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Single-cell gene expression in Her2-specific CAR T cells in osteosarcoma mouse model.

ABSTRACT: Purpose: To compare cell states between control Her2-BBz and JUN-overexpression Her2-BBz human CAR T cells in a mouse model of osteosarcoma. Methods: 8-week old NSG mice were implanted with 1 million 143B osteosarcoma tumor cells intramuscularly in the right leg. 14 days post tumor implantation, mice were infused intravenously with 10 million human CAR+ T cells via tail vein injection. Before T cell transfer, mice were randomized for tumor size and treated with either control Her2-BBz or JUN-overexpressing Her2-BBz CAR T cells. T cells were 70% CAR+ at infusion for both groups. 14 days post T cell treatment (day 28 post tumor engraftment), 6 mice per group were euthanized, solid tumor tissue was collected, mechanically dissociated, and single cell suspensions were labeled with human-CD45 antibody and viability dye. Live hCD45+ tumor-infiltrating lymphocytes were sorted from each tumor and 6 samples were pooled for each group. ~35,000-40,000 sorted live cells (counted post sort) for each group were delivered to the Stanford Functional Genomics Facility for 3' single-cell RNA-sequencing on the 10X Genomics platform. Results: We found that substantially more JUN-overexpressing Her2-BBz CAR T cells were within the G2/M and S phases of the cell cycle than control Her2-BBz CAR T cells, consistent with increased in vivo proliferation of JUN-Her2-BBz CAR T cells. Furthermore, JUN-Her2-BBz CAR T cells also demonstrated a more activated transcriptional program (as measured by IL2RA and CD38), while many exhaustion-associated genes were downregulated (PDCD1, BTLA, TIGIT, CD200, ENTPD1, NR4A2) compared to control Her2-BBz CAR T cells. Finally, a small cluster of cells characterized by high IL7R expression (IL7R+ KLF2+ CD27+ TCF7+ SELL+) was apparent in JUN-overexpressing but not in control Her2-BBz CAR T cells, consistent with maintenance of a memory-like population capable of self-renewal. Conclusions: This study provides insights into cell states that could explain the improved efficacy of JUN-overexpression Her2-BBz CAR T cells compared to control Her2-BBz CAR T cells in a mouse model of osteosarcoma.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136805 | GEO | 2019/10/01

REPOSITORIES: GEO

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Project description:CAR T cell therapy has revolutionized the treatment of hematologic malignancies, but its efficacy in solid tumors remains limited due to the immunosuppressive nature of the tumor microenvironment and the inability of T cells to persist and traffic to the tumor site. While current strategies focus on enhancing CAR T cell activity through costimulatory molecules and cytokines, a critical yet often overlooked factor is the competition for nutrients between tumor cells and T cells in the nutrient-deprived tumor microenvironment. To address this challenge, we employed a selective metabolic refueling (MR) strategy by providing T cells with inosine as an alternative fuel source for growth and functionality. In this study, we engineered CAR T cells to co-express a membrane-bound CD26 and a cytoplasmic adenosine deaminase 1 (ADA1) fused to an anti-CD3 scFv. ADA1 irreversibly converts both intracellular and extracellular adenosine to inosine, overcoming adenosine-mediated immunosuppression and providing T cells with inosine for growth. The inclusion of an anti-CD3 scFv fusion partner and overexpressing CD26 boosts ADA1 capture in a membrane proximal manner, providing inosine for T cells and minimizing feeding the tumor cells. We demonstrate that ADA1 is conditionally secreted in an autocrine manner in response to CD3/CD26 stimulation and that it activates CAR T cells through trans-signaling in a tumor-specific manner. In addition, we show that, compared to unmodified CAR T cells, CD26-overexpressing CAR T cells have better migration capacity and are less susceptible to TGF-β1 suppression. Finally, we demonstrate that, in mice models of human hepatocellular carcinoma (GPC3-MR-CAR) and human non-small cell lung cancer (HER2-MR-CAR), metabolically refueled CAR T cells (MR-CAR) are more efficient in reducing tumor growth than unmodified CAR T cells. Thus, selective refueling CAR T cells using ADA1 and CD26 holds promise for improving the efficacy of CAR T cell therapy of solid tumors.

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Single-cell gene expression in Her2-specific CAR T cells in osteosarcoma mouse model.

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