Engineered polypeptides
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.
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.