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Publication Number
US-11459572-B2
Publication Date
2022-10-04
Expiration Date
Abstract
Control Devices are disclosed including RNA destabilizing elements (RDE), RNA control devices, and destabilizing elements (DE) combined with Chimeric Antigen Receptors (CARs) or other transgenes in eukaryotic cells. Multicistronic vectors are also disclosed for use in engineering host eukaryotic cells with the CARs and transgenes under the control of the control devices. These control devices can be used to optimize expression of CARs in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered after stimulation of the CAR on the eukaryotic cell.
Core Innovation
The invention provides controlling a transgene in a primary T-cell comprising a chimeric antigen receptor. A heterologous nucleic acid includes a promoter operably linked to a polynucleotide encoding the transgene, where the transgene-encoding polynucleotide is operably linked to an RNA degradation element. The RNA degradation element is an AU rich element, and the heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RNA degradation element.
The primary T-cell is exposed to a ligand for the chimeric antigen receptor, where the ligand is an antigen found on a target cell. Binding of the ligand activates the primary T-cell and changes a metabolic state of the primary T-cell. After the change in metabolic state, the amount of polypeptide made from the transgene is increased, enabling payload expression to be controlled in response to CAR activation.
The document further describes RNA destabilizing elements and RNA control devices integrated with CARs, including variants referred to as Smart CAR embodiments. RNA destabilizing elements are placed in UTRs or introns and include AU/U-rich elements and microRNA-site engineered configurations, and modular RNA control devices incorporate an aptamer sensor and regulatory components, including ribozymes.
Claims Coverage
Three inventive features are presented in the independent claim framework. Dependent claims refine this by specifying particular CAR ligand/target-cell pairings, defined payloads, and sources of the RNA degradation element.
Au rich RNA degradation element fused transgene transcript
A primary T-cell comprising a chimeric antigen receptor is provided with a heterologous nucleic acid in which a promoter is operably linked to a polynucleotide encoding the transgene that is operably linked to a RNA degradation element, wherein the RNA degradation element is an AU rich element.
Metabolic-state change after CAR ligand binding
The primary T-cell is exposed to a ligand for the chimeric antigen receptor, wherein the ligand is an antigen found on a target cell, and wherein binding activates the primary T-cell and thereby changes a metabolic state of the primary T-cell.
Increased transgene polypeptide after metabolic-state change
The transgene is expressed so that the amount of polypeptide made from the transgene is increased after the change in metabolic state of the primary T-cell.
Ligand/target-cell pairing for CAR activation
The CAR ligand is specified for particular target cell types by selecting tumor-associated antigen ligands and corresponding target cells, including NY-ESO-1 with a melanoma cell and CD33 with an AML cell.
Payload selection from defined cytokines/biologics
The transgene payload is selected from IL-2, IL-12, IL-15, IL-18, or TNFα.
RNA degradation element derived from specific 3'-UTRs
The RNA degradation element is defined as being from a 3'-UTR of INFg or from a 3'-UTR of IL6.
The inventive coverage centers on using an AU rich RNA degradation element fused to a CAR T-cell transgene transcript, activating the T-cell through CAR ligand binding to change metabolic state, and producing increased transgene-derived polypeptide after that metabolic change, with dependent refinements specifying particular ligand/target-cell pairings, defined payload choices, and specific RDE sources.
Stated Advantages
Increased amount of polypeptide made from the transgene after the change in metabolic state of the primary T-cell.
Balance tumor killing versus normal-tissue toxicity.
Reduce tumor lysis syndrome (TLS).
Reduce cytokine release syndrome (CRS).
Reduce exhaustion.
Support on-off switching control of CAR/DE-CAR/Side-CAR polypeptide levels.
Documented Applications
Target cell recognition and CAR T-cell activation using an antigen ligand found on a target cell, including melanoma-associated targeting (NY-ESO-1 to melanoma) and AML targeting (CD33 to AML), as part of the described transgene control approach.
Therapeutic use cases for engineered immune effector cell control using RDE-based regulation linked to metabolic state, including delivery of payloads such as cytokines, checkpoint inhibitors, cytotoxics, enzymes, and imaging agents, as described in the provided content.
Control of expression and effector activity for CAR T-cells responding to CAR ligand binding on target cells, with described support for balancing tumor killing versus normal-tissue toxicity and reducing CRS/TLS and exhaustion.
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