Engineered polypeptides

Patent metadata

Field	Value
Publication number	US-10457717-B2
Application number	US-201815934744-A
Country	US
Kind code	B2
Filing date	Mar 23, 2018
Priority date	Feb 17, 2017
Publication date	Oct 29, 2019
Grant date	Oct 29, 2019

Abstract

Provided herein are polypeptides that bind to a blood-brain barrier (BBB) receptor, methods of generating such polypeptides, and methods of using the polypeptides to target a composition to a BBB receptor-expressing cell, e.g., for transport across the BBB. Also provided herein are transferrin receptor (TfR) constructs that comprise a monomeric TfR apical domain or one or more portions of the TfR apical domain which have been circularly permuted relative to the full-length TfR sequence.

First claim

What is claimed is:

1. A transferrin receptor (TfR) construct comprising from N- to C-terminus:
(a) a first polypeptide comprising a C-terminal fragment of a TfR apical domain;
(b) an optional linker; and
(c) a second polypeptide comprising an N-terminal fragment of the TfR apical domain,
wherein the first polypeptide, the optional linker, and the second polypeptide are fused in a tandem series.

2. The TfR construct of claim 1 , wherein the last amino acid of the first polypeptide is fused to the first amino acid of the second polypeptide.

3. The TfR construct of claim 1 , wherein the first polypeptide comprises a sequence having at least 90% sequence identity or up to seven amino acid changes relative to the sequence of SEQ ID NO:427.

4. The TfR construct of claim 1 , wherein the second polypeptide comprises a sequence having at least 90% sequence identity or up to sixteen amino acid changes relative to the sequence of SEQ ID NO:428.

5. The TfR construct of claim 1 , wherein the TfR apical domain comprises the sequence of SEQ ID NO:107 or SEQ ID NO:108.

6. The TfR construct of claim 1 , comprising:
(a) the first polypeptide comprising a sequence having at least 90% sequence identity or up to seven amino acid changes relative to the sequence of SEQ ID NO:427;
(b) the optional linker; and
(c) the second polypeptide comprising a sequence having at least 90% sequence identity or up to sixteen amino acid changes relative to the sequence of SEQ ID NO:428.

7. The TfR construct of claim 6 , wherein the first polypeptide at the C-terminus further comprises a sequence having at least 90% sequence identity or up to five amino acid changes relative to the sequence of SEQ ID NO:429 or a fragment thereof.

8. The TfR construct of claim 6 , wherein the second polypeptide at the C-terminus further comprises a sequence having at least 90% sequence identity or up to five amino acid changes relative to the sequence of SEQ ID NO:434 or a fragment thereof.

9. The TfR construct of claim 6 , wherein the first polypeptide at the N-terminus further comprises a sequence having at least 90% sequence identity or up to five amino acid changes relative to the sequence of SEQ ID NO:439 or a fragment thereof.

10. The TfR construct of claim 6 , wherein the second polypeptide at the N-terminus further comprises a sequence having at least 90% sequence identity or up to five amino acid changes relative to the sequence of SEQ ID NO:444 or a fragment thereof.

11. The TfR construct of claim 6 , wherein the first polypeptide at the C-terminus further comprises the sequence of SEQ ID NO:431 and/or the first polypeptide at the N-terminus further comprises the sequence of SEQ ID NO:441.

12. The TfR construct of claim 6 , wherein the second polypeptide at the C-terminus further comprises the sequence of SEQ ID NO:436 and/or wherein the second polypeptide at the N-terminus further comprises the sequence of SEQ ID NO:446.

13. The TfR construct of claim 1 , wherein the first polypeptide comprises a sequence having at least 90% sequence identity or up to ten amino acid changes relative to the sequence of SEQ ID NO:449.

14. The TfR construct of claim 1 , wherein the second polypeptide comprises a sequence having at least 90% sequence identity or up to twenty amino acid changes relative to the sequence of SEQ ID NO:450.

15. The TfR construct of claim 1 , wherein the TfR construct comprises the first polypeptide having the sequence of SEQ ID NO:449 or SEQ ID NO:451, and the second polypeptide having the sequence of SEQ ID NO:450 or SEQ ID NO:452, wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.

16. The TfR construct of claim 1 , wherein the linker is 1 to 10 amino acids in length or comprises a protein loop domain.

17. The TfR construct of claim 16 , wherein the N- and C-termini of the protein loop domain are less than 5 Å apart.

18. The TfR construct of claim 1 , wherein the TfR construct further comprises a purification peptide and/or a cleavage peptide.

19. The TfR construct of claim 1 , wherein the TfR construct is soluble.

20. An isolated, recombinant TfR apical domain construct comprising an amino acid sequence having at least about 90% identity to any one of SEQ ID NOS:109, 110, 301, 468, and 469.

21. The TfR apical domain construct of claim 20 , wherein the TfR apical domain construct comprises the amino acid sequence of any one of SEQ ID NOS:109, 110, 301, 468, and 469.

22. An isolated polynucleotide comprising a nucleotide sequence encoding the TfR construct of claim 1 .

23. A vector comprising the polynucleotide of claim 22 .

24. A method of identifying an agent that binds the apical domain of a TfR, comprising:
(a) contacting the TfR construct of claim 1 with the agent; and
(b) determining whether the agent binds to the TfR construct.

25. The method of claim 24 , wherein the agent is a polypeptide or a protein.

26. The method of claim 24 , wherein the agent is a modified Fc polypeptide, a modified Fc polypeptide dimer, or an antibody.

27. The TfR construct of claim 1 , wherein the C-terminal fragment of the TfR apical domain comprises 25 to 55 amino acids.

28. The TfR construct of claim 1 , wherein the N-terminal fragment of the TfR apical domain comprises 75 to 120 amino acids.

29. The TfR construct of claim 27 , wherein the N-terminal fragment of the TfR apical domain comprises 75 to 120 amino acids.

Assignees

• Denali Therapeutics Inc

Inventors

• Chen Xiaocheng
• Kariolis Mihalis
• Wells Robert C

Classifications

• C07K14/70582 Primary

  CD71 · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• G01N33/6854

  Immunoglobulins · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title