Artificial antibody polypeptides
First Claim
1. A fibronectin type III (Fn3) polypeptide monobody comprising a plurality of Fn3 β
- -strand domain sequences that are linked to a plurality of loop region sequences, wherein one or more of the monobody loop region sequences vary by deletion, insertion or replacement of at least two amino acids from the corresponding loop region sequences in wild-type Fn3, and wherein the β
-strand domains of the monobody have at least a 50% total amino acid sequence homology to the corresponding amino acid sequence of wild-type Fn3'"'"'s β
-strand domain sequences.
5 Assignments
0 Petitions
Accused Products
Abstract
A fibronectin type III (Fn3) polypeptide monobody, a nucleic acid molecule encoding said monobody, and a variegated nucleic acid library encoding said monobody, are provided by the invention. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform said methods. Further provided is a method of identifying the amino acid sequence of a polypeptide molecule capable of binding to a specific binding partner (SBP) so as to form a polypeptide:SSP complex, and a method of identifying the amino acid sequence of a polypeptide molecule capable of catalyzing a chemical reaction with a catalyzed rate constant, kcat, and an uncatalyzed rate constant, kuncat, such that the ratio of kcat/kuncat is greater than 10.
-
Citations
73 Claims
-
1. A fibronectin type III (Fn3) polypeptide monobody comprising a plurality of Fn3 β
- -strand domain sequences that are linked to a plurality of loop region sequences,
wherein one or more of the monobody loop region sequences vary by deletion, insertion or replacement of at least two amino acids from the corresponding loop region sequences in wild-type Fn3, and wherein the β
-strand domains of the monobody have at least a 50% total amino acid sequence homology to the corresponding amino acid sequence of wild-type Fn3'"'"'s β
-strand domain sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)
- -strand domain sequences that are linked to a plurality of loop region sequences,
-
11. A method of preparing a fibronectin type III (Fn3) polypeptide monobody comprising the steps of:
-
a) providing a DNA sequence encoding a plurality of Fn3 β
-strand domain sequences that are linked to a plurality of loop region sequences wherein at least one loop region contains a unique restriction enzyme site;
b) cleaving the DNA sequence at the unique restriction site;
c) inserting into the restriction site a DNA segment known to encode a peptide capable of binding to a specific binding partner (SBP) or a transition state analog compound (TSAC) so as to yield a DNA molecule comprising the insertion and the DNA sequence of (a); and
d) expressing the DNA molecule so as to yield polypeptide monobody. - View Dependent Claims (14)
-
-
12. A method of preparing a fibronectin type III (Fn3) polypeptide monobody comprising the steps of:
-
(a) providing a replicatable DNA sequence encoding a plurality of Fn3 β
-strand domain sequences that are linked to a plurality of loop region sequences, wherein the nucleotide sequence of at least one loop region is known;
(b) preparing polymerase chain reaction (PCR) primers sufficiently complementary to the known loop sequence so as to be hybridizable under PCR conditions, wherein at least one of the primers contains a modified nucleic acid sequence to be inserted into the DNA;
(c) performing polymerase chain reaction using the DNA sequence of (a) and the primers of (b);
(d) annealing and extending the reaction products of (c) so as to yield a DNA product; and
(e) expressing the polypeptide monobody encoded by the DNA product of (d).
-
-
13. A method of preparing a fibronectin type III (Fn3) polypeptide monobody comprising the steps of:
-
a) providing a replicatable DNA sequence encoding a plurality of Fn3 β
-strand domain sequences that are linked to a plurality of loop region sequences, wherein the nucleotide sequence of at least one loop region is known;
b) performing site-directed mutagenesis of at least one loop region so as to create a DNA sequence comprising an insertion mutation; and
c) expressing the polypeptide monobody encoded by the DNA sequence comprising the insertion mutation.
