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Spatially controlled enhanced anti-tumor efficacy via CAR T cells engineered with a CTLA-4-based autoregulated circuit

ABSTRACT: The widespread application of CAR T cell therapy to solid tumors is hindered by antigen expression heterogeneity, on-target off-tumor toxicity, and limited functional persistence of CAR T cells. Next generation engineering strategies aiming to overcome these hurdles rely on multiple antigen-targeting approaches that fail to simultaneously address all the limitations and/or the use of synthetic receptor transcriptional circuits, which suffer from slow activation kinetics. Here, we leveraged the unique trafficking properties of CTLA-4, regulated by a rapid internalization process, to create CTLA-4-based activation-inducible antigen receptors (CAVI-Rs). CAVI-Rs were exclusively expressed on T cells upon activation with remarkably fast on/off kinetics and self-sustainable expression. In in vitro and in vivo models of heterogeneous tumors, CAR+CAVI-R dual-engineered T cells demonstrated enhanced cytotoxicity and tumor-selective, spatially restricted functionality. Additionally, intermittent co-expression of the CAVI-R lead to improved T cell metabolic and transcriptional fitness, resulting in superior expansion, persistence and anti-tumor efficacy. The CAVI-R provides a novel, simple, safe, potent, and fully-human approach to expand CAR T cell applicability while improving anti-tumor outcomes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE310581 | GEO | 2025/11/23

REPOSITORIES: GEO

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Project description:Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies.

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Spatially controlled enhanced anti-tumor efficacy via CAR T cells engineered with a CTLA-4-based autoregulated circuit

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