Look-through mutagenesis for developing altered polypeptides with enhanced properties
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Publication Number
US-9012369-B2
Publication Date
2015-04-21
Expiration Date
Abstract
A method of mutagenesis by which a predetermined amino acid is introduced into each and every position of a selected set of positions in a preselected region (or several different regions) of a polypeptide to produce a library of polypeptide analogs is disclosed. The method is based on the premise that certain amino acids play a crucial role in the structure and function of proteins and thus is capable of identifying and distinguishing functional amino acid residues (“hot spots”) from non-functional amino acids residues (“cold spots”) within a polypeptide or portion thereof. Libraries can be generated which contain only desired polypeptide analogs and are of reasonable size for screening. The libraries can be used to study the role of specific amino acids in polypeptide structure and function and to develop new or improved polypeptides such as antibodies, antibody fragments, single chain antibodies, enzymes, and ligands.
Core Innovation
The invention addresses look-through mutagenesis (LTM) in which a predetermined amino acid is systematically introduced into each position of a defined polypeptide region to create libraries of single-substitution-per-analog variants. The method then distinguishes functional amino acid residues from non-functional amino acid residues by screening polypeptide analogs for an alteration in a measurable property. Functional residues contributing to the measurable property are identified and distinguished from non-functional residues that do not contribute to the measurable property.
An improved LTM2 workflow separates functional hot spots from non-functional cold spots before restricting further mutagenesis. This workflow eliminates undesired library noise by restricting which residues are carried forward for mutagenesis based on the screening results. The approach is positioned relative to restrictive site-directed mutagenesis and inclusive saturation, random, and WTM mutagenesis, emphasizing the distinction between functional and non-functional residues to guide exclusive mutagenization.
The framework includes generating an expression library using polynucleotides that encode the polypeptide or portion thereof with defined codon criteria for the selected amino acids, expressing the library to produce polypeptide analogs, and screening the analogs for alterations in a measurable property. The disclosed workflow supports creating combinatorial beneficial mutation libraries by combining beneficial substitutions identified through the LTM2 distinction process. The document further describes antibody-focused embodiments involving CDRs and antibody fragments, as well as catalytic-enzyme and catalytic antibody embodiments.
Claims Coverage
The explicitly provided claim content includes one independent claim directed to a workflow that selects residue(s), generates a variant polynucleotide expression library under defined codon rules, expresses to produce polypeptide analogs, and screens for alterations in a measurable property to distinguish functional from non-functional residues. The claim requires that functional residues are determined suitable for mutagenesis and non-functional residues are determined unsuitable, with one or more functional residues exclusively mutagenized. Dependent claims refine the polypeptide type, defined region, predetermined amino acid set, mutagenesis approach, measurable properties, and expression-display formats.
Distinguishing functional from non-functional amino acid residues by exclusive mutagenesis
Selecting amino acid residue(s) within a polypeptide; determining an amino acid residue to be substituted for the selected residue(s); synthesizing polynucleotides collectively representing possible variant amino acid substitutions under codon criteria for a defined region; generating an expression library; expressing the library to produce polypeptide analogs; screening analogs for alteration in a measurable property to identify functional amino acid residue(s) contributing to the measurable property and distinguishing them from non-functional amino acid residue(s); determining functional residue(s) suitable for mutagenesis and non-functional residue(s) unsuitable; and exclusively mutagenizing the functional amino acid residue(s).
Codon-limited expression library representing single predetermined amino acid substitutions
Synthesizing polynucleotides encoding the polypeptide or portion thereof wherein each polynucleotide contains at each codon position either a codon required for the amino acid residue of the polypeptide or a codon for one of the predetermined amino acid residues, and wherein each polynucleotide contains no more than one codon for the predetermined amino acid residue, thereby generating an expression library containing the polynucleotides.
Selection and screening based on alteration in a measurable property
Screening the polypeptide analogs for an alteration in a measurable property such that one or more functional amino acid residues within the polypeptide, or portion thereof, is identified as contributing to the measurable property and therefore distinguished from a non-functional amino acid residue.
Mutagenized hot spots after distinguishing functional residues from cold spots
Identifying one or more functional amino acid residues as contributing to the measurable property and determining those functional residues are suitable for mutagenesis while non-functional residues are determined unsuitable, with one or more functional amino acid residues exclusively mutagenized.
Antibody CDR-defined region for functional residue distinction
Performing the method where the defined region includes a CDR or part of a CDR selected from CDR1–CDR6 or a combination thereof.
Predetermined amino acid set for substitutions
Performing the method wherein the predetermined amino acid residue is selected from a specified set of amino acids.
Mutagenesis approach selected from LTM and WTM
Carrying out the method using mutagenesis selected from look-through mutagenesis (LTM), walk-through mutagenesis (WTM), or a combination of both.
Measurable property selected from protein or enzymatic property types
Where the measurable property is one of binding specificity, binding avidity, binding affinity, Fc receptor binding, glycosylation, complement binding, half-life stability, solubility, thermal stability, catalytic activity, or enzymatic activity.
Expression-display format for linking polynucleotide to polypeptide analog
Linking a polynucleotide to a polypeptide analog using an expression-display method selected from ribosome display, polysome display, phage display, prokaryotic display, yeast display, eukaryotic cell display, or arrayed library display.
The core claim centers on distinguishing functional amino acid residues from non-functional amino acid residues by generating a codon-limited expression library of polypeptide analogs, screening for alterations in a measurable property, and then exclusively mutating only the functional residues. Dependent refinements include defining the region in antibody CDRs, constraining the predetermined amino acid set, selecting mutagenesis approaches, specifying property types for screening, and selecting expression-display technologies to link polynucleotides to polypeptide analogs.
Stated Advantages
Distinguishes functional amino acid residues from non-functional amino acid residues so that functional residues are suitable for mutagenesis while non-functional residues are unsuitable.
Restricts further mutagenesis to functional hot spots, eliminating undesired library noise and improving signal-to-noise in the library.
Documented Applications
Antibody-focused embodiments using antibody CDRs and antibody fragments, including Fv and scFv, for identifying residues that contribute to measurable properties associated with binding or kinetics.
Catalytic-enzyme and catalytic antibody embodiments involving screening for catalytic activity or enzymatic activity to distinguish functional from non-functional residues.
Combinatorial beneficial mutation library design that combines beneficial substitutions identified through the LTM2 distinction process.
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