Engineered transferrin receptor binding polypeptides

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.

What is claimed is: 
     
         1 . A polypeptide comprising a CH2 domain that specifically binds to a transferrin receptor, wherein the CH2 domain comprises:
 (a) five, six, seven, eight, or nine substitutions at a set of amino acid positions comprising 47, 49, 56, 58, 59, 60, 61, 62, and 63;   (b) five, six, seven, eight, nine, or ten substitutions at a set of amino acid positions comprising 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72;   (c) five, six, seven, eight, nine, or ten substitutions at a set of amino acid positions comprising 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73; or   (d) five, six, seven, eight, or nine substitutions at a set of amino acid positions comprising 45, 47, 49, 95, 97, 99, 102, 103, and 104,   and wherein the substitutions and the positions are determined with reference to amino acids 4-113 of SEQ ID NO:1.   
     
     
         2 . The polypeptide of  claim 1 , wherein, in part (a), the CH2 domain comprises Glu at position 60 and/or Trp at position 61. 
     
     
         3 . The polypeptide of  claim 1 , wherein, in part (a), the CH2 domain comprises at least one, two, three, four, five, six, seven, eight, or nine positions selected from the following: position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; position 49 is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; position 56 is Asp, Pro, Met, Leu, Ala, Asn, or Phe; position 58 is Arg, Ser, Ala, or Gly; position 59 is Tyr, Trp, Arg, or Val; position 60 is Glu; position 61 is Trp or Tyr; position 62 is Gln, Tyr, His, Ile, Phe, Val, or Asp; and position 63 is Leu, Trp, Arg, Asn, Tyr, or Val. 
     
     
         4 . The polypeptide of  claim 3 , wherein the CH2 domain comprises Glu, Gly, Gln, Ser, Ala, Asn, or Tyr at position 47; Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49; Asp, Pro, Met, Leu, Ala, or Asn at position 56; Arg, Ser, or Ala at position 58; Tyr, Trp, Arg, or Val at position 59; Glu at position 60; Trp at position 61; Gln, Tyr, His, Ile, Phe, or Val at position 62; and/or Leu, Trp, Arg, Asn, or Tyr at position 63. 
     
     
         5 . The polypeptide of  claim 3 , wherein the CH2 domain comprises Arg at position 58; Tyr or Trp at position 59; Glu at position 60; Trp at position 61; and/or Arg or Trp at position 63. 
     
     
         6 . The polypeptide of  claim 1 , wherein, in part (a), the CH2 domain has at least 80% identity to amino acids 4-113 of any one of SEQ ID NOS:47-62. 
     
     
         7 . The polypeptide of  claim 1 , wherein, in part (a), the CH2 domain comprises amino acids 47-49 and amino acids 56-63 of any one of SEQ ID NOS:47-62. 
     
     
         8 . The polypeptide of  claim 7 , wherein the CH2 domain comprises amino acids 47-49 and amino acids 56-63 and has at least 90% identity to amino acids 4-113 of any one of SEQ ID NOS:47-62. 
     
     
         9 . The polypeptide of  claim 1 , wherein, in part (a), the CH2 domain comprises amino acids 47-63 of any one of SEQ ID NOS:47-62. 
     
     
         10 . The polypeptide of  claim 1 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:47-62 without the first three amino acids “PCP” at the amino-terminal end. 
     
     
         11 . The polypeptide of  claim 10 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:47-62. 
     
     
         12 . The polypeptide of  claim 1 , wherein, in part (b), the CH2 domain comprises Pro at position 43, Glu at position 68, and/or Tyr at position 70. 
     
     
         13 . The polypeptide of  claim 1 , wherein, in part (b), the CH2 domain comprises at least one, two, three, four, five, six, seven, eight, nine, or ten positions selected from the following: position 39 is Pro, Phe, Ala, Met, or Asp; position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu; position 42 is Pro, Val, Ala, Thr, or Asp; position 43 is Pro, Val, or Phe; position 44 is Trp, Gln, Thr, or Glu; position 68 is Glu, Val, Thr, Leu, or Trp; position 70 is Tyr, His, Val, or Asp; position 71 is Thr, His, Gln, Arg, Asn, or Val; and position 72 is Tyr, Asn, Asp, Ser, or Pro. 
     
