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Publication Number
US-11912987-B2
Publication Date
2024-02-27
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 relates to a CRISPR/Cas9-based target identification platform for discovering genes or pathways that govern immune effector ability to generate anti-tumor responses. Edited receptor-engineered immune effector cells that comprise a chimeric antigen receptor (CAR) are used together with engineered tumor cells that comprise an extracellular protein target recognized by the CAR.
During co-culturing of the engineered tumor cells and the edited T cells, edited T cells that accumulate are isolated as a readout. The gRNA library is used to identify relevant resistance, suppression, or immune-evasion genes or pathways by sequencing and enriching or depleting gRNAs from the accumulating edited population.
The disclosed approach is described as enabling genome-wide or subgenome-scale screening of immune effector function in the tumor context. The workflow is implemented with CRISPR/Cas9 formats including spCas9 or saCas9 and CRISPRi or CRISPRa using dCas9 fusion proteins, and is described with activator cells and antigen-presenting cells (APC) that express target antigen and may include immunosuppressive molecules.
The document further describes using extracellular protein targets such as HER2/ERBB2, EGFR, CD19, and CD20, and validating results under immunosuppression mediated by factors including PD-L1. It also states feasibility using disseminated and subcutaneous NSG mouse models, together with sequencing and guide enrichment strategies.
Claims Coverage
Independent claim clm-00001 covers one core inventive concept: selecting accumulating CAR-bearing edited T cells during co-culture with engineered tumor cells presenting a cognate extracellular protein target, using Cas9 together with a gRNA library targeting genomic loci in the edited T cells.
Co-culturing CAR-bearing Cas9/gRNA-edited T cells with engineered tumor cells presenting an extracellular protein target
Engineered tumor cells that comprise an extracellular protein are co-cultured with edited T cells that comprise a chimeric antigen receptor (CAR) having an extracellular target-binding domain that binds the extracellular protein, where the edited T cells comprise a Cas9 protein and a gRNA library that comprises gRNAs targeting one or more genomic loci in the edited T cells.
Isolating accumulating edited T cells during co-culture
Edited T cells that accumulate during the co-culturing are isolated.
The key inventive elements are the CAR-mediated co-culture between engineered tumor cells expressing an extracellular protein and Cas9/gRNA-edited T cells, followed by isolating the accumulating edited T cell population as the basis for identifying relevant genomic loci.
Stated Advantages
Documented Applications
No documented applications found
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