Strong Cytotoxicity Research Articles

Characterization of a 3S PRAME VLD-Specific T Cell Receptor and Its Use in Investigational Medicinal Products for TCR-T Therapy of Patients with Myeloid Malignancies

Background/Objectives: MDG1011 is an autologous TCR-T therapy developed as a treatment option for patients with myeloid malignancies, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). It is specific for the target antigen PReferentially expressed Antigen in MElanoma (PRAME). The recombinant TCR used in MDG1011 recognizes PRAME100–108 VLD-peptide presented by HLA-A*02:01-encoded surface molecules. Methods: Two preclinical batches of MDG1011, produced from enriched CD8+ T cells of healthy donors, underwent rigorous evaluation of on-target and off-target recognition of tumor cells and test cells representing healthy tissues. MDG1011 investigational medicinal products (IMPs) were produced for 13 patients. VLD-TCR surface expression was assessed using dual-marker flow cytometry using TCR V-beta-specific antibody and VLD/HLA-A2-specific multimer. Functionality was assessed by interferon-gamma (IFN-γ) secretion and cell-mediated cytotoxicity of target cells. Results: Preclinical MDG1011 batches displayed strong VLD-TCR expression, cytokine secretion, and cytotoxicity after antigen-specific activation, while showing no signals of on-target/off-tumor or off-target recognition. All IMPs had good VLD-TCR expression as well as functionality after activation by multiple target cells. Conclusions: Preclinical studies demonstrated that MDG1011 displayed key 3S attributes of high specificity, sensitivity, and safety required for regulatory approval of a first-in-human (FIH) clinical study of patients with myeloid malignancies (CD-TCR-001: ClinicalTrials.gov Identifier: NCT03503968). MDG1011 IMP manufacturing was successful at 92%, even including heavily pretreated elderly patients with very advanced disease. The IMPs applied in nine patients all displayed antigen-specific functionality. Elsewhere, clinical study results for MDG1011 showed no dose-limiting toxicity and signs of biological and/or clinical activity in several patients.

Depletion of alloreactive B cells by drug- resistant chimeric alloantigen receptor T cells to prevent transplant rejection.

Antibody-mediated rejection (AMR) remains a major complication after solid organ transplantation (SOT). Current treatment options are inefficient and result in drastic impairment of the general immunity. To selectively eliminate responsible alloreactive B cells characterized by anti-donor-HLA B-cell receptors (BCRs), we generated T cells overcoming rejection by antibodies (CORA-Ts) engineered with a novel chimeric receptor comprising a truncated donor-HLA molecule as antigen recognition domain. As proof-of-concept, CORA receptors based on HLA-A*02 were developed. In co-cultures with anti-HLA-A*02 B-cell lines, CORA-Ts were specifically activated, released pro-inflammatory mediators, and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A*02 antibody release. Significant reduction of growth of an anti-HLA-A*02 B-cell line could be confirmed using an in vivo mouse model. Modification of the CORA receptor effectively abrogated T-cell binding, thereby avoiding T-cell sensitization. Additionally, using CRISPR/Cas9-mediated knockout of the FKBP12 gene, CORA-Ts were able to resist immunosuppressive treatment with tacrolimus, thereby allowing high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically eliminate alloreactive, anti-HLA B cells, thus selectively preventing anti-HLA antibody release even under immunosuppressive conditions. This suggests CORA-Ts as potent approach to combat AMR and improve long-term graft survival in SOT patients while preserving their overall B-cell immunity.

Royal Jelly's Strong Selective Cytotoxicity Against Lung Malignant Cells and Macromolecular Alterations in Cells Observed by FTIR Spectroscopy.

