Conditionally active biological proteins
Inventors
Short, Jay M. • Chang, Hwai Wen • Frey, Gerhard
Assignees
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Publication Number
US-12110611-B2
Publication Date
2024-10-08
Expiration Date
Abstract
Methods of generating conditionally active biologic proteins, in particular therapeutic or diagnostic proteins, which are more active at an aberrant condition than at a normal physiological condition. The methods include discovery methods using libraries of proteins and assays employing physiological concentrations of components of bodily fluids. The conditionally active biologic proteins may be further evolved, conjugated to other molecules, masked, reduced in activity by attaching a cleavable moiety. Criteria for selecting starting proteins for the discovery methods, as well as formats of the proteins are also disclosed.
Core Innovation
The disclosed approach prepares conditionally active antibodies or antibody fragments whose activity is inhibited in an assay at a normal physiological pH compared to the same activity in an assay at an aberrant pH. The described concept is pH-dependent conditional activity, where the decrease in activity is defined by performing parallel assays at a normal physiological pH and an aberrant pH.
The method selects a template antibody or antibody fragment from a wild-type antibody or antibody fragment, a mutated antibody or antibody fragment, and a fragment of a wild-type antibody or antibody fragment with a desired property. A DNA encoding the template is evolved using one or more evolutionary techniques to create mutant DNAs, the mutant DNAs are expressed to obtain mutant antibodies or antibody fragments, and mutants are identified by screening for decreased activity at the normal physiological pH compared to the aberrant pH.
Assays used for screening are performed in assay solutions containing at least one component at substantially the same concentration across the normal physiological pH assay and the aberrant pH assay. The partial disclosure specifies hydrogen sulfide and bicarbonate as example components in these solutions, supporting identification of conditionally active antibodies or antibody fragments.
Claims Coverage
The independent claim covers a complete workflow: selecting templates, evolving and expressing encoding DNA to create mutant antibodies or fragments, and screening mutants by comparing activity at a normal physiological pH versus an aberrant pH in assay solutions that contain specified components at substantially the same concentration. The inventive features center on pH-dependent conditional activity and assay-solution composition constraints.
PHD-dependent conditional activity screening workflow for antibodies
Preparing conditionally active antibodies or antibody fragments by evolving DNA encoding selected template antibodies or fragments to create mutant DNAs, expressing mutant DNAs to obtain mutant antibodies or fragments, and screening each mutant by assaying activity at a normal physiological pH and an aberrant pH to identify mutants exhibiting decreased activity at the normal physiological pH compared to the same activity at the aberrant pH.
Substantially matched assay-solution component concentrations for normal vs aberrant pH assays
Performing the assay under the normal physiological pH and the assay under the aberrant pH in assay solutions containing at least one component at substantially the same concentration, including assay-solution components selected from hydrogen sulfide and bicarbonate.
Across the claim scope, the core coverage is the combined evolution, expressing, and screening method that identifies antibodies or fragments with pH-dependent conditional activity, with the comparison assays configured so at least one component is present at substantially the same concentration in both normal and aberrant pH assay solutions.
Stated Advantages
Provides conditionally active antibodies or antibody fragments that exhibit a decrease in activity at a normal physiological pH compared to the same activity at an aberrant pH.
Documented Applications
No documented applications found
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