Methods for characterizing and engineering protein-protein interactions
Inventors
YOUNGER, David • Colby, David • Lopez, Randolph • Wittekind, Michael
Assignees
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Publication Number
US-11474111-B2
Publication Date
2022-10-18
Expiration Date
Abstract
Characterization of the binding dynamics at the interface between any two proteins that specifically interact plays a role in myriad biomedical applications. The methods disclosed herein provide for the high-throughput characterization of the specific interaction at the interface between two protein binding partners and the identification of functionally significant mutations of one or both protein binding partners. For example, the methods disclosed herein may be useful for epitope and paratope mapping of an antibody-antigen pair, which is useful for the discovery and development of novel therapies, vaccines, diagnostics, among other biomedical applications.
Core Innovation
The invention provides a method for identifying compensatory mutations between two mutant protein binding partners. A first library of first protein binding partners is expressed on a surface of a first plurality of haploid yeast cells, and the first library includes a first wild-type polypeptide and a first plurality of mutant polypeptides of the first wild-type polypeptide. A second library of second protein binding partners is expressed on a surface of a second plurality of haploid yeast cells, and the second library includes a second wild-type polypeptide and a second plurality of mutant polypeptides of the second wild-type polypeptide.
The method cultures the first and second populations of haploid yeast cells such that diploid yeast cells are produced if the first and second protein binding partners interact. For each protein binding partner of the first library and each protein binding partner of the second library, it measures an observed affinity value. It then identifies compensatory mutations based on the observed affinity value, selecting one or more pairs of the first and second protein binding partners that have an observed affinity value substantially different from the observed affinity value between the first wild-type polypeptide and the second wild-type polypeptide.
The approach is directed to comparing affinity relationships across mutant and wild-type polypeptides, and it enables identification of mutant pairs whose binding differs substantially from the wild-type pair. In supported examples, it is applied to mutational libraries and compensatory mutation pairs, including correlation of compensatory residues with interface proximity using a crystal structure. The technique is also described for epitope/paratope mapping and for inferring structure or proximity based on binding behavior.
Claims Coverage
The document describes one independent method claim and several dependent claims. The core claim includes a framework for measuring observed affinity values across two mutant protein binding partner libraries on haploid yeast, culturing to generate diploid yeast when binding occurs, and identifying compensatory mutation pairs whose affinity is substantially different from the wild-type pair affinity, using observed affinity value comparisons between mutant–wild-type and mutant–mutant polypeptide combinations.
Identifying compensatory mutation pairs via observed affinity comparison
The method identifies compensatory mutations between two mutant protein binding partners by providing first and second mutant-containing libraries expressed on haploid yeast surfaces, culturing to produce diploid yeast cells when the binding partners interact, measuring an observed affinity value for each protein binding partner combination, and identifying one or more pairs having an observed affinity value substantially different than the observed affinity value between the first wild-type polypeptide and the second wild-type polypeptide.
User-directed mutagenesis produces mutant polypeptides
The first plurality of mutant polypeptides and the second plurality of mutant polypeptides are produced by user-directed mutagenesis.
Identifying three or more compensatory mutations
The method further identifies three or more compensatory mutations.
Selecting specific substantially different affinity patterns for compensatory pairs
The identifying is characterized by comparing observed affinity values between mutant polypeptides and wild-type polypeptides across the first plurality and the second plurality, selecting compensatory pairs with an observed affinity value substantially different than the observed affinity value between the first wild-type polypeptide and the second wild-type polypeptide.
Mapping antibody paratope species using a defined mutation
A mutation in one polypeptide from a first set of mutant polypeptides defines an antibody paratope for an scFv, Fab, or VHH species.
Receptor species in the first library
Every protein binding partner in the first library is a receptor species.
Across the independent claim and dependents, the inventive concept centers on generating binding-dependent diploid yeast from two haploid yeast-displayed mutant partner libraries, measuring observed affinity values for combinations, and identifying compensatory mutation pairs based on substantial differences relative to the wild-type pair affinity. Dependent claims further constrain mutant generation (user-directed mutagenesis), the number of compensatory mutations identified (three or more), the affinity comparison framework, and specific biological contexts such as antibody paratope mapping (scFv/Fab/VHH) and receptor species in the first library.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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