Gut microbiome for predicting immune checkpoint blockade-associated adverse events.

BACKGROUND: The impact of the gut microbiome on the initiation and intensity of immune-related adverse events (irAEs) prompted by immune checkpoint inhibitors (ICIs) is widely acknowledged. Nevertheless, there is inconsistency in the gut microbial associations with irAEs reported across various studies. METHODS: We performed a comprehensive analysis leveraging a dataset that included published microbiome data (n = 317) and in-house generated data from 16S rRNA and shotgun metagenome samples of irAEs (n = 115). We utilized a machine learning-based approach, specifically the Random Forest (RF) algorithm, to construct a microbiome-based classifier capable of distinguishing between non-irAEs and irAEs. Additionally, we conducted a comprehensive analysis, integrating transcriptome and metagenome profiling, to explore potential underlying mechanisms. RESULTS: We identified specific microbial species capable of distinguishing between patients experiencing irAEs and non-irAEs. The RF classifier, developed using 14 microbial features, demonstrated robust discriminatory power between non-irAEs and irAEs (AUC = 0.88). Moreover, the predictive score from our classifier exhibited significant discriminative capability for identifying non-irAEs in two independent cohorts. Our functional analysis revealed that the altered microbiome in non-irAEs was characterized by an increased menaquinone biosynthesis, accompanied by elevated expression of rate-limiting enzymes menH and menC. Targeted metabolomics analysis further highlighted a notably higher abundance of menaquinone in the serum of patients who did not develop irAEs compared to the irAEs group. CONCLUSIONS: Our study underscores the potential of microbial biomarkers for predicting the onset of irAEs and highlights menaquinone, a metabolite derived from the microbiome community, as a possible selective therapeutic agent for modulating the occurrence of irAEs.

PLA2G2A+ cancer-associated fibroblasts mediate pancreatic cancer immune escape via impeding antitumor immune response of CD8+ cytotoxic T cells.

Our previous research defined a novel metabolic cancer associated fibroblasts subset (meCAFs) enriched in loose-type pancreatic ductal adenocarcinoma (PDAC) and related to CD8+ T cells accumulation. Consistently, the abundance of meCAFs was associated with poor prognosis but better immunotherapy responses in PDAC patients. However, the metabolic characteristic of meCAFs and its cross-talk with CD8+ T cells remain to be elucidated. In this study, we identified PLA2G2A as a marker of meCAFs. In particular, the abundance of PLA2G2A+ meCAFs was positively related to the accumulation of total CD8+ T cells and negatively correlated with clinical outcomes of PDAC patients and infiltration of intratumoral CD8+ T cells. We demonstrated that PLA2G2A+ meCAFs substantially attenuated the antitumor ability of tumor infiltrating CD8+ T cells and facilitated tumor immune escape in PDAC. Mechanistically, PLA2G2A regulated the function of CD8+ T cells as a pivotal soluble mediator via MAPK/Erk and NF-κB signaling pathways. In conclusion, our study identified the unrecognized role of PLA2G2A+ meCAFs in promoting tumor immune escape by impeding the antitumor immune function of CD8+ T cells, and strongly suggested PLA2G2A as a promising biomarker and therapeutic target for immunotherapy in PDAC.

Plasma Transforming Ni(OH)2 Nanosheets into Porous Nickel Nitride Sheets for Alkaline Hydrogen Evolution.

Nickel nitride (Ni3N) is a superior hydrogen evolution reaction (HER) catalyst where the nitrogen source is usually ammonia and the reaction temperature is high during the synthesis process. Herein, we employed an innovative method to obtain three-dimensional porous nickel nitride nanosheets on Ni foam (Ni3N/NF) by transforming Ni(OH)2 nanosheets in N2-H2 glow discharge plasma. The obtained Ni3N/NF displays a high HER activity with a small overpotential of 44 mV and a low Tafel slope of 46 mV dec-1, which is competitive to a Pt/C catalyst. Both the test data and simulation results prove that active ions and radicals in plasma play essential roles in achieving the facile nitridation, as well as building a nanostructured morphology over the Ni3N/NF surface. The unique synthesis method opens new avenues for metal nitrides of HER catalysts and beyond.