Several nutraceuticals, food, and cosmetic products can be developed using royal jelly. It is known for its potential health benefits, including its ability to boost the immune system and reduce inflammation. It is rich in vitamins, minerals, and antioxidants, which can improve general health. Royal jelly (RJ) is also being studied as a potential therapeutic agent for cancer and other chronic diseases. It is effective in reducing tumor growth and stimulating immunity. In this study, we investigated the effects of royal jelly on cancerous A549 cells and healthy MRC-5 cells at various doses ranging from 1.25 to 10 mg/ml. Royal jelly's anti-proliferative effect was evaluated by MTT and SRB assay for 48 h. The induction of necrosis and apoptosis was assessed by flow cytometry as well. The relative amounts of major molecules in Royal jelly were determined by FTIR spectroscopy to identify key functional groups and molecular structures. In addition, this technique was used for the first time to detect changes in the macromolecular composition of lung cells treated with royal jelly. Thus, it provided insights into the relative abundance of proteins, lipids, and carbohydrates, which could correlate with their bioactive properties. The antiproliferative effect of Royal jelly was found to be selective on A549 cells in a dose-dependent manner with an IC50 of 9.26 mg/mL, with no cytotoxic effects on normal MRC-5 cells. Moreover, Royal jelly induced predominantly necrotic cell death in A549 cells, %39.10 at 4 mg/ml and %57.88 at 10 mg/ml concentrations. However, the necrosis rate in MRC-5 cells was quite low, at 9.16% and 20.44% at the same doses. Royal jelly showed dose-dependent selective cytotoxicity toward A549 cells, whereas it exhibited no apparent cytotoxicity in MRC-5 cells. In order to identify the biomolecular changes induced by royal jelly, we used two unsupervised chemometric pattern recognition algorithms (PCA and HCA) on the preprocessed sample FTIR spectra to determine the effects of royal jelly on cell biochemistry. According to PCA and HCA results, RJ treatments especially affected biomolecules in A549 cells. The total spectral band variances in the PCA loading spectra were calculated for understanding biomolecular alterations. These plots revealed profound changes in the lipid, protein, and nucleic acid content of RJ-applied lung cells, primarily identifying RJ and H2O2 treated groups for A549 cells. Ultimately, the selective cytotoxicity of royal jelly toward A549 cancerous cells suggests that royal jelly may be a promising therapeutic agent for identifying innovative lung cancer treatment strategies. Additionally, understanding the molecular alterations induced by royal jelly could guide the development of novel cancer treatments that exploit its bioactive properties. This could lead to more effective and safer therapies.

Benzimidazole Derivatives in Breast Cancer: Target-Specific Therapeutic Breakthroughs.

Despite ongoing advancements in drug design and developments, breast cancer remains a serious and devastating disease and is ranked as the second most common illness in women. Breast cancer rates have increased significantly during the last 40 years. This necessitates the development of novel treatment techniques. Currently, chemotherapy is the primary mode of treatment for breast cancer; however, its toxicity to normal cells and drug resistance are considered the main obstacles. Researchers are looking for novel anti-breast cancer medication classes to improve cancer therapy efficacy and survival rates. Using non-targeting medicines in a 'one-size-fits-all' strategy can harm healthy cells and may not be effective for all patients. Thus, now, the treatment of breast cancer is exploring targeted-based therapy. The tactics involved in this therapy may improve patient survival rates, but their extended usage can lead to significant side effects and medication resistance. Targeted therapy enables precision medicine by targeting particular oncogenic markers in malignancies. Because of their strong cytotoxicity against several cancer cell types, heterocyclic compounds play an important role in the development of therapeutically effective anticancer drugs. Benzimidazole derivatives have grown in favour of anti-breast cancer medicines in recent years due to their broad biological characteristics and therapeutic applications. This review provides healthcare professionals and researchers with an overview of current breakthroughs (2019-2024) in benzimidazole derivatives as breast cancer-targeted therapy, based on the perspectives of leading experts. We have illuminated the diverse and evolving landscape of hybridized benzimidazole, along with its biological targets and anti-breast cancer activity. Further, we also have compiled the various ongoing clinical trials of benzimidazole scaffolds as anti-breast cancer agents. A detailed illustration of the structure-activity connection with special emphasis is provided. The effort may help to develop potent, selective, and effective drugs to combat breast cancer.