     
         14 . The polypeptide of  claim 13 , wherein the CH2 domain comprises:
 (i) Pro, Phe, or Ala at position 39; Gln, Pro, Arg, Lys, Ala, or Ile at position 40; Thr, Ser, Gly, Met, Val, Phe, or Trp at position 41; Pro, Val, or Ala at position 42; Pro at position 43; Trp or Gln at position 44; Glu at position 68; Tyr at position 70; Thr, His, or Gln at position 71; and/or Tyr, Asn, Asp, or Ser at position 72;   (ii) Met at position 39; Leu or Glu at position 40; Trp at position 41; Pro at position 42; Val at position 43; Thr at position 44; Val or Thr at position 68; His at position 70; His, Arg, or Asn at position 71; and/or Pro at position 72; or   (iii) Asp at position 39; Asp at position 40; Leu at position 41; Thr at position 42; Phe at position 43; Gln at position 44; Val or Leu at position 68; Val at position 70; Thr at position 71; and/or Pro at position 72.   
     
     
         15 . The polypeptide of  claim 1 , wherein, in part (b), the CH2 domain has at least 80% identity to amino acids 4-113 of any one of SEQ ID NOS:63-85. 
     
     
         16 . The polypeptide of  claim 1 , wherein, in part (b), the CH2 domain comprises amino acids 39-44 and amino acids 68-72 of any one of SEQ ID NOS:63-85. 
     
     
         17 . The polypeptide of  claim 16 , wherein the CH2 domain comprises amino acids 39-44 and amino acids 68-72 and has at least 90% identity to amino acids 4-113 of any one of SEQ ID NOS:63-85. 
     
     
         18 . The polypeptide of  claim 1 , wherein, in part (b), the CH2 domain comprises amino acids 39-72 of any one of SEQ ID NOS:63-85. 
     
     
         19 . The polypeptide of  claim 1 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:63-85 without the first three amino acids “PCP” at the amino-terminal end. 
     
     
         20 . The polypeptide of  claim 19 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:63-85. 
     
     
         21 . The polypeptide of  claim 1 , wherein, in part (c), the CH2 domain comprises at least one, two, three, four, five, six, seven, eight, nine, or ten positions selected from the following: position 41 is Val or Asp; position 42 is Pro, Met, or Asp; position 43 is Pro or Trp; position 44 is Arg, Trp, Glu, or Thr; position 45 is Met, Tyr, or Trp; position 65 is Leu or Trp; position 66 is Thr, Val, Ile, or Lys; position 67 is Ser, Lys, Ala, or Leu; position 69 is His, Leu, or Pro; and position 73 is Val or Trp. 
     
     
         22 . The polypeptide of  claim 21 , wherein the CH2 domain comprises:
 (i) Val at position 41; Pro at position 42; Pro at position 43; Arg or Trp at position 44; Met at position 45; Leu at position 65; Thr at position 66; Ser at position 67; His at position 69; and/or Val at position 73; or   (ii) Asp at position 41; Met or Asp at position 42; Trp at position 43; Glu or Thr at position 44; Tyr or Trp at position 45; Trp at position 65; Val, Ile, or Lys at position 66; Lys, Ala, or Leu at position 67; Leu or Pro at position 69; and/or Trp at position 73.   
     
     
         23 . The polypeptide of  claim 1 , wherein, in part (c), the CH2 domain has at least 80% identity to amino acids 4-113 of any one of SEQ ID NOS:86-90. 
     
     
         24 . The polypeptide of  claim 1 , wherein, in part (c), the CH2 domain comprises amino acids 41-45 and amino acids 65-73 of any one of SEQ ID NOS:86-90. 
     
     
         25 . The polypeptide of  claim 24 , wherein the CH2 domain comprises amino acids 41-45 and amino acids 65-73 and has at least 90% identity to amino acids 4-113 of any one of SEQ ID NOS:86-90. 
     
     
         26 . The polypeptide of  claim 1 , wherein, in part (c), the CH2 domain comprises amino acids 41-73 of any one of SEQ ID NOS:86-90. 
     
