Universal fibronectin type III binding-domain libraries

Abstract

Walk-through mutagenesis and natural-variant combinatorial fibronectin Type III (FN3) polypeptide libraries are described, along with their method of construction and use. Also disclosed are a number of high binding affinity polypeptides selected by screening the libraries against a variety of selected antigens.

1 . A method of forming a library of fibronectin Type 3 (FN3) domain polypeptides useful in screening for the presence of one or more polypeptides having a selected binding or enzymatic activity, comprising
 (i) aligning BC, DE, and FG amino acid loop sequences in a collection of native fibronectin Type 3 domain polypeptides,   (ii) segregating the aligned loop sequences according to loop length,   (iii) for a selected loop and loop length from step (ii), performing positional amino acid frequency analysis to determine the frequencies of amino acids at each loop position,   (iv) for each loop and loop length analyzed in step (iii), identifying at each position a conserved or selected semi-conserved consensus amino acid and other natural-variant amino acids,   (v) for at least one selected loop and loop length, forming:   (1) a library of walk-through mutagenesis sequences expressed by a library of coding sequences that encode, at each loop position, the consensus amino acid, and if the consensus amino acid has a occurrence frequency equal to or less than a selected threshold frequency of at least 50%, a single common target amino acid and any co-produced amino acids, or   (2) a library of natural-variant combinatorial sequences expressed by a library of coding sequences that encode at each loop position, a consensus amino acid and, if the consensus amino acid has a frequency of occurrence equal to or less than a selected threshold frequency of at least 50%, other natural variant amino acids, including semi-conserved amino acids and variable amino acids whose occurrence rate is above a selected minimum threshold occurrence at that position, or their chemical equivalents,   (vi) incorporating the library of coding sequences into framework FN3 coding sequences to form an FN3 expression library, and   (vi) expressing the FN3 polypeptides of the expression library.   
     
     
         2 . The method of  claim 1 , wherein the given threshold frequency is 100%. 
     
     
         3 . The method of  claim 1 , wherein the given threshold frequency is a selected frequency between 50-95%. 
     
     
         4 . The method of  claim 1 , wherein step (ii) segregates the loops and loop lengths into the group consisting of BC/11, BC/14, BC/15, DE/6, FG/8, and FG/11. 
     
     
         5 . The method of  claim 5 , wherein the library formed has a library of walk-through mutagenesis sequences formed at each of the loops and loop lengths selected from the group consisting of BC/11, BC/14, BC/15, DE/6, FG/8, and FG11, and from each of the common target amino selected from the group consisting of lysine, glutamine, aspartic acid, tyrosine, leucine, praline, serine, histidine, and glycine. 
     
     
         6 . The method of  claim 1 , wherein the library formed has a library of natural-variant combinatorial sequences at a combination of loops and loop lengths selected from loops BC and DE, BC and FG, and DE and FG loops, where the BC loop is selected from one of BC/11, BC/14, and BC/15, the DE loop is DE/6, and the FG loop is selected from one of FG/8, and FG11. 
     
     
         7 . The method of  claim 6 , wherein the given threshold is 100%, unless the loop amino acid position contains only one dominant and one variant amino, and the dominant and variant amino have side chains with similar physiochemical properties, in which case the given threshold is 90%. 
     
     
         8 . The method of  claim 6 , wherein the two loops in the selected combination of loops and loop lengths has an average diversity of between 10 5  and 10 7 , or where the average number of different amino acids at each position is equal to or less than 5. 
     
     
         9 . The method of  claim 1 , wherein said polypeptides have the wildtype amino acid sequences in beta-strand regions A, AB, B, C, CD, D, E, EF, F, and G of the 14 th  fibronectin Type III module of human fibronecton. 
     
     
         10 . A walk-through mutagenesis library of fibronectin Type 3 (FN3) domain polypeptides useful in screening for the presence of one or more polypeptides having a selected binding or enzymatic activity, said polypeptides comprising:
 (a) regions A, AB, B, C, CD, D, E, EF, F, and G having wildtype amino acid sequences of a selected native fibronectin Type 3 polypeptide, and   (b) loop regions BC, DE, and FG having selected lengths, where at least one selected loop region of a selected length contains a library of walk through mutagenesis sequences expressed by a library of coding sequences that encode, at each loop position, a conserved or selected semi-conserved consensus amino acid and, if the consensus amino acid has an occurrence frequency equal to or less than a selected threshold frequency of at least 50%, a single common target amino acid and any co-produced amino acids.   
     
     
         11 . The library of  claim 10 , wherein the given threshold frequency is 100%. 
     
     
         12 . The library of  claim 10 , wherein the given threshold frequency is a selected frequency between 50-95%. 
     
     
         13 . The library of  claim 10 , wherein loops and loop lengths are selected from the group consisting of BC/11, BC/14, BC/15, DE/6, FG/8, and FG11, and which has a library of walk-through mutagenesis sequences formed at each of the loops and loop lengths selected from the group consisting of BC/11, BC/14, BC/15, DE/6, FG/8, and FG11. 
     
     
         14 . The library  claim 13 , which has a library of walk-through mutagenesis sequences formed from each of the common target amino selected from the group consisting of lysine, glutamine, aspartic acid, tyrosine, leucine, praline, serine, histidine, and glycine. 
     
