Non-radioactive cytotoxicity assays
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Publication Number
US-12196759-B2
Publication Date
2025-01-14
Expiration Date
Abstract
Described herein are methods for assessing cytotoxicity of an agent. The methods include providing a target cell that has been engineered to express intracellularly a reporter that is not expressed endogenously by the target cell, exposing the target cell to an agent capable of modulating cytotoxicity and assaying the activity of the reporter, wherein a change in reporter activity relative to a reference value is indicative of the agent being able to modulate cytotoxicity of the target cell.
Core Innovation
A Matador cytotoxicity assay is based on a target cell engineered to express an intracellular non-secretory thermostable reporter. The target cell is exposed to an agent capable of modulating cytotoxicity, and cytotoxicity is assessed by measuring the level of the reporter. In the disclosed embodiments, the reporter is a thermostable beetle luciferase.
The approach addresses reporter instability during cytotoxicity assay conditions, including stability under 37°C culture and cytotoxicity conditions for extended durations. The intracellular non-secretory thermostable reporter is resistant to protease and redox changes associated with cell death, supporting usable reporter signals during the assay. Reporter signal is measured relative to reference values and is described for detection of cytotoxicity at low cell numbers.
The broader Matador cytotoxicity assay framework describes single-reporter and multi-reporter target cell formats, luciferase substrate and light generation principles, and reporter delivery using different vector types and non-viral methods. Broad assay applications are described, including cytotoxicity assessment for CAR-T and NK contexts, ADCC, bispecific antibodies, ADCs, drug screening and potency QA, and patient-sample and in vivo monitoring.
Claims Coverage
The identified independent claim defines a cytotoxicity assessment method using a target cell engineered to express an intracellular non-secretory thermostable reporter, specifically a thermostable beetle luciferase, with measurement of reporter level to assess cytotoxicity modulation. The dependent claims add five further inventive features relating to thermostability, sequence identity, source species, assay location, and substrate combinations for light generation.
Assessing cytotoxicity using intracellular non-secretory thermostable reporter
Exposing a target cell engineered to express an intracellular non-secretory thermostable reporter to an agent capable of modulating cytotoxicity; measuring the level of the reporter; wherein the reporter is a thermostable beetle luciferase.
Thermostable reporter stability at assay temperature
The reporter remains stable during assay conditions at 37°C for more than 15 minutes.
Thermostable beetle luciferase sequence identity constraint
The beetle luciferase contains an amino acid sequence that is SEQ ID NO: 50-56 or is 85-99% identical to that sequence.
Beetle luciferase source species constraint
The beetle luciferase is obtained from Photinus pyralis, Photuris pennsylvanica, or Pyrophorus plagiothalamus.
Reporter assayed in cell media versus cell supernatant free of cells
Assaying a reporter in either cell media containing target cells or in a cell supernatant that is free of target cells.
Two-reporter luciferase substrate combinations for light generation
Two luciferase reporters generate light using D-luciferin or a synthetic analog together with coelenterazine or imidazopyrazinone or a synthetic analog, pNNP with coelenterazine or imidazopyrazinone or synthetic analogs, or D-luciferin or synthetic analog together with pNNP.
Across the identified independent claim and dependent refinements, the core coverage centers on using a target cell engineered to express an intracellular non-secretory thermostable reporter and measuring reporter level to assess cytotoxicity modulation. Dependent features further constrain thermostability at 37°C, reporter amino acid identity, source species, the assay location in cell media or cell supernatant free of cells, and representative substrate combinations for two-reporter light generation.
Stated Advantages
Enables reporter stability under cytotoxicity assay conditions, including stability at 37°C for more than 15 minutes.
Provides resistance to protease and redox changes during cell death to support reliable intracellular reporter readouts.
Detecting cytotoxicity at low cell numbers.
Documented Applications
Assessing cytotoxicity in CAR-T and NK contexts.
Assessing cytotoxicity in ADCC contexts, including bispecific antibodies and ADCs.
Drug screening and potency QA using cytotoxicity reporter readouts.
Patient-sample and in vivo monitoring.
Monitoring cytotoxicity in Matador cytotoxicity assay contexts, including CAR-T potency monitoring and in vivo monitoring concepts.
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