-
-
15. A variegated nucleic acid library encoding Fn3 polypeptide monobodies comprising a plurality of nucleic acid species each comprising a plurality of loop regions, wherein the species encode a plurality of Fn3 β
- -strand domain sequences that are linked to a plurality of loop region sequences,
wherein one or more of the loop region sequences vary by deletion, insertion or replacement of at least two amino acids from corresponding loop region sequences in wild-type Fn3, and wherein the β
-strand domain sequences of the monobody have at least a 50% total amino acid sequence homology to the corresponding amino acid sequences of β
-strand domain sequences of the wild-type Fn3. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 36, 37, 38, 39)
- -strand domain sequences that are linked to a plurality of loop region sequences,
-
31. A method of preparing a variegated nucleic acid library encoding Fn3 polypeptide monobodies having a plurality of nucleic acid species each comprising a plurality of loop regions, wherein the species encode a plurality of Fn3 β
- -strand domain sequences that are linked to a plurality of loop region sequences, wherein one or more of the loop region sequences vary by deletion, insertion or replacement of at least two amino acids from corresponding loop region sequences in wild-type Fn3, and wherein the β
-strand domain sequences of the monobody have at least a 50% total amino acid sequence homology to the corresponding amino acid sequences of β
-strand domain sequences of the wild-type Fn3, comprising the steps ofa) preparing an Fn3 polypeptide monobody having a predetermined sequence;
b) contacting the polypeptide with a specific binding partner (SBP) so as to form a polypeptide;
SSP complex wherein the dissociation constant of the said polypeptide;
SBP complex is less than 10−
6 moles/liter;
c) determining the binding structure of the polypeptide;
SBP complex by nuclear magnetic resonance spectroscopy or X-ray crystallography; and
d) preparing the variegated nucleic acid library, wherein the variegation is performed at positions in the nucleic acid sequence which, from the information provided in (c), result in one or more polypeptides with improved binding to the SBP.
- -strand domain sequences that are linked to a plurality of loop region sequences, wherein one or more of the loop region sequences vary by deletion, insertion or replacement of at least two amino acids from corresponding loop region sequences in wild-type Fn3, and wherein the β
-
33. A method of preparing a variegated nucleic acid library encoding Fn3 polypeptide monobodies having a plurality of nucleic acid species each comprising a plurality of loop regions, wherein the species encode a plurality of Fn3 β
- -strand domain sequences that are linked to a plurality of loop region sequences, wherein one or more of the loop region sequences vary by deletion, insertion or replacement of at least two amino acids from corresponding loop region sequences in wild-type Fn3, and wherein the β
-strand domain sequences of the monobody have at least a 50% total amino acid sequence homology to the corresponding amino acid sequences of β
-strand domain sequences of the wild-type Fn3, comprising the steps ofa) preparing an Fn3 polypeptide monobody having a predetermined sequence, wherein the polypeptide is capable of catalyzing a chemical reaction with a catalyzed rate constant, kcat, and an uncatalyzed rate constant, kuncat, such that the ratio of kcat/kuncat is greater than 10;
b) contacting the polypeptide with an immobilized or separable transition state analog compound (TSAC) representing the approximate molecular transition state of the chemical reaction;
c) determining the binding structure of the polypeptide;
TSAC complex by nuclear magnetic resonance spectroscopy or X-ray crystallography; and
d) preparing the variegated nucleic acid library, wherein the variegation is performed at positions in the nucleic acid sequence which, from the information provided in (c), result in one or more polypeptides with improved binding to or stabilization of the TSAC.
- -strand domain sequences that are linked to a plurality of loop region sequences, wherein one or more of the loop region sequences vary by deletion, insertion or replacement of at least two amino acids from corresponding loop region sequences in wild-type Fn3, and wherein the β
- 40. A binding protein comprising a fibronectin type III domain having at least one randomized loop as compared to a corresponding wild-type fibronectin, wherein the protein binds to a ligand that is different from the ligand that binds to the corresponding wild-type fibronectin.
- 56. A method for generating a protein comprising a fibronectin type III domain that is pharmaceutically acceptable to a mammal, the method comprising removing an integrin-binding domain from the fibronectin type III domain.
Specification