Methods of engineering transferrin receptor binding polypeptides

A method of generating one or more variant polypeptides that specifically bind transferrin receptor protein and transport across the endothelium of a cell, comprising:

• (a) identifying a contiguous surface exposed register of about 7 to 15 amino acids of a native polypeptide for mutation;
• (b) generating one or more polynucleotides comprising a nucleotide sequence that encodes for a library of sequence variants of the native polypeptide, wherein the nucleotide sequence encodes for one or more mutations in the identified register of the native polypeptide;
• (c) expressing the one or more polynucleotides to produce the library of sequence variants;
• (d) screening the library of sequence variants to identify one or more variant polypeptides that specifically bind transferrin receptor protein;
• and
• (e) isolating the variant polypeptides,
• wherein the identified register of the native polypeptide does not bind transferrin receptor protein;
• and the isolated variant polypeptides exhibit substantially improved cellular uptake compared to the native polypeptide.