Engineered Luminescent Oncolytic Vaccinia Virus Activation of Photodynamic-Immune Combination Therapy for Colorectal Cancer.

Oncolytic virus therapy is currently regarded as a promising approach in cancer immunotherapy. It has greater therapeutic advantages for colorectal cancer that is prone to distant metastasis. However, the therapeutic efficacy and clinical application of viral agents alone for colorectal cancer remain suboptimal. In this study, an engineered oncolytic vaccinia virus (OVV-Luc) that expresses the firefly luciferase gene is developed and loaded Chlorin e6 (Ce6) onto the virus surface through covalent coupling, resulting in OVV-Luc@Ce6 (OV@C). The OV@C infiltrates tumor tissue and induces endogenous luminescence through substrate catalysis, resulting in the production of reactive oxygen species. This unique system eliminates the need for an external light source, making it suitable for photodynamic therapy (PDT) in deep tissues. Moreover, this synergistic effect between PDT and viral immunotherapy enhances dendritic cell maturation, macrophage polarization, and reversal of the immunosuppressive microenvironment. This synergistic effect has the potential to convert a "cold" into a "hot" tumor, it offers valuable insights for clinical translation and application.

Tumor-targeted delivery of copper-manganese biomineralized oncolytic adenovirus for colorectal cancer immunotherapy.

Oncolytic viral therapy (OVT) is a novel anti-tumor immunotherapy approach, specifically replicating within tumor cells. Currently, oncolytic viruses are mainly administered by intratumoral injection. However, achieving good results for distant metastatic tumors is challenging. In this study, a multifunctional oncolytic adenovirus, OA@CuMnCs, was developed using bimetallic ions copper and manganese. These metal cations form a biomineralized coating on the virus's surface, reducing immune clearance. It is known that viruses upregulate the expression of PD-L1. Copper ions in OA@CuMnCs can decrease the PD-L1 expression of tumor cells, thereby promoting immune cell-related factor release. This process involves antigen presentation and the combination of immature dendritic cells, transforming them into mature dendritic cells. It changes "cold" tumors into "hot" tumors, further inducing immunogenic cell death. While oncolytic virus replication requires oxygen, manganese ions in OA@CuMnCs can react with endogenous hydrogen peroxide. This reaction produces oxygen, enhancing the virus's replication ability and the tumor lysis effect. Thus, this multifunctionally coated OA@CuMnCs demonstrates potent amplification in immunotherapy efficacy, and shows great potential for further clinical OVT. STATEMENT OF SIGNIFICANCE: Oncolytic virus therapy (OVs) is a new anti-tumor immunotherapy method that can specifically replicate in tumor cells. Although the oncolytic virus can achieve a therapeutic effect on some non-metastatic tumors through direct intratumoral injection, there are still three major defects in the treatment of metastatic tumors: immune response, hypoxia effect, and administration route. Various studies have shown that the immune response in vivo can be overcome by modifying or wrapping the surface protein of the oncolytic virus. In this paper, a multifunctional coating of copper and manganese was prepared by combining the advantages of copper and manganese ions. The coating has a simple preparation method and mild conditions, and can effectively enhance tumor immunotherapy.

Integrated lithium niobate microwave photonic processing engine.

Integrated microwave photonics (MWP) is an intriguing technology for the generation, transmission and manipulation of microwave signals in chip-scale optical systems1,2. In particular, ultrafast processing of analogue signals in the optical domain with high fidelity and low latency could enable a variety of applications such as MWP filters3-5, microwave signal processing6-9 and image recognition10,11. An ideal integrated MWP processing platform should have both an efficient and high-speed electro-optic modulation block to faithfully perform microwave-optic conversion at low power and also a low-loss functional photonic network to implement various signal-processing tasks. Moreover, large-scale, low-cost manufacturability is required to monolithically integrate the two building blocks on the same chip. Here we demonstrate such an integrated MWP processing engine based on a 4 inch wafer-scale thin-film lithium niobate platform. It can perform multipurpose tasks with processing bandwidths of up to 67 GHz at complementary metal-oxide-semiconductor (CMOS)-compatible voltages. We achieve ultrafast analogue computation, namely temporal integration and differentiation, at sampling rates of up to 256 giga samples per second, and deploy these functions to showcase three proof-of-concept applications: solving ordinary differential equations, generating ultra-wideband signals and detecting edges in images. We further leverage the image edge detector to realize a photonic-assisted image segmentation model that can effectively outline the boundaries of melanoma lesion in medical diagnostic images. Our ultrafast lithium niobate MWP engine could provide compact, low-latency and cost-effective solutions for future wireless communications, high-resolution radar and photonic artificial intelligence.

Functional diversification and dynamics of CAR-T cells in patients with B-ALL.

