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Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity

ABSTRACT: Major challenges of CAR-T cell therapy include poor antigen sensitivity and cell persistence. Here we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered CD3e motif, EB6I, to the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. EB6I CAR can form a mature immunological synapse with CD2 corolla to transduce efficient antigen and costimulatory signaling, while its tonic signaling remains at low levels. Functionally, EB6I CAR-T cells exhibited improved signaling and cytoxicity against low-antigen tumor and persistent tumor killing function. In multiple primary and relapsed murine tumor models, EB6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a new CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE278512 | GEO | 2024/10/05

REPOSITORIES: GEO

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Project description: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.

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Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity

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