     
         15 . A natural-variant combinatorial library of fibronectin Type 3 (FN3) domain polypeptides useful in screening for the presence of one or more polypeptides having a selected binding or enzymatic activity, said polypeptides comprising:
 (a) regions A, AB, B, C, CD, D, E, EF, F, and G having wildtype amino acid sequences of a selected native fibronectin Type 3 polypeptide, and   (b) loop regions BC, DE, and FG having selected lengths, where at least one selected loop region of a selected length contains a library of natural-variant combinatorial sequences expressed by a library of coding sequences that encode at each loop position, a conserved or selected semi-conserved consensus amino acid and, if the consensus amino acid has a frequency of occurrence equal to or less than a selected threshold frequency of at least 50%, other natural variant amino acids, including semi-conserved amino acids and variable amino acids whose occurrence rate is above a selected minimum threshold occurrence at that position, or their chemical equivalents.   
     
     
         16 . The library of  claim 15 , which has a library of natural-variant combinatorial sequences at a combination of loops and loop lengths selected from loops BC and DE, BC and FG, and DE and FG loops, where the BC loop is selected from one of BC/11, BC/14, and BC/15, the DE loop is DE/6, and the FG loop is selected from one of FG/8, and FG11. 
     
     
         17 . The library of  claim 15 , which has at two of the loop combinations BC and DE, BC and FG, and DE and FG, beneficial mutations identified by screening a universal combinatorial library containing amino acid variants in the two loop combination, and at the third loop, identified by FG, DE, and BC, respectively, a library of natural variant combinatorial sequences at a third loop and lop length identified by BC/11, BC/14, and BC/15, DE/6, or FG/8, and FG11. 
     
     
         18 . The library method of  claim 17 , wherein each of the two selected loops have an average diversity of between 10 5  and 10 7 . 
     
     
         19 . The library of  claim 15 , which has a given threshold is 100%, unless the loop amino acid position contains only one dominant and one variant amino, and the dominant and variant amino have side chains with similar physiochemical properties, in which case the given threshold is 90%. 
     
     
         20 . The library of  claim 15 , wherein said polypeptides have the wildtype amino acid sequences in beta-strand regions A, AB, B, C, CD, D, E, EF, F, and G of the 14 th  fibronectin Type III module of human fibronecton. 
     
     
         21 . The library of  claim 15 , wherein said polypeptides have the wildtype amino acid sequences in regions A, AB, B, C, CD, D, E, EF, F, and G of the 10 th  fibronectin Type III module of human fibronecton. 
     
     
         22 . The library of  claim 15 , wherein the BC loop length is 11, and has the amino acid sequence identified by SEQ ID NOS: 43 or 49. 
     
     
         23 . The library of  claim 15 , wherein the BC loop length is 14, and has the amino acid sequence identified by SEQ ID. NOS: 44 or 50. 
     
     
         24 . The library of  claim 15 , wherein the BC loop length is 15, and has the amino acid sequence identified by SEQ ID. NOS: 45 or 51. 
     
     
         25 . The library of  claim 15 , wherein the DE loop length is 6, and has the amino acid sequence identified by SEQ ID. NOS: 46 or 52. 
     
     
         26 . The library of  claim 15 , wherein the FG loop length is 8, and has the amino acid sequence identified by SEQ ID. NOS: 47, for the first N-terminal six amino acids, or SEQ ID NO:53. 
     
     
         27 . The library of  claim 15 , wherein FG loop length is 11, and has the amino acid sequence identified by SEQ ID. NO: 48, for the first N-terminal nine amino acids, or SEQ ID NO:54. 
     
     
         28 . The library of  claim 15 , wherein the polypeptides are encoded by an expression library selected from the group consisting of a ribosome display library, a polysome display library, a phage display library, a bacterial expression library, and a yeast display library. 
     
     
         29 . An expression library of polynucleotides encoding the library of polypeptides of  claim 15 , and produced by synthesizing polynucleotides encoding one or more beta-strand framework regions and one or more loop regions wherein the polynucleotides are predetermined, wherein the polynucleotides encoding said regions further comprise sufficient overlapping sequence whereby the polynucleotide sequences, under polymerase chain reaction (PCR) conditions, are capable of assembly into polynucleotides encoding complete fibronectin binding domains. 
     
     
         30 . A method of identifying a polypeptide having a desired binding affinity with respect to a selected antigen, comprising
 reacting the natural-variant combinatorial library of FN3 polypeptides of  claim 15  with the selected antigen, and   screening the FM3 polypeptides to select those having a desired binding affinity with respect to the selected antigen   
     
     
         31 . The method of  claim 30 , wherein the method further comprises the step of identifying the polynucleotide that encodes the selected fibronectin binding domain. 
     
     
         32 . A TNF-α binding protein having a K d  binding constant equal to or greaterthan 0.1 μM and having a sequence selected from SEQ ID NOS: 55-63. 
     
     
         33 . A VEGF binding protein having a K d  binding constant equal to or greater than 0.1 μM and having a sequence selected from SEQ ID NOS: 64-67. 
     
     
         34 . A HMGB1 binding protein having a K d  binding constant equal to or greater than 0.1 μM and having a sequence selected from SEQ ID NOS: 67-81.