Engineering a controllable and reversible switch for CAR-based cellular immunotherapies via a genetic code expansion system

BackgroundAs one of the most promising adoptive cell therapies, CAR-T cell therapy has achieved notable clinical effects in patients with hematological tumors. However, several treatment-related obstacles remain in CAR-T therapy, such as cytokine release syndrome, neurotoxicity, and high-frequency recurrence, which severely limit the long-term effects and can potentially be fatal. Therefore, strategies to increase the controllability and safety of CAR-T therapy are urgently needed.MethodsIn this study, we engineered a genetic code expansion-based therapeutic system to achieve rapid CAR protein expression and regulation in response to cognate unnatural amino acids at the translational level. When the unnatural amino acid N-ε-((tert-butoxy) carbonyl)-l-lysine (BOCK) is absent, the CAR protein cannot be completely translated, and CAR-T is “closed”. When BOCK is present, complete translation of the CAR protein is induced, and CAR-T is “open”. Therefore, we investigated whether the BOCK-induced device can control CAR protein expression and regulate CAR-T cell function using a series of in vitro and in vivo experiments.ResultsFirst, we verified that the BOCK-induced genetic code expansion system enables the regulation of protein expression as a controllable switch. We subsequently demonstrated that when the system was combined with CAR-T cells, BOCK could effectively and precisely control CAR protein expression and induce CAR signaling activation. When incubated with tumor cells, BOCK regulated CAR-T cells cytotoxicity in a dose-dependent manner. Our results revealed that the presence of BOCK enables the activation of CAR-T cells with strong anti-tumor cytotoxicity in a NOG mouse model. Furthermore, we verified that the BOCK-induced CAR device provided NK cells with controllable anti-tumor activity, which confirmed the universality of this device.ConclusionsOur study systematically demonstrated that the BOCK-induced genetic code expansion system effectively and precisely regulates CAR protein expression and controls CAR-T cell anti-tumor effects in vitro and in vivo. We conclude that this controllable and reversible switch has the potential for more effective, secure, and clinically available CAR-based cellular immunotherapies.

Metabolomic approach in anticancer biomarker discovery of folious lichens

Purpose: Lichens are well-known as a source of pharmacologically active compounds. This includes anticancer compounds which have biomass constraints including using traditional techniques of lichen bioprospecting. This current study reports the use of cutting-edge metabolomics and a computational approach to discover anticancer biomarkers from Indonesian lichens. Methods: Seven lichen crude extracts were evaluated against cervical cell lines HeLa using a MTT assay and secondary metabolites were profiled and recorded via a GCMS protocol. A multivariate analysis OPLSDA was employed to determine anticancer biomarker of the lichens. A structure-based computational study against the HeLa cancer cell related protein targets (BCL-2 (4MAN), AKT-1 (4GV1), MCL-1 (5FDO), and BRAF (5VAM)) was used to determine the most potent biomarker. Results: The MTT assessment indicated the seven lichens possessed strong, medium and weak cytotoxicity. Multivariate analysis showed an OPLS-DA score plot with distinct separation among the strong, medium and weak cytotoxic groups. The biplot OPLS-DA and GC-MS analysis proposed 13 compounds of Parmelia caroliniana and 12 compounds of Physcia cf. millegrana as anticancer biomarker candidates. Docking experiments revealed 6-amino-3,4,7-triphenylpyrido[2',3':4,5]thieno[2,3-c]pyridazine 4 from P. caroliniana to possess the highest binding affinity against BCL-2 (4MAN), AKT-1 (4GV1), MCL-1 (5FDO), and BRAF (5VAM) proteins with affinity energy values of -10.0, -11.6, -10.4, -12.6, respectively. Conclusion: The study successfully revealed compound 4 as the anticancer biomarker against HeLa cell cancer of P. caroliniana in which can be further explored through in vitro and in vivo studies. Further, the metabolomic protocol established can be adapted as a tool for biomarker discoveries from other medicinal plants.

Folic Acid-Targeted Mixed Pluronic Micelles for Delivery of Triptolide.

