Universal fibronectin type III binding-domain libraries
First Claim
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.
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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.
178 Citations
34 Claims
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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. - View Dependent Claims (2, 3, 4, 6, 7, 8, 9)
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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.
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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:
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(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. - View Dependent Claims (11, 12, 13, 14)
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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:
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(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. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
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32. A TNF-α
- binding protein having a Kd binding constant equal to or greaterthan 0.1 μ
M and having a sequence selected from SEQ ID NOS;
55-63.
- binding protein having a Kd binding constant equal to or greaterthan 0.1 μ
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33. A VEGF binding protein having a Kd binding constant equal to or greater than 0.1 μ
- M and having a sequence selected from SEQ ID NOS;
64-67.
- M and having a sequence selected from SEQ ID NOS;
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34. A HMGB1 binding protein having a Kd binding constant equal to or greater than 0.1 μ
- M and having a sequence selected from SEQ ID NOS;
67-81.
- M and having a sequence selected from SEQ ID NOS;
Specification