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Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies [RNA-Seq]

ABSTRACT: Chimeric antigen receptor-engineered macrophages (CAR-Ms) hold great promise for solid tumor immunotherapy. The intracellular domains ICDs of CARs determine the phenotypic output of therapeutic macrophages but remain largely unexplored. Here, we constructed a library of CARs containing 131 unique signaling domains derived from native immune receptors and revealed 17 ICDs that enhanced macrophage phagocytosis, the inflammatory response and tumor infiltration in vitro and in vivo. We further developed a scalable 3’ barcode technology, CARode, to uniquely label and trace ICD variants in a large-scale combinatorial signaling domain CAR library and applied it to perform single-cell RNA sequencing and single-cell CAR analysis to measure the synergetic effects of ICD combinations in promoting macrophage activation. This platform includes a novel CD40-LY9-FCRL1 chimeric receptor that modulates the tumor microenvironment and improves solid tumor clearance. In conclusion, pooled screening facilitates the discovery of complex ICD constructs to program macrophage functions for therapeutic applications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE289148 | GEO | 2025/02/17

REPOSITORIES: GEO

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Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies [scRNA-seq]

Project description:Chimeric antigen receptor-engineered macrophages (CAR-Ms) hold great promise for solid tumor immunotherapy. The intracellular domains ICDs of CARs determine the phenotypic output of therapeutic macrophages but remain largely unexplored. Here, we constructed a library of CARs containing 131 unique signaling domains derived from native immune receptors and revealed 17 ICDs that enhanced macrophage phagocytosis, the inflammatory response and tumor infiltration in vitro and in vivo. We further developed a scalable 3’ barcode technology, CARode, to uniquely label and trace ICD variants in a large-scale combinatorial signaling domain CAR library and applied it to perform single-cell RNA sequencing and single-cell CAR analysis to measure the synergetic effects of ICD combinations in promoting macrophage activation. This platform includes a novel CD40-LY9-FCRL1 chimeric receptor that modulates the tumor microenvironment and improves solid tumor clearance. In conclusion, pooled screening facilitates the discovery of complex ICD constructs to program macrophage functions for therapeutic applications.

2025-02-17 | GSE289149 | GEO

Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies [scCAR-seq]

2025-02-17 | GSE289201 | GEO

Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence

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Chimeric antigen receptor with novel intracellular modules improves antitumor performance of T cells

Project description:The excessive cytokine release and limited persistence represent major obstacles to successful chimeric antigen receptor T (CAR-T) cell therapy in solid tumors. Conventional CARs employ an intracellular domain (ICD) from the ζ chain of CD3 as a signaling module, and it is largely unknown how alternative CD3 chains potentially contribute to CAR design. Here, we obtained a series of CAR-T cells against HER2 and mesothelin using a domain containing one immunoreceptor tyrosine-based activation motif from different CD3 subunits as the ICD of CARs. While these reconstituted CARs conferred sufficient antigen-specific cytolytic activity on equipped T cells, they elicited low tonic signal, ameliorated CAR-T cell exhaustion and facilitated memory differentiation. Intriguingly, the CD3ε-derived ICD outperformed others in generation of CAR-T cells that produced minimized cytokines. Mechanistically, CD3ε-based CARs displayed a restrained cytomembrane expression on engineered T cells, which was ascribed to endoplasmic reticulum retention mediated by the carboxyl terminal basic residues. The present study demonstrated the applicability of CAR reconstitution using signaling modules from different CD3 subunits, and depicted a novel pattern of CAR expression that reduces cytokine release, thus paving a way for generation of CAR-T cells with improved safety and persistence against diverse tumor antigens.

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The Second Generation of CAR-iMACs with Enhanced Polarization, CAR-mediated Efferocytosis and Superior Potency against Solid Tumors

Project description:Chimeric antigen receptor (CAR)-T cell therapies have shown great success in treating hematologic malignancies. Nonetheless, their therapeutic effect on solid tumors remains to be improved. Recently, macrophages have attracted great attention, given their ability to infiltrate solid tumors, phagocytize tumor cells as well as their immunomodulatory capacities. The first generation of CD3ζ-based CAR-macrophages demonstrated that the CAR could stimulate macrophage phagocytosis in a tumor antigen-dependent way. Here, we genetically engineered induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) with TLR4 intracellular TIR domain-containing CARs against EGFRvIII and GPC3, which yielded markedly enhanced antitumor effect in two different solid tumor models including glioblastoma, and hepatocellular carcinoma in which complete remission was achieved with CAR-iMACs alone or in combination with CD47 antibody. Moreover, the tandem CD3ζ-TIR-CAR, or the “second-generation” design of TIR-based dual signaling CAR, endowed iMACs with both target engulfment/efferocytosis capacity against antigen-expressing solid tumor cells, and potency of antigen-dependent M1 state polarization and M2 state resistance in an NF-κB dependent manner. We also illustrated a surprising mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we established the next generation CAR-iMACs equipped with orthogonal phagocytosis and polarization capacity for better antitumor functions in treating solid tumors.

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2015-01-14 | E-GEOD-64914 | biostudies-arrayexpress

Chimeric Antigen Receptor-Macrophage Extracellular Vesicles Reprogram Immune Microenvironment for Cancer Therapy

Project description:Immunotherapy has become a new milestone in cancer treatment, with chimeric antigen receptor (CAR)-T cell therapy proven to be an effective method for treating hematologic malignancies. However, its efficacy in solid tumors remains limited. Macrophages, the most infiltrative innate immune cells in the tumor microenvironment, are being genetically engineered to express CARs, rapidly emerging as a promising new therapy for non-hematologic malignancies. Yet, CAR- Macrophages therapy is associated with unique acute toxicities and is susceptible to immunosuppressive mechanisms. Here, we propose using CAR macrophages-derived extracellular vesicles (CAR-MEVs) as an optimization of CAR-Ms therapy. Compared to CAR-Ms cells, CAR-MEVs exhibit lower toxicity and enhanced drug delivery capabilities. In an in vivo liver cancer model, CAR-MEVs administration proved safer than CAR-Ms therapy, reducing tumor burden and prolonging overall survival. In a humanized mouse model, CAR-MEVs further demonstrated the ability to induce a pro-inflammatory tumor microenvironment and enhance anti-tumor T cell activity. Mechanistically, CAR-MEVs target tumor cells, inducing immunogenic cell death (ICD) and subsequently leading to neutrophil infiltration in tumor tissues. We demonstrated that the anti-tumor effects of CAR-MEVs depend on neutrophils and are partially reliant on inducible nitric oxide synthase (iNOS). This study supports the potential of macrophage extracellular vesicles as a novel therapeutic approach for solid tumors.

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Project description:Adoptive transfer of chimeric antigen receptor (CAR)-T cells is expected to become the first line of treatment for multiple malignancies, following the enormous success of anti-CD19 therapies. However, their mechanism of action is not fully understood, and clear guidelines for the design of safe and efficient receptors are missing. We hereby describe a systematic analysis of the CAR “interactome” in human primary T cells, which allowed us to identify molecular traits that influence CAR-T cell efficacy. Interactome analysis was based on immunoprecipitation of CARs followed by protein identification by mass spectrometry.

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