The present study aimed to explore an ideal delivery system for triptolide (TPL) by utilizing the thin-film hydration method to prepare drug-loaded, folate-modified mixed pluronic micelles (FA-F-127/F-68-TPL). Scanning electron microscopy and atomic force microscopy showed that the drug-loaded micelles had a spherical shape with a small particle size, with an average of 30.7 nm. Cell viability experiments showed that FA-F-127/F-68-TPL significantly reduced HepG2 cell viability, exhibiting strong cytotoxicity. Its cytotoxicity was markedly enhanced compared with bare TPL. Nile red (Nr) was used as a model drug to prepare FA-F-127/F-68-Nr to further validate its tumor-targeting and cellular uptake capability. After coincubation with HepG2 cells, a multifunctional microplate reader showed that intracellular fluorescence intensity significantly increased, indicating that FA-F-127/F-68-Nr could more effectively enter the cells. A nude mouse model of subcutaneous hepatocellular carcinoma was constructed. Following tail vein injection of FA-F-127/F-68-Nr, the fluorescence imaging system showed that FA-F127/F-68-Nr could significantly target tumor tissue, and even if entering the small-sized tumor was challenging, it could be excreted through urine. Nude mice with subcutaneous hepatocellular carcinoma were treated with tail vein injections of FA-F-127/F-68-TPL (45 µg/kg) every other day for 21 days. The results showed that the growth of the transplanted tumors was significantly slowed, with no significant difference compared with bare TPL. In summary, the FA-F-127/F-68-TPL exhibits the advantages of low cost, excellent biological properties, active/passive targeting capabilities, notable cytotoxicity against liver cancer cells, and significant inhibition of transplanted hepatocellular carcinoma growth. Significantly, the FA-F-127/F-68-TPL, despite challenges in targeting tumors with an insignificant EPR effect, can be efficiently excreted via the kidneys, thereby preventing the release of the drug during prolonged circulation and potential damage to normal tissues. Therefore, FA-F-127/F-68-TPL represents a promising antitumor drug delivery system.

Organometallic Half-Sandwich Complexes of 8-Hydroxyquinoline-Derived Mannich Bases with Enhanced Solubility: Targeting Multidrug Resistant Cancer.

Drug resistance is a major obstacle in cancer treatment. Herein, four novel organometallic complexes, with the general formula [Ru(η6-p-cymene)(HL)Cl]Cl and [Rh(η5-C5Me5)(HL)Cl]Cl, were developed to target multidrug-resistant (MDR) cancer cells, where HL denotes 8-hydroxyquinoline-derived Mannich bases (HQCl-pyr and HQCl-pip). The aim of the complexation was to obtain compounds with improved drug-like properties. The complexes were comprehensively characterized by various spectroscopic methods in terms of their structure, solution speciation and interaction with human serum albumin. The structure of [Rh(η5-C5Me5)(HQCl-pip)Cl]Cl was analyzed by X-ray crystallography. The complexes were found to be highly stable in solution and in various biological matrices, showing enhanced solubility compared with the ligands and significant binding ability to albumin via coordination. The Rh(η5-C5Me5) complexes exhibited strong cytotoxicity against MDR MES-SA/Dx5 cell lines (IC50 = 0.19 and 0.22 μM), demonstrating high MDR-selectivity. Ganglioside-functionalized nanoparticles with the most promising ligand HQCl-pip and its Rh(η5-C5Me5) complex were prepared to enhance the bioavailability. The nanocarriers showed faster drug release at acidic pH than at pH 7.4, and could retain the cytotoxicity and selectivity of the loaded compounds. The encapsulated Rh(η5-C5Me5) complex of HQCl-pip has been identified as an optimal candidate for the pharmacological development of MDR-selective compounds.

Discovery of anti-SARS-CoV-2 S2 protein antibody CV804 with broad-spectrum reactivity with various beta coronaviruses and analysis of its pharmacological properties in vitro and in vivo.

