Methods of engineering transferrin receptor binding polypeptides

Patent metadata

Field	Value
Publication number	US-11912778-B2
Application number	US-202217888406-A
Country	US
Kind code	B2
Filing date	Aug 15, 2022
Priority date	Feb 17, 2017
Publication date	Feb 27, 2024
Grant date	Feb 27, 2024

Abstract

Provided herein are polypeptides that bind to a transferrin receptor, methods of generating such polypeptides, and methods of using the polypeptides to target a composition to a transferrin receptor-expressing cell.

First claim

What is claimed is:

1. A method of generating one or more variant polypeptides that specifically bind transferrin receptor protein and transport across the blood brain barrier, comprising:
(a) identifying a contiguous surface exposed register of about 7 to 15 amino acids of a native polypeptide for mutation;
(b) generating one or more polynucleotides comprising a nucleotide sequence that encodes for a library of sequence variants of the native polypeptide, wherein the nucleotide sequence encodes for one or more mutations in the identified register of the native polypeptide;
(c) expressing the one or more polynucleotides to produce the library of sequence variants;
(d) screening the library of sequence variants to identify one or more variant polypeptides that specifically bind transferrin receptor protein; and
(e) isolating the variant polypeptides,
wherein the identified register of the native polypeptide does not bind transferrin receptor protein; and the isolated variant polypeptides exhibit substantially improved brain uptake compared to the native polypeptide.

2. The method of claim 1 , wherein the register comprises about 7 to 15 amino acids from a region of the native polypeptide selected from the group consisting of a beta sheet, loop, hinge, parallel beta sheets, less structured residues, two loops, and a combination thereof.

3. The method of claim 1 , wherein the amino acids of the register in step (a) do not bind an antigen in the native polypeptide.

4. The method of claim 1 , further comprising:
(f 1 ) identifying at least one position having conserved amino acids within the register among the isolated variant polypeptides;
(g 1 ) generating one or more polynucleotides comprising an encoding region for a library of sequence variants of the isolated variant polypeptides, wherein the encoding region includes mutations to encode for an amino acid substitution, insertion, or deletion within the register at the positions other than the conserved amino acid positions; and
(h 1 ) repeating steps (c) through (e).

5. The method of claim 1 , further comprising:
(f 2 ) identifying about 4 to 7 amino acid positions near the register among the isolated variant polypeptides for mutation;
(g 2 ) generating one or more polynucleotides comprising an encoding region for a library of sequence variants of the isolated variant polypeptides, wherein the encoding region includes mutations to encode for an amino acid substitution, insertion, or deletion at the about 4 to 7 positions; and
(h 2 ) repeating steps (c) through (e).

6. The method of claim 1 , wherein step (b) comprises:
(b 1 ) generating first polynucleotide fragments comprising a first encoding region, wherein the first encoding region includes mutations to encode for an amino acid substitution, insertion, or deletion within the register at the first half of the register positions;
(b 2 ) generating second polynucleotide fragments comprising a second encoding region, wherein the second encoding region includes mutations to encode for an amino acid substitution, insertion, or deletion within the register at the second half of the register positions; and
(b 3 ) shuffling the first and second polynucleotide fragments during PCR amplification to mix the first and second encoding regions and form a library of sequence variants for expression.

7. The method of claim 1 , further comprising:
(f 3 ) generating first polynucleotide fragments comprising a first encoding region within the register among the isolated variant polypeptides for mutation, wherein the first encoding region includes mutations to encode for an amino acid substitution, insertion, or deletion within the register at the first half of the register positions;
(g 3 ) generating second polynucleotide fragments comprising a second encoding region within the register among the isolated variant polypeptides for mutation, wherein the second encoding region includes mutations to encode for an amino acid substitution, insertion, or deletion within the register at the second half of the register positions;
(h 3 ) shuffling the first and second polynucleotide fragments during PCR amplification to mix the first and second encoding regions and form a library of sequence variants for expression; and
(i 3 ) repeating steps (c) through (e).

8. The method of claim 1 , further comprising:
(f 4 ) identifying one position near the register among the isolated variant polypeptides for mutation;
(g 4 ) generating one or more polynucleotides comprising an encoding region for a library of sequence variants of the isolated variant polypeptides, wherein the encoding region includes an NNK mutation at the position near the register; and
(h 4 ) repeating steps (c) through (e).

9. The method of claim 1 , further comprising:
(f 5 ) identifying about 4 to 7 amino acid positions adjacent to the register positions among the isolated variant polypeptides for mutation;
(g 5 ) generating one or more polynucleotides comprising an encoding region for a library of sequence variants of the isolated variant polypeptides, wherein the encoding region includes NNK mutations at the about 4 to 7 positions adjacent to the register; and
(h 5 ) repeating steps (c) through (e).

10. The method of claim 1 , further comprising:
(f 6 ) identifying one position within the register among the isolated variant polypeptides for mutation;
(g 6 ) generating one or more polynucleotides comprising an encoding region for a library of sequence variants of the isolated variant polypeptides, wherein the encoding region includes an NNK mutation at the position within the register; and
(h 6 ) repeating steps (c) through (e).

11. The method of claim 4 , further comprising:
(i 1 ) repeating steps (f 1 )-(h 1 ) until one or more of the isolated variant polypeptides bind transferrin receptor protein with an affinity of 500 nM or higher.

12. The method of claim 1 , wherein one or more of the isolated variant polypeptides exhibit improved brain uptake compared to the native polypeptide by greater than 10-fold.

13. The method of claim 1 , wherein one or more of the isolated variant polypeptides have about 100-fold or greater affinity for a transferrin receptor protein compared to an unrelated target when assayed under the same affinity assay conditions.

14. The method of claim 1 , wherein the mutated register in one or more of the isolated variant polypeptides comprises a sequence selected from the group consisting of YGTEW (SEQ ID NO:115), YxTEWSS (SEQ ID NO:636), and TxxExxxxF.

15. The method of claim 1 , wherein step (d) comprises contacting the library of sequence variants with a transferrin receptor protein in the presence of transferrin.

16. The method of claim 15 , wherein step (d) further comprises selecting one or more variant polypeptides that do not compete with transferrin for binding to the transferrin receptor protein.

17. The method of claim 1 , wherein one or more of the variant polypeptides selectively bind an apical domain of the transferrin receptor protein.

Assignees

• Denali Therapeutics Inc

Inventors

• Chen Xiaocheng
• Dennis Mark S
• Kariolis Mihalis
• Silverman Adam P
• Srivastava Ankita
• Watts Ryan J
• Wells Robert C
• Zuchero Joy Yu

Classifications

• C07K16/2881 Primary

  against CD71 · CPC title

• C07K16/005

  constructed by phage libraries · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C40B20/00

  Methods specially adapted for identifying library members · CPC title

• C40B30/04

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title