Engineered transferrin receptor binding polypeptides
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.
1. A polypeptide comprising a CH3 domain that specifically binds to a transferrin receptor, wherein the CH3 domain comprises five, six, seven, or eight substitutions in a set of amino acid positions comprising 118, 119, 120, 122, 210, 211, 212, and 213; and (i) wherein the substitutions and the positions are determined with reference to amino acids 114-220 of SEQ ID NO:1 and (ii) the CH3 domain has at least 80% identity to amino acids 114-220 of any one of SEQ ID NOS:30-46.
2. The polypeptide of claim 1 , wherein the CH3 domain comprises Gly at position 210; Phe at position 211; and/or Asp at position 213.
3. The polypeptide of claim 1 , wherein the CH3 domain comprises at least one position selected from the following: position 118 is Phe or Ile; position 119 is Asp, Glu, Gly, Ala, or Lys; position 120 is Tyr, Met, Leu, Ile, or Asp; position 122 is Thr or Ala; position 210 is Gly; position 211 is Phe; position 212 is His, Tyr, Ser, or Phe; and position 213 is Asp.
4. The polypeptide of claim 1 , wherein the CH3 domain comprises Gly at position 210 and Phe at position 211.
5. The polypeptide of claim 1 , wherein the CH3 domain comprises Gly at position 210 and Asp at position 213.
6. The polypeptide of claim 1 , wherein the CH3 domain comprises Phe at position 211 and Asp at position 213.
7. The polypeptide of claim 1 , wherein the CH3 domain comprises Gly at position 210; Phe at position 211; and Asp at position 213.
8. The polypeptide of claim 1 , wherein the CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from the following: position 118 is Phe or Ile; position 119 is Asp, Glu, Gly, Ala, or Lys; position 120 is Tyr, Met, Leu, Ile, or Asp; position 122 is Thr or Ala; position 210 is Gly; position 211 is Phe; position 212 is His, Tyr, Ser, or Phe; and position 213 is Asp.
9. The polypeptide of claim 1 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of any one of SEQ ID NOS:30-46.
10. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:30 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:30.
11. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:31 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:31.
12. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:32 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:32.
13. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:33 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:33.
14. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:34 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:34.
15. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:35 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:35.
16. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:36 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:36.
17. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:37 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:37.
18. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:38 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:38.
19. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:39 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:39.
20. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:40 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:40.
21. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:41 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:41.
22. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:42 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:42.
23. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:43 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:43.
24. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:44 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:44.
25. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:45 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:45.
26. The polypeptide of claim 9 , wherein the CH3 domain comprises amino acids 118-122 and amino acids 210-213 of SEQ ID NO:46 and has at least 90% identity to amino acids 114-220 of SEQ ID NO:46.
27. The polypeptide of claim 1 , wherein the CH3 domain comprises amino acids 118-213 of any one of SEQ ID NOS:30-46.
28. The polypeptide of claim 1 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:30-46 without the first three amino acids “PCP” at the amino-terminal end.
29. The polypeptide of claim 28 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:30-46.
30. The polypeptide of claim 1 , wherein the CH3 domain further comprises (i) a Trp at position 139 or (ii) a Ser at position 139, an Ala at position 141, and a Val at position 180, wherein the amino acid positions are determined with reference to SEQ ID NO: 1.
31. The polypeptide of claim 1 , wherein the polypeptide is joined to a CH2 domain.
32. The polypeptide of claim 31 , wherein the CH2 domain contains one or both of the following sets of modifications with reference to the amino acid sequence of SEQ ID NO:1:
(a) Ala at position 7 and at position 8; and
(b) Tyr at position 25, Thr at position 27, and Glu at position 29.
33. The polypeptide of claim 32 , wherein set (a) further comprises Gly at position 102.
34. The polypeptide of claim 31 , wherein the polypeptide is further joined to a Fab.
35. A method for engineering a CH3 domain to specifically bind to a transferrin receptor, the method comprising:
(a) engineering a polynucleotide that encodes the CH3 domain to have at least five amino acid substitutions at a set of amino acid positions comprising 118, 119, 120, 122, 210, 211, 212, and 213,
wherein the substitutions and positions are determined with reference to amino acids 114-220 of SEQ ID NO:1;
(b) expressing a polypeptide comprising the engineered CH3 domain; and
(c) determining whether the engineered CH3 domain binds to the transferrin receptor.
36. The method of claim 35 , wherein the polypeptide comprising the engineered CH3 domain is expressed as a soluble protein.
37. The method of claim 35 , wherein the steps of expressing the polypeptide comprising the engineered CH3 domain and determining whether the engineered CH3 domain binds to the transferrin receptor are performed using a display system.
38. The method of claim 37 , wherein the display system is a cell surface display system, a viral display system, an mRNA display system, a polysomal display system, or a ribosomal display system.