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 method of identifying an agent that binds a transferrin receptor (TfR) apical domain, the method comprising:
(a) contacting a TfR construct comprising a monomeric circularly permuted TfR apical domain with the agent, wherein the TfR construct does not include a protease-like domain or helical domain of the TfR; and
(b) determining whether the agent binds to the TfR construct by ELISA, surface plasmon resonance, flow cytometry, kinetic exclusion assay, fluorescence-activated cell sorting, or BioLayer interferometry.
2. The method of claim 1 , wherein the agent is a polypeptide or a protein.
3. The method of claim 1 , wherein the agent is a modified Fc polypeptide, a modified Fc polypeptide dimer, or an antibody.
4. The method of claim 1 , wherein the TfR construct is expressed and displayed as a monomer on the surface of a phage or a cell.
5. The method of claim 1 , wherein the TfR construct comprises a sequence having at least 90% sequence identity to SEQ ID NO:427 or SEQ ID NO:428.
6. The method of claim 5 , wherein the TfR construct comprises the sequence of SEQ ID NO:427 or SEQ ID NO:428.
7. The method of claim 1 , wherein the TfR construct comprises a human or cynomolgus TfR apical domain.
8. The method of claim 1 , wherein the TfR construct comprises from N- to C-terminus:
(i) a first polypeptide comprising a C-terminal fragment of the TfR apical domain, wherein the C-terminal fragment of the TfR apical domain comprises 25 to 55 amino acids;
(ii) an optional linker; and
(iii) 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.
9. The method of claim 8 , wherein the last amino acid of the first polypeptide is fused to the first amino acid of the second polypeptide.
10. The method of claim 8 , wherein the N-terminal fragment of the TfR apical domain comprises 75 to 120 amino acids.
11. The method of claim 8 , wherein the first polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO:427 or the second polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO:428.
12. The method of claim 11 , wherein the first polypeptide comprises the sequence of SEQ ID NO:427 or the second polypeptide comprises the sequence of SEQ ID NO:428.
13. The method of claim 8 , wherein the linker is 1 to 10 amino acids in length or comprises a protein loop domain.
14. The method of claim 13 , wherein the N- and C-termini of the protein loop domain are less than 5 Å apart.
15. The method of claim 8 , wherein the TfR construct further comprises a purification peptide and/or a cleavage peptide.
16. The method of claim 1 , wherein the TfR construct is soluble.
17. A method of identifying an agent that binds a transferrin receptor (TfR) apical domain, the method comprising:
(a) contacting a TfR construct comprising a monomeric circularly permuted human or cynomolgus TfR apical domain with the agent, wherein the TfR construct does not include a protease-like domain or helical domain of the TfR; and
(b) determining whether the agent binds to the TfR construct.