The SARS-CoV-2 pandemic alerted the potential for significant harm due to future cross-species transmission of various animal coronaviruses to human. There is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses. In this study, we generated CV804, an antibody that binds to the S2 domain of SARS-CoV-2 spike protein, which is highly conserved across the coronavirus family and less susceptible to mutations. CV804 demonstrated broad cross-reactivities not only disease-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and with existing mutant strains of SARS-CoV-2 and but also with 20 representative animal-origin coronaviruses. CV804 exhibits strong antibody-dependent cellular cytotoxicity (ADCC) to SARS-CoV-2 spike protein expressed on cells in vitro, while completely lacks virus-neutralization activity. In animal models, CV804 suppressed disease progression caused by SARS-CoV-2 infection. Structural studies using HDX-MS combined with reactivity analysis with point mutants of recombinant spike proteins revealed that CV804 binds to a unique conformational epitope within the S2 domain of the spike proteins that is highly conserved among various coronaviruses. Overall, obtained data suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein displayed on the surface of infected cells and weakens the viral virulence by supporting the host immune cells' attack through ADCC activity in vivo. The CV804 epitope information revealed in this study is useful for designing pan-corona antibody therapeutics and universal coronavirus vaccines for preparing potential future pandemics.

Improved epirubicin delivery selectivity to bladder cancer achieved by functionalized hydroxyethyl starch-based prodrug.

Bladder instillation of chemo-therapeutic agents is common for bladder cancer (BC) treatment, however, due to the poor tissue selectivity of chemotherapeutic agents, this method suffers from bladder irritation or even chemical cystitis. Here, we designed a hydroxyethyl starch-based prodrug for epirubicin (EPI) using a pH-sensitive hydrazone linker and folate as the active targeting moiety (FA-HES-hyd-EPI) to achieve delivery selectivity. Prodrug micelles decorated with FA (FA-m), with diameter of 203.6 ± 7.1 nm and EPI loading of 5.80 ± 0.129 %, were prepared by self-assemble method. FA-m was found with improved cellular uptake and stronger cytotoxicity over EPI. An orthotopic BC model was established with FA receptor over-expression, and FA-m showed significantly higher accumulation at tissue and cell levels compared with the FA-free counterpart. Besides, the systemic exposure of FA-m was significantly lower than EPI (AUC0-t: 28.11 ± 7.23 ng/mL/h versus 11.60 ± 4.27 ng/mL/h). More importantly, FA-m showed stronger in vivo tumor growth inhibition with average tumor weight of 0.26 ± 0.21 mg (EPI group: 1.695 ± 0.62 mg), and better safety according to body weight and HE staining data. Overall, the combination of active targeting and pH responsive prodrug strategies was a feasible way to achieve selective EPI delivery to BC via instillation that improved both therapeutic effect and safety.

The role of imbalanced CD226/TIGIT on activated peripheral double-negative T cells in the pathogenesis of primary Sjögren's syndrome.

To explore whether the balance of CD226 and TIGIT is disturbed in CD3+CD56-TCRαβ+CD4-CD8- (DN) T cells and have a better understanding of the potential role of DN T cells in the pathogenesis of primary Sjögren's syndrome (pSS). The percentage of DN T cells as well as the expression of CD226 and TIGIT was identified by flowmetry. After in vitro stimulation, we further detected the expression of activation and cytotoxic marker, as well as intracellular cytokines secreted by DN T cells. DN T cells were found to expand in the peripheral blood of pSS patients (1.77±0.66%) and correlate with IgG (r=0.451, p<0.05), C3 (r=-0.438, p<0.05) and C4 (r=-0.470, p<0.05). Imbalanced CD226/TIGIT was observed on peripheral DN T cells of pSS patients, especially the overexpression of inhibitory immunoreceptor TIGIT. The expression ratio of TIGIT and CD226 on DN T cells was elevated in pSS patients and correlated with ESSDAI scores≥5 (r=0.743, p<0.05). Besides, these DN T cells were found to be activated and show strong cytotoxicity. The balance between CD226 and TIGIT on DN T cells was disturbed and correlated with the disease activity in pSS patients, which may be implicated in the pathogenesis of pSS.