     
         27 . The polypeptide of  claim 1 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:86-90 without the first three amino acids “PCP” at the amino-terminal end. 
     
     
         28 . The polypeptide of  claim 27 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:86-90. 
     
     
         29 . The polypeptide of  claim 1 , wherein, in part (d), the CH2 domain comprises Trp at position 103. 
     
     
         30 . The polypeptide of  claim 1 , wherein, in part (d), the CH2 domain comprises at least one, two, three, four, five, six, seven, eight, or nine positions selected from the following: position 45 is Trp, Val, Ile, or Ala; position 47 is Trp or Gly; position 49 is Tyr, Arg, or Glu; position 95 is Ser, Arg, or Gln; position 97 is Val, Ser, or Phe; position 99 is Ile, Ser, or Trp; position 102 is Trp, Thr, Ser, Arg, or Asp; position 103 is Trp; and position 104 is Ser, Lys, Arg, or Val. 
     
     
         31 . The polypeptide of  claim 30 , wherein the CH2 domain comprises Val or Ile at position 45; Gly at position 47; Arg at position 49; Arg at position 95; Ser at position 97; Ser at position 99; Thr, Ser, or Arg at position 102; Trp at position 103; and/or Lys or Arg at position 104. 
     
     
         32 . The polypeptide of  claim 1 , wherein, in part (d), the CH2 domain has at least 80% identity to amino acids 4-113 of any one of SEQ ID NOS:91-95. 
     
     
         33 . The polypeptide of  claim 1 , wherein, in part (d), the CH2 domain comprises amino acids 45-49 and amino acids 95-104 of any one of SEQ ID NOS:91-95. 
     
     
         34 . The polypeptide of  claim 33 , wherein the CH2 domain comprises amino acids 45-49 and amino acids 95-104 and has at least 90% identity to amino acids 4-113 of any one of SEQ ID NOS:91-95. 
     
     
         35 . The polypeptide of  claim 1 , wherein, in part (d), the CH2 domain comprises amino acids 45-104 of any one of SEQ ID NOS:91-95. 
     
     
         36 . The polypeptide of  claim 1 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:91-95 without the first three amino acids “PCP” at the amino-terminal end. 
     
     
         37 . The polypeptide of  claim 36 , wherein the polypeptide comprises the sequence of any one of SEQ ID NOS:91-95. 
     
     
         38 . The polypeptide of  claim 1 , wherein the CH2 domain further comprises one or both of the following sets of modifications with reference to the amino acid sequence of SEQ ID NO: 1:
 (i) Ala at position 7 and at position 8; and   (ii) Tyr at position 25, Thr at position 27, and Glu at position 29.   
     
     
         39 . The polypeptide of  claim 38 , wherein set (i) further comprises Gly at position 102. 
     
     
         40 . The polypeptide of  claim 1 , wherein the polypeptide is joined to a CH3 domain. 
     
     
         41 . The polypeptide of  claim 40 , wherein the CH3 domain 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. 
     
     
         42 . The polypeptide of  claim 40 , wherein the polypeptide is further joined to a Fab. 
     
     
         43 . A method for engineering a CH2 domain to specifically bind to a transferrin receptor, the method comprising:
 (a) engineering a polynucleotide that encodes the CH2 domain to have at least five amino acid substitutions at a set of amino acid positions comprising:
 (i) 47, 49, 56, 58, 59, 60, 61, 62, and 63; 
 (ii) 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72; 
 (iii) 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73; or 
 (iv) 45, 47, 49, 95, 97, 99, 102, 103, and 104; 
   wherein the substitutions and the positions are determined with reference to amino acids 4-113 of SEQ ID NO:1;   (b) expressing a polypeptide comprising the engineered CH2 domain; and   (c) determining whether the engineered CH2 domain binds to the transferrin receptor.   
     
     
         44 . The method of  claim 43 , wherein the steps of expressing the polypeptide comprising the engineered CH2 domain and determining whether the engineered CH2 domain binds to the transferrin receptor are performed using a display system. 
     
     
         45 . The method of  claim 44 , 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. 
     
     
         46 . The method of  claim 43 , wherein the polypeptide comprising the engineered CH2 domain is expressed as a soluble protein.