Hypoxia-Responsive CAR-T Cells Exhibit Reduced Exhaustion and Enhanced Efficacy in Solid Tumors.

Expanding the utility of chimeric antigen receptor (CAR)-T cells in solid tumors requires improving their efficacy and safety. Hypoxia is a feature of most solid tumors that could be used to help CAR-T cells discriminate tumors from normal tissues. In this study, we developed hypoxia-responsive CAR-T cells by engineering the CAR to be under regulation of hypoxia-responsive elements and selected the optimal structure (5H1P-CEA CAR), which can be activated in the tumor hypoxic microenvironment to induce CAR-T cells with high polyfunctionality. Hypoxia-responsive CAR T cells were in a "resting" state with low CAR expression under normoxic conditions. Compared with conventional CAR-T cells, hypoxia-responsive CAR-T cells maintained lower differentiation and displayed enhanced oxidative metabolism and proliferation during cultivation, and they sowed a capacity to alleviate the negative effects of hypoxia on T-cell proliferation and metabolism. Furthermore, 5H1P-CEA CAR-T cells exhibited decreased T-cell exhaustion and improved T-cell phenotype in vivo. In patient-derived xenograft models, hypoxia-responsive CAR-T cells induced more durable antitumor activity than their conventional counterparts. Overall, this study provides an approach to limit CAR expression to the hypoxic tumor microenvironment that could help to enhance CAR T-cell efficacy and safety in solid tumors. SIGNIFICANCE: Engineering CAR-T cells to upregulate CAR expression under hypoxic conditions induces metabolic reprogramming, reduces differentiation, and increases proliferation to enhance their antitumor activity, providing a strategy to improve efficacy and safety.

Mn-Anti-CTLA4-CREKA-Sericin Nanotheragnostics for Enhanced Magnetic Resonance Imaging and Tumor Immunotherapy.

The integration of magnetic resonance imaging (MRI), cGAS-STING, and anti-CTLA-4 (aCTLA-4) based immunotherapy offers new opportunities for tumor precision therapy. However, the precise delivery of aCTLA-4 and manganese (Mn), an activator of cGAS, to tumors remains a major challenge for enhanced MRI and active immunotherapy. Herein, a theragnostic nanosphere Mn-CREKA-aCTLA-4-SS (MCCS) is prepared by covalently assembling Mn2+, silk sericin (SS), pentapeptide CREKA, and aCTLA-4. MCCS are stable with an average size of 160 nm and is almost negatively charged or neutral at pH 5.5/7.4. T1-weighted images showed MCCS actively targeted tumors to improve the relaxation rate r1 and contrast time of MRI. This studies demonstrated MCCS raises reactive oxygen species levels, activates the cGAS-STING pathway, stimulates effectors CD8+ and CD80+ T cells, reduces regulatory T cell numbers, and increases IFN-γ and granzyme secretion, thereby inducing tumor cells autophagy and apoptosis in vitro and in vivo. Also, MCCS are biocompatible and biosafe. These studies show the great potential of Mn-/SS-based integrative material MCCS for precision and personalized tumor nanotheragnostics.

RUNX3 improves CAR-T cell phenotype and reduces cytokine release while maintaining CAR-T function.

CAR-T therapy has shown successful in the treatment of certain types of hematological malignancy, while the efficacy of CAR-T cell in treating solid tumors has been limited due to the exhaustion of CAR-T caused by the tumor microenvironment in solid tumors. Therefore, improving the exhaustion of CAR-T cell is one of the inspiring strategies for CAR-T treatment of solid tumors. As an important regulator in T cell immunity, the transcription factor RUNX3 not only negatively regulates the terminal differentiation T-bet gene, reducing the ultimate differentiation of T cells, but also increases the residency of T cells in non-lymphoid tissues and tumors. By overexpressing RUNX3 in CAR-T cells, we found that increasing the expression of RUNX3 maintained the low differentiation of CAR-T cells, further improving the exhaustion of CAR-T cells during antigen stimulation. In vitro, we found that RUNX3 could reduce the release of cytokines while maintaining CAR-T cells function. In re-challenge experiments, CAR-T cells overexpressing RUNX3 (Runx3-OE CAR-T) were safer than conventional CAR-T cells, while RUNX3 could also maintain the anti-tumor efficacy of CAR-T cells in vivo. Collectively, we found that Runx3-OE CAR-T cells can improve CAR-T phenotype and reduce cytokines release while maintaining CAR-T cells function, which may improve the safety of CAR-T therapy in clinical trials.

