Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts

a technology of conditionally active and eukaryotic cells, applied in the field of protein evolution and activity, to achieve the effect of increasing reducing the activity of the assay

Inactive Publication Date: 2020-07-09
BIOATLA LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach generates proteins, referred to as Mirac proteins, that are effectively inactivated under normal conditions but active at specific conditions, offering therapeutic benefits by limiting activity duration and avoiding adverse effects, suitable for treatments like thrombolytic therapy and autoimmune disorders.

Problems solved by technology

However, engineering or evolving a protein to be inactive or virtually inactive (less than 10% activity and especially 1% activity) at its wild type operating condition, while maintaining activity equivalent or better than its wild type condition at new conditions, requires that the destabilizing mutation(s) co-exist with activity increasing mutations that do not counter the destabilizing effect.

Method used

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  • Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts
  • Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts
  • Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts

Examples

Experimental program
Comparison scheme
Effect test

example 1

escription of a Multiwall Assay (for Example, 96-Well Assay) for Temperature Mutants

[0527]Fluorescent substrate is added to each well of a multiwall plate, at both wild-type and new, lower reaction temperatures (for example, either 37° C. or 25° C. as mentioned above) for an appropriate time period. Fluorescence is detected by measuring fluorescence in a fluorescent plate reader at appropriate excitation and emission spectra (for example, 320 nm exitation / 405 nm emission). Relative fluorescence units (RFU) are determined. Supernatant from wild type molecule and plasmid / vector transformed cells are used as positive and negative controls. Duplicate reactions are performed for each sample, reaction temperature, and positive and negative control.

[0528]Mutants that are active at the lower temperature (for example, the mutants active at 25° C.) and that have a decrease in activity at the wild type temperature (for example, a 10%, 20%, 30%, 40% or more decrease in activity at 37° C.), thus...

example 2

escription of a Different Assay Format for Confirmation of Activity (for Example, a 14-mL Assay) for Temperature Mutants

[0529]Mutants that are identified as temperature sensitive primary hits are expressed in 14 ml culture tubes and their enzymatic activity is measured at wild type (for example, 37° C.) and the lower temperature (for example, 25° C.). Protein is expressed and purified as described above for the multiwall format, with the exception that the expression is performed in different format (14 ml tubes) rather than the multiwall (96-well plate) format.

[0530]Each mutant supernatant is transferred to a multiwall plate, for example a 96-well microplate. Fluorescent substrate is added to each tube at the indicated reaction temperatures (wild-type, lower temperature) for a required period of time. Wild-type molecules are used as a positive control and supernatant from cells transformed with only vector is used as a negative control. Fluorescence is detected by measuring fluores...

example 3

escription of Further Evolution of Hits Discovered

[0533]If desired, a new, combinatorial variant library is generated from all or selected mutant hits previously identified. The new library can be designed to contain every possible combination of amino acid variants for each of the selected mutants, and rescreened as described for new hits.

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Abstract

A method of preparing a conditionally active biologic protein by selecting a wild-type biologic protein, evolving the DNA which encodes the wild-type biologic protein using one or more evolutionary techniques to create mutant DNAs, expressing the mutant DNAs in a eukaryotic cell production host to obtain a mutant protein, subjecting the mutant protein and the wild-type protein to an assay under a normal physiological condition and to an assay under an aberrant condition, selecting a conditionally active mutant protein which exhibits at least one of: (a) a decrease in activity in the assay at the normal physiological condition compared to the wild-type protein, and (b) an increase in activity in the assay under the aberrant condition compared to the wild-type protein; and producing the conditionally active biologic protein in the same eukaryotic cell production host used in the expression step.

Description

RELATED APPLICATION DATA[0001]This application is a continuation of U.S. patent application Ser. No. 15 / 329,491, filed on Aug. 29, 2017, which, in turn is a 371 continuation of International Application No. PCT / US15 / 48258, filed Sep. 3, 2015, which, in turn, claims the benefit of U.S. Provisional Application No. 62 / 045,207, filed Sep. 3, 2014, the entire disclosures of which are hereby incorporated by reference as if set forth fully herein.FIELD OF THE DISCLOSURE[0002]This disclosure relates to the fields of protein evolution and activity. Specifically, this disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which generated proteins are reversibly or irreversibly virtually inactivated at normal physiological conditions typically encountered by the wild-type protein but active at other conditions. For example, evolved proteins are virtually inactive at body temperature, but are active at low...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12N15/10C07K16/00C07K14/435C12P21/00C12N15/80C07K16/46
CPCC07K14/435C07K16/46C07K2317/94C07K2317/52C07K2317/14C07K2317/31C07K2317/92C07K16/00C12P21/00C07K2317/622C12N15/80C12N15/1058C12N5/0638C12N15/85
InventorSHORT, JAY M.
OwnerBIOATLA LLC