Yang Li

The efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy is suboptimal in most cancers, necessitating further improvement in their therapeutic actions. However, enhancing antitumor T cell response inevitably confers an increased risk of cytokine release syndrome associated with monocyte-derived interleukin-6 (IL-6). Thus, an approach to simultaneously enhance therapeutic efficacy and safety is warranted. Here, we develop a chimeric cytokine receptor composed of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant that constitutively activates the JAK-STAT pathway (G6/7R or G6/7R-M452L). CAR-T cells with G6/7R efficiently absorb and degrade monocyte-derived IL-6 in vitro. The G6/7R-expressing CAR-T cells show superior expansion and persistence in vivo, resulting in durable antitumor response in both liquid and solid tumor mouse models. Our strategy can be widely applicable to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

Background While chimeric antigen receptor (CAR)-T cell therapy exhibits a robust therapeutic efficacy against B-cell malignancies and multiple myeloma, its efficacy and safety have not been established for acute myeloid leukemia (AML) and solid tumors due to the paucity of established target antigens. Some AML and solid tumor cells express tumor necrosis factor (TNF), which is initially expressed on the cell surface prior to shedding. Methods In this study, we obtained monoclonal antibodies against the N-terminal fragment of TNF (TNF-NTF) that remains on the cell surface after shedding. We then generated CAR-T cells to target TNF-NTF using the antibody sequence. To enhance the therapeutic efficacy of TNF-NTF CAR-T cells, we further engineered the previously developed chimeric cytokine receptor consisting of GP130, IL6R, and constitutively active IL7R with the M452L mutation (G6/7R). Results TNF-NTF CAR-T cells efficiently lysed TNF-expressing leukemia cells in vitro , while showing limited antitumor efficacy in vivo due to poor expansion and persistence. Activated T cells upregulate TNF, which was recognized by TNF-NTF CAR-T cells and led to fratricide. Genetic knockout (KO) of TNF significantly enhanced the viability and proliferation of TNF-NTF CAR-T cells, while slightly reducing their cytotoxic activity. In addition, ectopic expression of G6/7R improved the effector function of TNF-NTF CAR-T cells through constitutive activation of janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling. The G6/7R-expressing TNF -KO TNF-NTF CAR-T cells exhibited superior persistence and durable antileukemic efficacy in vivo compared with parental CAR-T cells. We also confirmed that TNF-NTF CAR-T cells can target primary AML cells, including a leukemia-initiating population with colony-forming capacity. Unlike CD33, targeting TNF-NTF did not show cytotoxicity against normal hematopoietic stem/progenitor cells. Finally, we demonstrated the curative efficacy of G6/7R TNF -KO TNF-NTF CAR-T cells against TNF-expressing ovarian tumor cells in vivo . Conclusions Our studies highlight TNF-NTF as a promising cell surface target for CAR-T cell therapy that can be applied to AML as well as solid tumors.

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The low efficiency of somatic cell nuclear transfer (SCNT) severely limits its application in animal cloning and regenerative medicine. To address this core scientific challenge, this study aims to explore a chemical reprogramming strategy that enhances the division rate of SCNT embryos during early developmental stages prior to transfer by pre-treating donor cells. Leveraging the role of small molecules in regulating cellular reprogramming, we designed a combination of small-molecule compounds (including 8 μM TranylcyprominT, 5 μM EPZ00477, 400 μM VPA, 8 μM Repsox, 1.2 μM PD0325901, 0.4 μM CHIR99021, 0.2 μM DZNeP, 8 μM Y-27632, and 1.2 μM UNC) to pre-treat donor cells, followed by embryo reconstruction and in vitro culture. Results demonstrated that this chemical treatment significantly improved embryo cleavage rates (35.59% vs. 46.15%). The combination of small molecules significantly upregulates the expression of core pluripotency genes (NANOG, SOX2, OCT4) and histone acetyltransferase TIP60 in donor cells. In summary, this study not only demonstrates the efficacy of chemical reprogramming in enhancing the early developmental capacity of SCNT embryos in large mammals but also lays a solid foundation for further elucidating its molecular mechanisms.