Combined Antitumor Effects of Sorafenib and GPC3-CAR T Cells in Mouse Models of Hepatocellular Carcinoma.

Our previous study indicated that GPC3-targeted chimeric antigen receptor (CAR) T cell therapy has a high safety profile in patients with hepatocellular carcinoma (HCC). However, the response rate requires further improvement. Here, we analyzed the combined effect of GPC3-CAR T cells and sorafenib in both immunocompetent and immunodeficient mouse models of hepatocellular carcinoma. In immunocompetent mouse model, mouse CAR (mCAR) T cells induced regression of small tumors (approximately 130 mm3 tumor volume) but had no effect on large, established tumors (approximately 400 mm3 tumor volume). Sorafenib, at a subpharmacologic but not a pharmacologic dose, augmented the antitumor effects of mCAR T cells, in part by promoting IL12 secretion in tumor-associated macrophages (TAMs) and cancer cell apoptosis. In an immunodeficient mouse model, both subpharmacologic and pharmacologic doses of sorafenib had limited impacts on the function of human CAR (huCAR) T cells in vitro and showed synergistic effects with huCAR T cells in vivo, which can at least partially be ascribed to the upregulated tumor cell apoptosis induced by the combined treatment. Thus, this study applied two of the most commonly used mouse models for CAR T cell research and demonstrated the clinical potential of combining sorafenib with GPC3-targeted CAR T cells against HCC.

Armored Inducible Expression of IL-12 Enhances Antitumor Activity of Glypican-3-Targeted Chimeric Antigen Receptor-Engineered T Cells in Hepatocellular Carcinoma.

Adoptive immunotherapy based on chimeric antigen receptor-modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived-armored glypican-3 (GPC3)-specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12-armored GPC3-CAR-T cells could broaden the application of CAR-T-based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

Plasma enabled non-thermal phosphorization for nickel phosphide hydrogen evolution catalysts.

While nickel phosphide is a highly attractive catalyst for the hydrogen evolution reaction, its preparation requires either high temperature or the use of highly toxic PH3 directly or indirectly. Herein, we demonstrate that H2 plasma activated red phosphorus enables the synthesis of self-supported Ni2P nanosheet (Ni2P/NF) arrays on commercial nickel foam from a NiO/NF precursor at only 250 °C. Highly reactive atomic H in the plasma induces dissociation of the P4 molecules, yielding an acid stable electrode with excellent hydrogen evolution activity and good mechanical strength.

Target-Dependent Expression of IL12 by synNotch Receptor-Engineered NK92 Cells Increases the Antitumor Activities of CAR-T Cells.

IL12 is an immune-stimulatory cytokine for key immune cells including T cells and NK cells. However, systemic administration of IL12 has serious side effects that limit its clinical application in patients. Recently, synthetic Notch (synNotch) receptors have been developed that induce transcriptional activation and deliver therapeutic payloads in response to the reorganization of specific antigens. NK92 cell is a human natural killer (NK) cell line which has been developed as tools for adjuvant immunotherapy of cancer. Here, we explored the possibility of using synNotch receptor-engineered NK92 cells to selectively secrete IL12 at the tumor site and increase the antitumor activities of chimeric antigen receptor (CAR)-modified T cells. Compared with the nuclear factor of activated T-cells (NFATs) responsive promoter, which is another regulatory element, the synNotch receptor was better at controlling the expression of cytokines. NK92 cells transduced with the GPC3-specific synNotch receptor could produce the proinflammatory cytokine IL12 (GPC3-Syn-IL12-NK92) in response to GPC3 antigen expressed in cancer cells. In vivo GPC3-Syn-IL12-NK92 cells controlling IL12 production could enhance the antitumor ability of GPC3-redirected CAR T cells and increase the infiltration of T cells without inducing toxicity. Taken together, our results demonstrated that IL12 supplementation by synNotch-engineered NK92 cells could secrete IL12 in a target-dependent manner, and promote the antitumor efficiency of CAR-T cells. Local expression of IL12 by synNotch-engineered NK92 cells might be a safe approach to enhance the clinical outcome of CAR-T cell therapy.

Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma.