Based on sodium alginate coatings and dendritic copolymeric modification of curcumin delivery system: pH-sensitive nanospheres and strong tumor cytotoxicity

This study aims to construct a novel drug delivery strategy to address the poor bioavailability and biostability of curcumin. A curcumin delivery strategy, basing on post-polymerization modification of poly(2-vinyl-4,4-dimethyl azlactone) to obtain conjugates of curcumin and dendritic polymers, combined with sodium alginate coating is reported. The curcumin-polymer conjugates were shown to have good fluorescence properties with fluorescence quantum yields of 0.486 and 0.470, respectively. The composites were characterized as spherical nanoparticles with a desirable particle size of 221.7 nm and massive negatively charged surfaces. These aesthetic properties make it an acceptable drug delivery and release vehicle. In vitro release experiments showed that release of curcumin from the conjugates was controllable and acid-sensitive, which is expected to guide delivery targeting to cancer sites. In addition, biodistribution studies of the gastrointestinal tract of mice showed high levels of exposure. The results of cell imaging showed that conjugates have strong permeability to cancer cell membranes. They have been shown to have strong targeting and inhibitory effects on a variety of cancer cells, with an inhibition rate of up to about 90 %. Therefore, such novel vector designs show great potential in drug delivery mechanism research and cancer therapy.

Unraveling SARS-CoV-2 Host-Response Heterogeneity through Longitudinal Molecular Subtyping.

Hospitalized COVID-19 patients exhibit diverse immune responses during acute infection, which are associated with a wide range of clinical outcomes. However, understanding these immune heterogeneities and their links to various clinical complications, especially long COVID, remains a challenge. In this study, we performed unsupervised subtyping of longitudinal multi-omics immunophenotyping in over 1,000 hospitalized patients, identifying two critical subtypes linked to mortality or mechanical ventilation with prolonged hospital stay and three severe subtypes associated with timely acute recovery. We confirmed that unresolved systemic inflammation and T-cell dysfunctions were hallmarks of increased severity and further distinguished patients with similar acute respiratory severity by their distinct immune profiles, which correlated with differences in demographic and clinical complications. Notably, one critical subtype (SubF) was uniquely characterized by early excessive inflammation, insufficient anticoagulation, and fatty acid dysregulation, alongside higher incidences of hematologic, cardiac, and renal complications, and an elevated risk of long COVID. Among the severe subtypes, significant differences in viral clearance and early antiviral responses were observed, with one subtype (SubC) showing strong early T-cell cytotoxicity but a poor humoral response, slower viral clearance, and greater risks of chronic organ dysfunction and long COVID. These findings provide crucial insights into the complex and context-dependent nature of COVID-19 immune responses, highlighting the importance of personalized therapeutic strategies to improve both acute and long-term outcomes.

Cloning and Functional Analysis of Skin Host Defense Peptides from Yakushima Tago's Brown Frog (Rana tagoi yakushimensis) and Development of Serum Endotoxin Detection System.

Background/Objective: Amphibian skin is a valuable source of host defense peptides (HDPs). This study aimed to identify HDPs with novel amino acid sequences from the skin of Rana tagoi yakushimensis and analyze their functions. Methods: cDNAs encoding HDP precursors were cloned and sequenced using RT-PCR and 3'-RACE. The novel HDPs were synthesized to evaluate their antimicrobial activity, antioxidant activity, and cytotoxicity. Antimicrobial activity was evaluated by way of broth microdilution and endotoxin- and β-glucan-binding capacity using an enzyme-linked endotoxin binding assay (ELEBA) and a modified ELEBA, respectively. Results: Nine cDNAs encoding precursors for various HDP families, including temporin, ranatuerin-2, brevinin-1, amurin-9, and a novel yakushimin peptide, were identified. Brevinin-1TYa exhibited antibacterial activity against Staphylococcus aureus, and brevinin-1TYa and amurin-9TYa induced morphological changes in Escherichia coli and S. aureus. Yakushimin-TYa, amurin-9TYa, and brevinin-1TYa showed concentration-dependent antibacterial effects against the plant pathogens Xanthomonas oryzae pv. oryzae and Clavibacter michiganensis subsp. michiganensis. Amurin-9TYa demonstrated strong binding affinity to lipopolysaccharide, lipoteichoic acid, and β-glucan, exhibited antioxidant activity, and lacked cytotoxicity, making it a promising therapeutic candidate. Moreover, brevinin-1TYa showed strong cytotoxicity, whereas yakushimin-TYa exhibited weak cytotoxicity. Conclusions: These findings highlight the potential of these peptides, particularly amurin-9TYa, for future applications as antimicrobial and therapeutic agents.

