Armed with IL-2 based fusion protein improves CAR-T cell fitness and efficacy against solid tumors.

Chimeric antigen receptor T (CAR-T) cell therapy is regarded as a potent immunotherapy and has made significant success in hematologic malignancies by eliciting antigen-specific immune responses. However, response rates of CAR-T cell therapy against solid tumors with immunosuppressive microenvironments remain limited. Co-engineering strategies are advancing methods to overcome immunosuppressive barriers and enhance antitumor responses. Here, we engineered an IL-2 mutein co-engineered CAR-T for the improvement of CAR-T cells against solid tumors and the efficient inhibition of solid tumors. We equipped the CAR-T cells with co-expressing both tumor antigen-targeted CAR and a mutated human interleukin-2 (IL-2m), conferring enhanced CAR-T cells fitness in vitro, reshaped immune-excluded TME, enhanced CAR-T infiltration in solid tumors, and improved tumor control without significant systemic toxicity. Overall, this subject demonstrates the universal CAR-T cells armed strategy for the development and optimization of CAR-T cells against solid tumors.

Noninvasive Imaging OX40+ Activated T Cells Provides Early Warning of Rheumatoid Arthritis.

PURPOSE: The goal of this study was to develop an imaging probe-IRDye-680RD-OX40 mAb-that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA. METHODS: OX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups. RESULTS: NIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring. CONCLUSION: The results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.

Astragaloside IV - mediated endothelial progenitor cell exosomes promote autophagy and inhibit apoptosis in hyperglycemic damaged endothelial cells via miR-21/PTEN axis.

INTRODUCTION: As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells. MATERIALS AND METHODS: After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN. RESULTS: HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes. CONCLUSIONS: AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.

Fluorophore-conjugated 4-1BB antibody enables early detection of T-cell responses in inflammatory arthritis via NIRF imaging.

PURPOSE: We first developed a 4-1BB-targeted optical probe, named IRDye-680RD-4-1BB mAb (monoclonal antibody), and evaluated its value for the detection of 4-1BB+ activated T cells in vivo as well as the diagnosis of rheumatoid arthritis (RA) in an adjuvant-induced arthritis (AIA) mouse model. METHODS: The 4-1BB expression pattern was analysed by flow cytometry and immunofluorescence (IF) staining. The 4-1BB mAb was conjugated with IRDye-680RD NHS ester, and characterized via fluorescence spectrum. A cell-binding assay was also performed to assess the interaction of this probe with activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed at 6, 24, 48, 72, and 96 h after probe administration. RESULTS: 4-1BB expression was highly upregulated during the pathogenesis of RA. Good colocalization was also observed between CD3 and 4-1BB by IF staining and t-SNE (T-distributed stochastic neighbour embedding) analysis, which indicates that 4-1BB was mainly expressed on T cells. Compared to the control group, a significantly higher signal was observed in the right hind paw (RP) of mice with AIA at all time points. The ex vivo biodistribution study results were consistent with the in vivo NIRF imaging results, which validated the accuracy of the region of interest (ROI) measurements. The sensitivity against 100% specificity observed in the receiver operator characteristic (ROC) curve analysis could distinguish the AIA group from the control group at all time points, indicating the value of IRDye-680RD-4-1BB mAb for RA diagnosis. CONCLUSION: We successfully developed a novel optical imaging probe, named IRDye-680RD-4-1BB mAb, for tracking 4-1BB+ activated T cells in vivo, and 4-1BB NIRF imaging is a promising strategy for noninvasively detecting the pathogenesis of RA.

Hypoxia-responsive nanomaterials for tumor imaging and therapy.

Hypoxia is an important component of tumor microenvironment and plays a pivotal role in cancer progression. With the distinctive physiochemical properties and biological effects, various nanoparticles targeting hypoxia had raised great interest in cancer imaging, drug delivery, and gene therapy during the last decade. In the current review, we provided a comprehensive view on the latest progress of novel stimuli-responsive nanomaterials targeting hypoxia-tumor microenvironment (TME), and their applications in cancer diagnosis and therapy. Future prospect and challenges of nanomaterials are also discussed.