Understanding of cellular evolution and molecular programs of chimeric antigen receptor-engineered (CAR)-T cells post-infusion is pivotal for developing better treatment strategies. Here, we construct a longitudinal high-precision single-cell transcriptomic landscape of 7,578 CAR-T cells from 26 patients with B cell acute lymphoblastic leukemia (B-ALL) post-infusion. We molecularly identify eight CAR-T cell subtypes, including three cytotoxic subtypes with distinct kinetics and three dual-identity subtypes with non-T cell characteristics. Remarkably, long-term remission is coincident with the dominance of cytotoxic subtypes, while leukemia progression is correlated with the emergence of subtypes with B cell transcriptional profiles, which have dysfunctional features and might predict relapse. We further validate in vitro that the generation of B-featured CAR-T cells is induced by excessive tumor antigen stimulation or suppressed TCR signaling, while it is relieved by exogenous IL-12. Moreover, we define transcriptional hallmarks of CAR-T cell subtypes and reveal their molecular changes along computationally inferred cellular evolution in vivo. Collectively, these results decipher functional diversification and dynamics of peripheral CAR-T cells post-infusion.

Photodynamic immunotherapy using ADU-S100-modified nanoparticles to treat triple-negative breast cancer.

Aim: STING agonists in immunotherapy show great promise and are currently in clinical trials. Combinations of STING agonists with other therapies remain underexplored. This study aimed to combine STING agonist-mediated immunotherapy with photodynamic therapy to treat breast cancer. Methods: STING agonist (ADU-S100)-functionalized porphyrin-based nanoparticles (NP-AS) were prepared and their antitumor properties in terms of cell apoptosis/necrosis and immune activation in triple-negative breast cancer were evaluated. Results: NP-AS induced tumor cell apoptosis/necrosis, activated the innate immune response and exhibited useful antitumor effects. Conclusion: NP-AS effectively treated breast cancer.

Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) treatment remains challenging. CD70 was reported as a promising AML-specific antigen. Preclinically, CAR T-cell with single-chain-variable fragment (scFv) or truncated CD27 targeting CD70 has been reported to treat AML. However, various disadvantages including spontaneous exhaustion, proteinase-mediated loss of functional receptors, and high immunogenicity, limited its further application to clinical settings. Alternatively, the single-variable domain on heavy chain (VHH), also known as nanobodies, with comparable binding ability and specificity, provides an optional solution. METHOD: We generated CD70 knocked-out novel nanobody-based anti-CD70-CAR T-cells (nb70CAR-T) with two different VHHs for antigen detection. Next, we detected the CD70 expression on primary AML blasts by flow cytometry and associated the efficacy of nb70CAR-T with the target antigen density. Finally, epigenetic modulators were investigated to regulate the CD70 expression on AML cells to promote the functionality of nb70CAR-T. RESULTS: Our nb70CAR-T exhibited expected tumoricidal functionality against CD70-expressed cell lines and primary AML blasts. However, CD70 expression in primary AML blasts was not consistently high and nb70CAR-T potently respond to an estimated 40.4% of AML patients when the CD70 expression level was over a threshold of 1.6 (MFI ratio). Epigenetic modulators, Decitabine and Chidamide can up-regulate CD70 expression on AML cells, enhancing the treatment efficacy of nb70CAR-T. CONCLUSION: CD70 expression in AML blasts was not fully supportive of its role in AML targeted therapy as reported. The combinational use of Chidamide and Decitabine with nb70CAR-T could provide a new potential for the treatment of AML.

Extracellular Vesicles Expressing CD19 Antigen Improve Expansion and Efficacy of CD19-Targeted CAR-T Cells.

Background: CAR-T cell therapy is effective in the treatment of certain hematological malignancies, and the expansion and functional persistence of CAR-T cells in vivo are crucial to clinical efficacy. The aim of this study was to investigate the potential of extracellular vesicles (EVs) modified with the CAR antigen to promote the efficacy of CAR-T cells in vivo. Methods: We generated HEK293T-derived EVs to present the CD19 antigen as the CAR target. In vitro, EVs expressing CD19 antigen (CD19 EVs) were co-incubated with anti-CD19 CAR-T cells. Then, proliferation, cytokine secretion, CD107a expression, tumor killing, subsets, and immune checkpoint expression were measured to assess CAR-T cell function. After infusion of CD19 EVs pretreated CAR-T cells into a lymphoma xenograft mouse model, flow cytometry and digital PCR were used to measure the expansion of CAR-T cells, and tumor volumes were continuously monitored to assess the anti-tumor efficacy of CAR-T cells in vivo. Another mouse model was created to investigate the effect of in vivo injection of CD19 EVs on the functional persistence of CAR-T cells, and safety was determined by histopathology of the main organs. Results: CD19 EVs activated CAR-T cells in an antigen-specific and dose-dependent manner and promoted the selective expansion and cytokine secretion of co-cultured CAR-T cells. Specifically, CD19 EVs preferably increased the expansion of the CAR-T subpopulation with a high surface CD19-CAR density and consequently enhanced the anti-tumor activity of CAR-T cells. Futhermore, CD19-EVs-primed CAR-T cells achieved superior proliferation and anti-tumor effects in a mouse model with lymphoma xenograft. In vivo administration of CD19 EVs promoted the functional persistence of CAR-T cells in the xenograft mouse model. Conclusion: Our findings indicate that antigen-expressing EVs can be utilized as a boost to improve CAR-T cell efficacy in vitro and in vivo.