Cancer immunotherapy has made unprecedented breakthrough in the fields of chimeric antigen receptor-redirected T (CAR T) cell therapy and immune modulation. Combination of CAR modification and the disruption of endogenous inhibitory immune checkpoints on T cells represent a promising immunotherapeutic modality for cancer treatment. However, the potential for the treatment of hepatocellular carcinoma (HCC) has not been explored. In this study, the gene expressing the programmed death 1 receptor (PD-1) on the Glypican-3 (GPC3)-targeted second-generation CAR T cells employing CD28 as the co-stimulatory domain was disrupted using the CRISPR/Cas9 gene-editing system. It was found that, in vitro, the CAR T cells with the deficient PD-1 showed the stronger CAR-dependent anti-tumor activity against native programmed death 1 ligand 1-expressing HCC cell PLC/PRF/5 compared with the wild-type CAR T cells, and meanwhile, the CD4 and CD8 subsets, and activation status of CAR T cells were stable with the disruption of endogenous PD-1. Additionally, the disruption of PD-1 could protect the GPC3-CAR T cells from exhaustion when combating with native PD-L1-expressing HCC, as the levels of Akt phosphorylation and anti-apoptotic protein Bcl-xL expression in PD-1 deficient GPC3-CAR T cells were significantly higher than those in wild-type GPC3-CAR T cells after coculturing with PLC/PRF/5. Furthermore, the in vivo anti-tumor activity of the CAR T cells with the deficient PD-1 was investigated using the subcutaneous xenograft tumor model established by the injection of PLC/PRF/5 into NOD-scid-IL-2Rγ-/- (NSG) mice. The results indicated that the disruption of PD-1 enhanced the in vivo anti-tumor activity of CAR T cells against HCC, improved the persistence and infiltration of CAR T cells in the NSG mice bearing the tumor, and strengthened the inhibition of tumor-related genes expression in the xenograft tumors caused by the GPC3-CAR T cells. This study indicates the enhanced anti-tumor efficacy of PD-1-deficient CAR T cells against HCC and suggests the potential of precision gene editing on the immune checkpoints to enhance the CAR T cell therapies against HCC.

Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1-CH3 fusion protein.

Our recent clinical study demonstrated that glypican-3 (GPC3)-specific chimeric antigen receptor-modified T (CAR-T) cells are a promising treatment for hepatocellular carcinoma (HCC). However, the interaction of programmed cell death 1 (PD-1) and PD-L1-mediated T-cell inhibition is involved in immune evasion in a wide range of solid tumors, including HCC. To overcome this problem, we introduced a fusion protein composed of a PD-1 extracellular domain and CH3 from IgG4 into GPC3-specific CAR-T cells (GPC3-28Z) to block the PD-1/PD-L1 pathway. GPC3-specific CAR-T cells carrying the PD-1-CH3 fusion protein (sPD1) specifically recognized and lysed GPC3-positive HCC cells. The proliferation capacity of GPC3-28Z-sPD1 T cells after weekly stimulation with target cells was much higher than that of control GPC3-28Z T cells. Additionally, the coexpression of sPD1 could protect CAR-T cells from exhaustion when incubated with target cells, as phosphorylated AKT and Bcl-xL expression levels were higher in GPC3-28Z-sPD1 T cells than in GPC3-28Z cells. Importantly, in two HCC tumor xenograft models, GPC3-28Z-sPD1 T cells displayed a significantly higher tumor suppression capacity than GPC3-28Z T cells. In addition, an increased number of CD3+ T cells in the circulation and tumors and increased granzyme B levels and decreased Ki67 expression levels in the tumors were observed in the mice treated with GPC3-28Z-sPD1 T cells. Together, these data indicated that GPC3-specific CAR-T cells carrying sPD1 show promise as a treatment for patients with HCC.

Development of GPC3-Specific Chimeric Antigen Receptor-Engineered Natural Killer Cells for the Treatment of Hepatocellular Carcinoma.

