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Publication Number
US-11718844-B2
Publication Date
2023-08-08
Expiration Date
Abstract
This disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which are reversibly or irreversibly inactivated at the wild type normal physiological conditions. For example, evolved proteins are virtually inactive at body temperature, but are active at lower temperatures.
Core Innovation
The invention relates to Mirac proteins, which are conditionally active biologic proteins engineered from wild-type proteins. The engineered proteins are created by evolving encoding DNA into mutant DNAs, expressing the mutant DNAs to obtain mutant proteins, and screening to identify variants with condition-dependent activity under a normal physiological condition and an aberrant physiological condition that deviates from a normal range at a site of action.
A selected Mirac protein exhibits decreased activity under the normal physiological condition and increased activity under the aberrant condition. For reversibility, activity under the aberrant condition is assessed again by comparison to the activity under the corresponding normal physiological condition, including optional reversibility by returning to the normal condition. The normal physiological condition and the aberrant condition may be selected from temperature, pH, osmotic pressure, oxidative stress, osmolality, and electrolyte concentration.
The described approach is applied to therapeutic proteins including enzymes and antibodies, including thrombolytic therapy proteins and antibodies/biologic response modifiers directed to inflammatory response mediators. The document further includes pharmaceutical composition and kit/pharmaceutical formulations using pharmaceutically acceptable carriers, and provides extensive definitions and implementation details for evolution, mutagenesis, expression, screening assays, and antibody-based detection, including example assay workflows and screening methodology for a selected variant.
Claims Coverage
The provided excerpt contains one independent claim. The claim focuses on preparing a conditionally active therapeutic antibody using evolved mutant DNAs, expressing mutant antibodies, screening under paired normal and aberrant physiological conditions, and selecting antibodies with increased binding at the aberrant condition, decreased binding at the normal condition versus a parent antibody, and reversibly activated activity under the aberrant condition.
Evolution of mutant antibody DNA for antigen binding
Evolving a DNA which encodes a parent antibody that binds the antigen using one or more evolutionary techniques to create mutant DNAs.
Expression of mutant DNAs to obtain conditionally active antibodies
Expressing the mutant DNAs to obtain mutant antibodies including at least one said conditionally active therapeutic antibody.
Paired screening under normal and aberrant physiological conditions
Subjecting the mutant antibodies to a screening assay under a value of a normal physiological condition within a normal range at a site of administration and a screening assay under a value of an aberrant condition deviating from a value within a normal range at a normal tissue or normal organ at the site of action.
Reversibility screening via second assay under normal physiological condition
Subjecting mutant antibodies determined to be active under the value of the aberrant condition to a second screening assay under the value of the normal physiological condition to determine if the activity under the value of the aberrant condition is reversible.
Selection of conditionally active antibody with increased aberrant binding and decreased normal binding
Selecting from the mutant antibodies the conditionally active therapeutic antibody that exhibits increased binding activity to the antigen at the aberrant condition compared to the binding activity at the normal physiological condition; decreased binding activity under the normal physiological condition compared to binding activity of the parent antibody; and reversibly activated at the aberrant condition.
Using the same selected condition pair for normal and aberrant activity modulation
Defining the normal physiological condition and the aberrant condition as a same condition selected from temperature, pH, osmotic pressure, oxidative stress, osmolality, and electrolyte concentration.
The inventive core is the combination of evolutionary creation of mutant antibody DNAs, expression to produce mutant antibodies, screening for activity or binding using paired normal versus aberrant physiological conditions, and selecting antibodies that show increased binding under aberrant conditions, decreased binding under normal conditions versus the parent antibody, and reversibility of the aberrant-condition activation.
Stated Advantages
Conditionally active therapeutic antibodies exhibit increased binding activity to the antigen under the aberrant physiological condition compared to the normal physiological condition.
Selected antibodies show decreased binding activity under the normal physiological condition compared to the parent antibody.
Selected antibodies have reversibly activated activity at the aberrant condition.
Documented Applications
Therapeutic proteins, including conditionally active therapeutic antibodies used in contexts where a normal physiological condition differs from an aberrant physiological condition at the site of action.
Thrombolytic therapy contexts described in the document, including thrombolytic proteins such as streptokinase, urokinase, and tPA variants activated under aberrant conditions.
Cardiovascular and shock-related disease contexts described in the document, including stroke, heart attack, hypovolemic shock, and Reynaud’s phenomenon.
Cancer and angiogenesis-related contexts described in the document, including angiostatin variants activated at low pH.
Autoimmune/inflammatory mediator contexts described in the document using conditionally active antibodies/biologic response modifiers directed to IL-6/IL-6R, TNF-alpha, IL-23, and IL-12.
Tissue permeability-related context described in the document using hyaluronidase variants to improve tissue permeability while limiting off-target activity.
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