Methods of engineering transferrin receptor binding polypeptides
Inventors
Chen, Xiaocheng • Dennis, Mark S. • Kariolis, Mihalis • Silverman, Adam P. • SRIVASTAVA, Ankita • Watts, Ryan J. • Wells, Robert C. • Zuchero, Joy Yu
Assignees
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Publication Number
US-12162948-B2
Publication Date
2024-12-10
Expiration Date
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.
Core Innovation
The invention relates to generating variant polypeptides that specifically bind transferrin receptor protein and transport across the endothelium of a cell. A native polypeptide is used as the starting point, where a contiguous surface exposed register of about 7 to 15 amino acids is identified for mutation and does not bind transferrin receptor protein in the native polypeptide.
The invention generates one or more polynucleotides encoding a library of sequence variants, with mutations in the identified register, and expresses the library to produce sequence variants. The library is screened to identify variant polypeptides that specifically bind transferrin receptor protein, and the variant polypeptides are isolated. The isolated variant polypeptides exhibit substantially improved cellular uptake compared to the native polypeptide.
The provided description includes Fc-engineered transferrin receptor-binding polypeptides in which Fc CH3 or CH2 domains are modified by substitutions at defined surface-exposed amino-acid sets, including CH2-CH3 Fc fusion formats, dimer/monovalent configurations, and optional Fab fusion to therapeutic targets. It further includes screening and selection contexts involving TfR in the presence of transferrin, binding affinity constraints, and example mutated-register sequence motifs such as YGTEW (SEQ ID NO:115), YxTEWSS (SEQ ID NO:636), and TxxExxxxF.
Claims Coverage
The independent claim coverage centers on one multistep method directed to mutating a contiguous surface exposed register that does not bind transferrin receptor protein, generating and expressing a variant library, screening for transferrin receptor binding, and isolating variants with substantially improved cellular uptake. Dependent claims add position-specific mutagenesis, fragment shuffling, screening in the presence of transferrin, an affinity threshold, and example register motifs.
Mutate a contiguous surface exposed register not binding transferrin receptor
Identifying a contiguous surface exposed register of about 7 to 15 amino acids of a native polypeptide for mutation, wherein the identified register of the native polypeptide does not bind transferrin receptor protein.
Generate and express a polynucleotide-encoded library of sequence variants with register mutations
Generating one or more polynucleotides comprising a nucleotide sequence that encodes a library of sequence variants of the native polypeptide, wherein the nucleotide sequence encodes one or more mutations in the identified register; expressing the one or more polynucleotides to produce the library of sequence variants.
Screen for transferrin receptor binding and isolate binding variant polypeptides
Screening the library of sequence variants to identify one or more variant polypeptides that specifically bind transferrin receptor protein; isolating the variant polypeptides.
Substantially improved cellular uptake of isolated variants
The isolated variant polypeptides exhibit substantially improved cellular uptake compared to the native polypeptide.
Select and mutagenize a position within the register and repeat
Further identifying a position within a register of isolated variant polypeptides for mutation; generating polynucleotides encoding a sequence-variant library that includes an NNK mutation at that position; repeating the screening and isolation steps.
Fragment shuffling during PCR to build the variant library
Generating first and second polynucleotide fragments carrying specified mutations in separate halves of a register; shuffling these fragments during PCR amplification to create a library of sequence variants for expression.
Iterative selection by binding affinity threshold
Repeating selection until isolated variant polypeptides bind the transferrin receptor protein with an affinity of 500 nM or higher.
Screening in the presence of transferrin
Contacting a library of sequence variants with a transferrin receptor protein in the presence of transferrin.
Use mutated-register sequence motifs
Variant polypeptides whose mutated register contains a sequence selected from YGTEW (SEQ ID NO:115), YxTEWSS (SEQ ID NO:636), or TxxExxxxF.
Across the claims, the inventive coverage centers on mutating a contiguous surface exposed register that does not bind transferrin receptor protein, building and expressing a library encoding register mutations, screening for transferrin receptor binding, and isolating variants showing substantially improved cellular uptake. Dependent claims further define specific mutagenesis and library construction approaches, screening conditions involving transferrin, an affinity-based repetition criterion, and example mutated-register sequence motifs.
Stated Advantages
Substantially improved cellular uptake compared to the native polypeptide.
Specifically bind transferrin receptor protein.
Transport across the endothelium of a cell.
Documented Applications
Transport across the endothelium of a cell by variant polypeptides that specifically bind transferrin receptor protein.
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