High-throughput screening of chimeric RNA-binding proteins for improved CAR T cell function

Genetically modified cell-based immunotherapy has been successful in multiple liquid tumors; however, the complex and immunosuppressive solid tumor microenvironment has limited the efficacy of cell-based therapies for solid tumors, which account for the majority of cancers. There is ongoing effort to achieve a more therapeutically effective T cell state by modulating the expression of endogenous genes to enhance chimeric antigen receptor (CAR) T cell activity against solid tumors and prevent T cell exhaustion. One promising strategy involves the use of RNA-binding proteins (RBPs), which regulate gene expression through mRNA splicing, export, stability, degradation, and translation. Recent research indicates that RBPs are crucial in T cell functions such as proliferation, cytokine production, inflammatory responses, and effector activities. However, genetic manipulation of endogenous RBPs has not been sufficient to produce improved effector phenotypes in CAR T cells for robust tumor clearance. To address this, we synthesized novel chimeric RBPs by combining effector and RNA-binding domains from different natural RBPs involved in immune response pathways, each tagged with a unique DNA barcode. We edited primary human T cells using CRISPR-Cas12a to integrate a library of nearly 12,000 chimeric RBPs and a CD19-28z CAR into the endogenous TRAC locus. In large scale pooled knockin screens, we repetitively co-cultured the library of edited CAR T cells with stress doses of Nalm6 target leukemia cells, and performed both acute and chronic antigen stimulation assays to assess CAR T cell persistence . The most effective chimeric RBPs were identified by comparing barcode readouts before versus after the acute or chronic stimulation assays, and linking the most abundant barcodes to enhanced CAR T cell proliferation. Multiple chimeric RBPs outperformed both natural RBPs and CAR only controls, with (N)YTHDF1-(RBD)ZFP36L1-(C)ZC3H12A emerging as a top hit. In subsequent functional validation assays, this synthetic protein induced unique single-cell transcriptomic signatures, and the edited cells demonstrated superior killing capacity and distinct phenotypic markers. These top chimeric RBPs represent promising candidates for further study, potentially leading to improved CAR T cell therapies that can overcome the challenges posed by solid tumors. This approach establishes a scalable platform for the discovery and screening of new synthetic genes for next-generation cell therapies.