T Cell Defects: New Insights Into the Primary Resistance Factor to CD19/CD22 Cocktail CAR T-Cell Immunotherapy in Diffuse Large B-Cell Lymphoma.

Despite impressive progress, a significant portion of patients still experience primary or secondary resistance to chimeric antigen receptor (CAR) T-cell immunotherapy for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). The mechanism of primary resistance involves T-cell extrinsic and intrinsic dysfunction. In the present study, a total of 135 patients of DLBCL treated with murine CD19/CD22 cocktail CAR T-therapy were assessed retrospectively. Based on four criteria (maximal expansion of the transgene/CAR-positive T-cell levels post-infusion [Cmax], initial persistence of the transgene by the CAR transgene level at +3 months [Tlast], CD19+ B-cell levels [B-cell recovery], and the initial response to CAR T-cell therapy), 48 patients were included in the research and divided into two groups (a T-normal group [n=22] and a T-defect [n=26] group). According to univariate and multivariate regression analyses, higher lactate dehydrogenase (LDH) levels before leukapheresis (hazard ratio (HR) = 1.922; p = 0.045) and lower cytokine release syndrome (CRS) grade after CAR T-cell infusion (HR = 0.150; p = 0.026) were independent risk factors of T-cell dysfunction. Moreover, using whole-exon sequencing, we found that germline variants in 47 genes were significantly enriched in the T-defect group compared to the T-normal group (96% vs. 41%; p<0.0001), these genes consisted of CAR structure genes (n=3), T-cell signal 1 to signal 3 genes (n=13), T cell immune regulation- and checkpoint-related genes (n=9), cytokine- and chemokine-related genes (n=13), and T-cell metabolism-related genes (n=9). Heterozygous germline UNC13D mutations had the highest intergroup differences (26.9% vs. 0%; p=0.008). Compound heterozygous CX3CR1I249/M280 variants, referred to as pathogenic and risk factors according to the ClinVar database, were enriched in the T-defect group (3 of 26). In summary, the clinical characteristics and T-cell immunodeficiency genetic features may help explain the underlying mechanism of treatment primary resistance and provide novel insights into CAR T-cell immunotherapy.

CAR19/22 T cell therapy in adult refractory Burkitt's lymphoma.

The treatment of refractory Burkitt's lymphoma (BL) is still a challenge. Although CAR-T cell therapy has achieved good responses in diffuse large B cell lymphoma, there is no case series report about the efficacy of CAR-T cell therapy in adult Burkitt's lymphoma. In this study, we evaluate the efficacy and safety of CAR19/22 T cell therapy in six refractory Burkitt's lymphoma cases with poor genetic prognostic factors. After CAR-T cell therapy, five cases had grade 1 and one had grade 3 cytokine release syndrome. Three patients achieved an objective response (3/6 50%), including two partial remission and one complete remission. One CR patient received allogeneic hematopoietic stem cell transplantation (HSCT) and one PR patient received CAR22/19-T cells following auto-HSCT, and they were still in remission at 37 and 22 months of follow-up, respectively. Interestingly, patients with bulky disease (case 2, 4 and 5) had higher levels of serum IL-2R, which was secreted by regulatory T cells, lower CAR lentiviral amplification and poorer prognosis with shorter survival time than cases with non-bulky disease. It is suggested that high tumor burden, more immune suppressive cells and limited CAR-T cell expansion might affect the efficacy of CAR-T cell therapy. CAR-T cell therapy in adult BL patients whose best response cannot achieve CR may need to bridge to other treatments (such as HSCT) early.

Role of leptin in the regulation of food intake in fasted mice.

Leptin is well acknowledged as an anorexigenic hormone that plays an important role in feeding control. Hypothalamic GABA system plays a significant role in leptin regulation on feeding and metabolism control. However, the pharmacological relationship of leptin and GABA receptor is still obscure. Therefore, we investigated the effect of leptin or combined with baclofen on the food intake in fasted mice. We detected the changes in hypothalamic c-Fos expression, hypothalamic TH, POMC and GAD67 expression, plasma insulin, POMC and GABA levels to demonstrate the mechanisms. We found that leptin inhibit fasting-induced increased food intake and activated hypothalamic neurons. The inhibitory effect on food intake induced by leptin in fasted mice can be reversed by pretreatment with baclofen. Baclofen reversed leptin's inhibition on c-Fos expression of PAMM in fasted mice. Therefore, these results indicate that leptin might inhibit fasting-triggered activation of PVN neurons via presynaptic GABA synaptic functions which might be partially blocked by pharmacological activating GABA-B. Our findings identify the role of leptin in the regulation of food intake.