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Publication Number
US-11078481-B1
Publication Date
2021-08-03
Expiration Date
Abstract
This invention describes a novel CRISPR/Cas9 target identification platform permitting the discovery of novel genes and pathways involved in the ability of T cells and NK cells to react against and generate an anti-tumor response.
Core Innovation
The invention provides a CRISPR/Cas9 target identification platform to discover genes or pathways governing resistance of receptor-engineered immune effector cells to tumor-mediated suppression or deactivation. The approach builds on co-culture of engineered immune effector cells with tumor cells expressing an extracellular protein target that binds the engineered extracellular target-binding domain on the immune cell surface.
Engineered CAR-T, CAR-NK, or CAR-NKT cells are modified with a Cas9 protein and a gRNA library so that a subpopulation of the immune cells is selectively edited at one or more locations of the CAR cell genome. After co-culture with tumor cells engineered to express the extracellular protein target, the edited immune cells that accumulate over the co-culture period are isolated and analyzed to determine which edits are associated with resistance to tumor suppression or deactivation.
The document further describes reciprocal engineering in which tumor or APC targets can be engineered and edited with Cas9 and a gRNA library to identify tumor genes that evade inhibition. The platform is exemplified with checkpoint-mediated immunosuppression screening and with in vivo feasibility in NSG mouse lymphoma models, and uses described extracellular targets and Cas9/dCas9 implementation options to enable identification of resistance and suppression mechanisms.
Claims Coverage
The partial claims include two independent claims that cover obtaining Cas9-and-gRNA-edited CAR T cells and Cas9-and-gRNA-edited CAR NK/NKT cells that accumulate during co-culture with engineered tumor cells and are edited to confer resistance to tumor suppression or deactivation. Each independent claim is supported by dependent refinements that specify extracellular protein targets and particular gene-editing and/or delivery configurations.
Obtaining CAR-T cells resistant to tumor suppression or deactivation via Cas9 and gRNA co-culture selection
Obtain a population of tumor cells engineered to express an extracellular protein target; obtain primary T cells engineered to express a CAR with an extracellular target-binding domain that binds the extracellular protein target on the tumor cells; engineer the CAR-T cells with a Cas9 protein and a gRNA library to selectively edit the CAR-T cell genome to form a subpopulation of edited CAR-T cells; co-culture the subpopulation of edited CAR-T cells with the population of tumor cells; and isolate the edited CAR-T cells that accumulate over the co-culture period, where the isolated CAR-T cells have been edited to make the CAR-T cells resistant to tumor suppression or deactivation.
Obtaining CAR-NK or CAR-NKT cells resistant to tumor suppression or deactivation via Cas9 and gRNA co-culture selection
Obtain a population of tumor cells engineered to express an extracellular protein target; obtain primary NK cells or NKT cells engineered to express a CAR with an extracellular target-binding domain that binds the extracellular protein target on the tumor cells; engineer the CAR-NK or CAR-NKT cells with a Cas9 protein and a gRNA library to selectively edit one or more locations of the CAR-NK cell genome or CAR-NKT cell genome to form a subpopulation of edited CAR-NK cells or edited CAR-NKT cells; co-culture the subpopulation of edited CAR-NK cells or edited CAR-NKT cells with the population of tumor cells; and isolate the edited CAR-NK cells or CAR-NKT cells that accumulate over the co-culture period, where the isolated CAR-NK cells or CAR-NKT cells have been edited to make the cells resistant to suppression or deactivation by the tumor.
Across the independent claims, the core inventive coverage is selecting, after co-culture, an edited subpopulation of receptor-engineered immune effector cells (CAR-T or CAR-NK/CAR-NKT) that accumulate and are edited to confer resistance to tumor suppression or deactivation, enabled by Cas9 plus a gRNA library targeted to the CAR cell genome. Dependent claims further specify particular extracellular protein targets and refinements such as co-culture duration and Cas9 delivery/encoding formats or dCas9 transcriptional activator domain options.
Stated Advantages
Enables obtaining T cells that are resistant to tumor suppression or deactivation.
Enables obtaining NK cells or NKT cells that are resistant to suppression or deactivation by tumor.
Enables resistance-associated edited immune cell subpopulations to be isolated based on accumulation over a co-culture period.
Documented Applications
Target identification for discovering genes or pathways governing resistance of receptor-engineered immune effector cells (CAR-T and CAR-NK/CAR-NKT) to tumor-mediated suppression or deactivation.
Checkpoint-mediated immunosuppression screening using described PD-L1 and PD1/PDCD1 contexts.
In vivo feasibility in NSG mouse lymphoma models.
Reciprocal engineering of tumor or APC targets to identify tumor genes that evade inhibition.
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