Simultaneous editing of TCR, HLA-I/II and HLA-E resulted in enhanced universal CAR-T resistance to allo-rejection.

Introduction: The major challenge for universal chimeric antigen receptor T cell (UCAR-T) therapy is the inability to persist for a long time in patients leading to inferior efficacy clinically. The objective of this study was to design a novel UCAR-T cell that could avoid the occurrence of allo-rejection and provide effective resistance to allogeneic Natural Killer (NK) cell rejection, together with the validation of its safety and efficacy ex vivo and in vivo. Methods: We prepared T-cell receptor (TCR), Human leukocyte antigen (HLA)-I/II triple-edited (TUCAR-T) cells and evaluated the anti-tumor efficacy ex vivo and in vivo. We measured the resistance of exogenous HLA-E expressing TUCAR-T (ETUCAR-T) to NK rejection by using an enhanced NK. Furthermore, we established the safety and efficacy of this regimen by treating Nalm6 tumor-bearing mice with a repeated high-dose infusion of ETUCAR-T. Moreover, we analyzed the effects of individual gene deficiency CAR-T on treated mice and the changes in the transcriptional profiles of different gene-edited T cells via RNA-Seq. Results: Data showed that HLA-II editing didn't impair the anti-tumor efficacy of TUCAR-T ex vivo and in vivo and we found for the first time that HLA-II deficiency could facilitate the persistence of CAR-T. Contrastively, as the most commonly eliminated target in UCAR-T, TCR deficiency was found to be a key disadvantageous factor for the shorter-term anti-tumor efficacy in vivo. Our study demonstrated ETUCAR-T could effectively resist allogeneic NK rejection ex vivo and in vivo. Discussion: Our research provided a potential and effective strategy for promoting the persistence of UCAR-T cells in clinical application. And it reveals the potential key factors of the poor persistence of UCAR-T along with new insights for future development.

Identification of hub genes in bladder cancer based on weighted gene co-expression network analysis from TCGA database.

BACKGROUND: Muscular invasive bladder cancer (MIBC) is a common malignant tumor in the world. Because of their heterogeneity in prognosis and response to treatment, biomarkers that can predict survival or help make treatment decisions in patients with MIBC are essential for individualized treatment. AIM: We aimed to integrate bioinformatics research methods to identify a set of effective biomarkers capable of predicting, diagnosing, and treating MIBC. To provide a new theoretical basis for the diagnosis and treatment of bladder cancer. METHODS AND RESULTS: Gene expression profiles and clinical data of MIBC were obtained by downloading from the Cancer Genome Atlas database. A dataset of 129 MIBC cases and controls was included. 2084 up-regulated genes and 2961 down-regulated genes were identified by differentially expressed gene (DEG) analysis. Then, gene ontology analysis was performed to explore the biological functions of DEGs, respectively. The up-regulated DEGs are mainly enriched in epidermal cell differentiation, mitotic nuclear division, and so forth. They are also involved in the cell cycle, p53 signaling pathway, PPAR signaling pathway, and so forth. The weighted gene co-expression network analysis yielded five modules related to pathological stages and grading, of which blue and turquoise were the most relevant modules for MIBC. Next, Using Kaplan-Meier survival analysis to identify further hub genes, the screening criteria at p ≤ .05, we found CNKSR1, HIP1R, CFL2, TPM1, CSRP1, SYNM, POPDC2, PJA2, and RBBP8NL genes associated with the progression and prognosis of MIBC patients. Finally, immunohistochemistry experiments further confirmed that CNKSR1 plays a vital role in the tumorigenic context of MIBC. CONCLUSION: The research suggests that CNKSR1, POPDC2, and PJA2 may be novel biomarkers as therapeutic targets for MIBC, especially we used immunohistochemical further to validate CNKSR1 as a therapeutic target for MIBC which may help to improve the prognosis for MIBC.