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential utilities have not been explored in hepatocellular carcinoma (HCC). Glypian-3 (GPC3) is a rational immunotherapeutic target for HCC. In this study, we developed GPC3-specific NK cells and explored their potential in the treatment of HCC. The NK-92/9.28.z cell line was established by engineering NK-92, a highly cytotoxic NK cell line with second-generation GPC3-specific CAR. Exposure of GPC3+ HCC cells to this engineered cell line resulted in significant in vitro cytotoxicity and cytokine production. In addition, soluble GPC3 and TGF-ß did not significantly inhibit the cytotoxicity of NK-92/9.28.z cells in vitro, and no significant difference in anti-tumor activities was observed in hypoxic (1%) conditions. Potent anti-tumor activities of NK-92/9.28.z cells were observed in multiple HCC xenografts with both high and low GPC3 expression, but not in those without GPC3 expression. Obvious infiltration of NK-92/9.28.z cells, decreased tumor proliferation, and increased tumor apoptosis were observed in the GPC3+ HCC xenografts. Similarly, efficient retargeting on primary NK cells was achieved. These results justified clinical translation of this GPC3-specific, NK cell-based therapeutic as a novel treatment option for patients with GPC3+ HCC.

A Fusion Receptor as a Safety Switch, Detection, and Purification Biomarker for Adoptive Transferred T Cells.

The incorporation of an endogenous safety switch represents a rational strategy for the control of toxicities following the administration of adoptive T cell therapies. An ideal safety switch should be capable of depleting the transferred T cells with minimal injury to normal tissues. We generated a fusion receptor by engineering a cryptic 806 epitope of human epidermal growth factor receptor (EGFR) into the N terminus of the full-length human folate receptor 1 (FOLR1), designated as FR806. The expression of FR806 allows transduced T cells to be targeted with CH12, a monoclonal antibody recognizing the 806 epitope, but not wild-type EGFR in healthy tissues. FR806, therefore, constitutes a specific cell-surface marker for the elimination of transduced T cells. We demonstrate that the antibody-drug conjugate (ADC) CH12-MMAF is efficiently internalized by FR806-expressing T cells and has the potential to eliminate them. Transfected T cells could, furthermore, be efficiently detected and purified using CH12 antibodies. In immuno-compromised mice, CH12-MMAF eliminated the majority of transferred T cells expressing FR806 and anti-CD19 chimeric antigen receptor (CAR). The selectivity for the 806 epitope and internalization capacity of FOLR1 makes FR806 an efficient safety switch, which may additionally be used as a detection and purification biomarker for human T cell immunotherapies.

EGFR modulates monounsaturated fatty acid synthesis through phosphorylation of SCD1 in lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR), a well-known oncogenic driver, contributes to the initiation and progression of a wide range of cancer types. Aberrant lipid metabolism including highly produced monounsaturated fatty acids (MUFA) is recognized as a hallmark of cancer. However, how EGFR regulates MUFA synthesis in cancer remains elusive. This is the focus of our study. METHODS: The interaction between EGFR and stearoyl-CoA desaturase-1 (SCD1) was detected byco-immunoprecipitation. SCD1 protein expression, stability and phosphorylation were tested by western blot. The synthesis of MUFA was determined by liquid chromatography-mass spectrometry. The growth of lung cancer was detected by CCK-8 assay, Annexin V/PI staining, colony formation assay and subcutaneous xenograft assay. The expression of activated EGFR, phosphorylated and total SCD1 was tested by immunohistochemistry in 90 non-small cell lung cancersamples. The clinical correlations were analyzed by Chi-square test, Kaplan-Meier survival curve analysis and Cox regression. RESULTS: EGFR binds to and phosphorylates SCD1 at Y55. Phosphorylation of Y55 is required for maintaining SCD1 protein stability and thus increases MUFA level to facilitate lung cancer growth. Moreover, EGFR-stimulated cancer growth depends on SCD1 activity. Evaluation of non-small cell lung cancersamples reveals a positive correlation among EGFR activation, SCD1 Y55 phosphorylation and SCD1 protein expression. Furthermore, phospho-SCD1 Y55 can serve as an independent prognostic factor for poor patient survival. CONCLUSIONS: Ourstudy demonstrates that EGFR stabilizes SCD1 through Y55 phosphorylation, thereby up-regulating MUFA synthesis to promote lung cancer growth. Thus, we provide the first evidence that SCD1 can be subtly controlled by tyrosine phosphorylation and uncover a previously unknown direct linkage between oncogenic receptor tyrosine kinase and lipid metabolism in lung cancer. We also propose SCD1 Y55 phosphorylation as a potential diagnostic marker for lung cancer.