Mirac proteins

摘要

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.

主权项

1. A method of preparing a conditionally active enzyme, the method comprising the steps of:
i. evolving DNA which encodes a wild-type mammalian enzyme using one or more evolutionary techniques to create mutant DNAs; ii. expressing the mutant DNAs to obtain at least one mutant enzyme which exhibits both: (a) a decrease in activity in an assay at a normal physiological condition that is within a normal range of the physiological condition at a site of administration of the conditionally active protein to a subject, or at a tissue or organ at a site of action of the conditionally active protein of a subject, when compared to the activity of the wild-type mammalian enzyme in the assay at the same normal physiological condition, and (b) an increase in activity in an assay under an aberrant condition that deviates from the normal range of the physiological condition at the site of administration of the conditionally active protein, or at the tissue or organ at the site of action of the conditionally active protein, when compared to the activity of the wild-type mammalian enzyme at the same aberrant condition; and iii. selecting the conditionally active enzyme from the at least one mutant enzyme which exhibits both: (a) the decrease in the activity in the assay at the normal physiological condition when compared to the activity of the wild-type mammalian enzyme in the assay at the same normal physiological condition, and (b) the increase in the activity in the assay under the aberrant condition when compared to the activity of the wild-type mammalian enzyme in the assay at the same aberrant condition; andwherein the normal physiological condition and aberrant condition are the same condition selected from the group consisting of temperature, pH, osmotic pressure, osmolality, oxidation and electrolyte concentration.