr-1 SEQ. ID. No.8, residues 68 through 98,  
 60A SEQ. ID. No.9, residues 68 through 98,  
 BMP-2A SEQ. ID. No.10, residues 67 through 97,  
 BMP3 SEQ. ID. No.11, residues 69 through 99,  
 BMP-4 SEQ. ID. No.12, residues 67 through 97,  
 BMP-5 SEQ. ID. No.13, residues 68 through 98,  
 BMP-6 SEQ. ID. No.14, residues 68 through 98,  
 Dorsalin SEQ. ID. No.15, residues 68 through 99,  
 OP-1 SEQ. ID. No.16, residues 68 through 98,  
 OP-2 SEQ. ID. No.17, residues 68 through 98, and  
 OP-3 SEQ. ID. No.18, residues 68 through 98.  
 
     
     
         13 . The construct of  claim 11 , wherein the finger 2 sequence is selected from the group of sequences consisting of 
 dpp SEQ. ID. No.6, residues 68 through 98,    Vg-1 SEQ. ID. No.7, residues 68 through 98,    Vgr-1 SEQ. ID. No.8, residues 68 through 98,    60A SEQ. ID. No.9, residues 68 through 98,    BMP-2A SEQ. ID. No.10, residues 67 through 97,    BMP-3 SEQ. ID. No.11, residues 69 through 99,    BMP-4 SEQ. ID. No.12, residues 67 through 97,    BMP-5 SEQ. ID. No.13, residues 68 through 98,    BMP-6 SEQ. ID. No . 14, residues 68 th rough 98,    Dorsalin SEQ. ID. No.15, residues 68 through 99,    OP-1 SEQ. ID. No.16, residues 68 through 98,    OP-2 SEQ. ID. No.17, residues 68 through 98, and    OP-3 SEQ. ID. No.18, residues 68 through 98.    
     
     
         14 . The construct of  claim 4 , wherein the finger 1 sequence is OP-1 finger 1 sequence [SEQ. ID. No.16, residues 2 through 29].  
     
     
         15 . The construct of  claim 4  or  14 , wherein the heel sequence is OP-1 heel sequence [SEQ. ID. No.16, residues 35 through 65].  
     
     
         16 . The construct of  claim 4  or  14 , wherein the finger 2 sequence is OP-1 finger 2 sequence [SEQ. ID. No.16, residues 68 through 98].  
     
     
         17 . The construct of  claim 15 , wherein the finger 2 sequence is OP-1 finger 2 sequence [SEQ. ID. No.16, residues 68 through 98].  
     
     
         18 . The construct of  claim 4 , wherein: 
 the finger 1 sequence is OP-1 finger 1 sequence [SEQ. ID. No.16, residues 2 through 29];    the heel sequence is OP-1 heel sequence [SEQ. ID. No.16, residues 35 through 65]; and    the finger 2 sequence is OP-1 finger 2 sequence [SEQ. ID. No.16, residues 68 through 98].    
     
     
         19 . The construct of  claim 1  wherein: 
 the finger 1 region comprises a sequence selected from the group consisting of 
 OP-1 finger 1 large peptide [SEQ. ID. No.16, residues 5 through 28],  
 OP-1 finger 1 medium peptide [SEQ. ID. No.16, residues 7 through 26], and  
 OP-1 finger 1 small peptide [SEQ. ID. No.16, residues 11 through 22];  
 
 the heel region comprises a sequence selected from the group consisting of 
 OP-1 heel large peptide [SEQ. ID. No.16, residues 49 through 64],  
 OP-1 heel medium peptide [SEQ. ID. No.16, residues 52 through 64], and  
 OP-1 heel small peptide [SEQ. ID. No.16, residues 56 through 62]; and  
 
 the finger 2 region comprises a sequence selected from the group consisting of 
 OP-1 finger 2 large peptide [SEQ. ID. No.16, residues 75 through 91],  
 OP-1 finger 2 medium peptide [SEQ. ID. No.16, residues 77 through 89], and  
 OP-1 finger 2 small peptide [SEQ. ID. No.16, residues 79 through 87].  
 
 
     
     
         20 . The construct of  claim 1  or  4 , wherein said finger 1 region, said heel region, and said finger 2 region are joined by a pair of polypeptide linkers to produce said single-chain amino acid construct.  
     
     
         21 . The construct of  claim 20 , wherein each of said linkers comprises between 3 and 13 amino acids.  
     
     
         22 . The construct of  claim 1 , wherein said regions are defined by amino acid sequences selected from the group consisting of 
 (a) F1-linker-F2-linker-H;    (b) F1-linker-H-linker-F2;    (c) F2-linker-F1-linker-H;    (d) F2-linker-H-linker-F1;    (e) H-linker-F1-linker-F2; and    (f) H-linker-F2-linker-F1, 
 wherein H comprises an alpha helical region comprising said heel region, F1 comprises a pair of anti-parallel β strand structures defining said finger 1 region, F2 comprises a pair of anti-parallel β strands structures defining said finger 2 region, each finger and heel region begins and ends with a cysteine residue, each linker comprises an amino acid sequence having 3-13 amino acids, and wherein group a) and b) optionally further comprise an N-terminal sequence.  
   
     
     
         23 . The construct of  claim 22 , wherein F1 comprises, in order from the N to C direction, a first β strand, a helical loop, a loop, and a second P strand antiparallel to the first β strand.  
     
     
         24 . The construct of  claim 22 , wherein F2 comprises, in order from the N to C direction, a first β strand, a loop, and a second β strand anti-parallel to the first β strand.  
     
     
         25 . The DNA encoding the amino acid sequence of the construct of  claim 1 ,  4 , or  22 .  
     
     
         26 . A cell line expressing a DNA of  claim 25.