Oral and tumor-targeting mixed micelles based on pegylated biotin and chitosan-based conjugate for breast cancer treatment

Oral drug delivery with tumor-targeting treatment persists as a challenge. In this study, a mixed polymeric micelle (PM) delivery system was designed to overcome the barriers to oral administration for tumor-targeting delivery of paclitaxel (PTX) for breast cancer treatment. D-α-Tocopheryl polyethylene glycol 1000 succinate-modified carboxymethyl chitosan-rhein (TCR) conjugate and Biotin-polyethylene glycol 2000-Biotin (BPB) conjugate were mixed to form TCR-BPB PMs. The particle size and polydispersity index of optimized PTX-loaded TCR-BPB PMs (PTX/TCR-BPB PMs) was 195.90 ± 7.63 nm and 0.08 ± 0.00, with a drug-loading capacity of 41.94 ± 2.47 %. In simulated gastroenteric fluid and blood environments, the PMs presented a sustained-release manner. PTX/TCR-BPB PMs were taken up by Caco-2 and 4T1 cells and displayed strong cytotoxicity in a time- and concentration-dependent manner. PTX/TCR-BPB PMs significantly improved intestinal absorption of PTX. The pharmacokinetics, tissue distribution, and in vivo imaging indicated that PTX/TCR-BPB PMs could enhance oral bioavailability of PTX, prolong the retention time of PTX in the blood, and enhance PTX tumor-targeting ability. PTX/TCR-BPB PMs enhance the antitumor efficacy of PTX and reduce its toxicity in normal organs. Therefore, TCR-BPB PMs are expected to serve as delivery carriers for the oral and tumor-targeting delivery of hydrophobic antitumor drugs.

Environmental Stability Determines the Cytotoxicity of Metal-Organic Frameworks to a Nitrogen-Fixing Bacterium Azotobacter vinelandii.

During widespread applications of metal-organic frameworks (MOFs), the environmental hazards and risks of MOFs have aroused great concerns. In this study, we aimed to reveal the importance of the environmental stability of MOFs on their toxicity. Two Zn-MOFs, namely, ZIF-8 with high aqueous stability and Zn-BDC with low aqueous stability, were compared directly in the toxicological evaluations of a nitrogen-fixing bacterium Azotobacter vinelandii. Zn-BDC showed strong cytotoxicity at 100 mg/L and higher, inducing growth inhibition, cell apoptosis, structural changes, oxidative damage, and, consequently, loss of nitrogen fixation ability. In contrast, ZIF-8 was nearly nontoxic to A. vinelandii. The transcriptome analysis showed that Zn-BDC directly disturbed the ribosome pathway and lowered the expression level of nitrogen-fixing nif cluster genes. On the other hand, ZIF-8 stress could regulate the flagellar assembly, siderophore group nonribosomal peptide biosynthesis, bacterial chemotaxis, and amino sugar and nucleotide sugar metabolism pathways to promote the cell growth of A. vinelandii. Beyond that, the toxicity of Zn-MOFs to A. vinelandii was associated with the release of Zn2+, but Zn-MOFs were less toxic than the mixtures of their starting materials. Overall, our results suggested that the environmental stability of Zn-MOFs determined their environmental toxicity through different molecular pathways. Designing stable MOFs is preferred due to environment-friendly considerations.

Targeting mitochondrial metabolism by the mitotoxin bromoxib in leukemia and lymphoma cells

Targeting mitochondrial metabolism represents a promising approach for cancer treatment. Here, we investigated the mitotoxic potential of the polybrominated diphenyl ether bromoxib, a natural compound isolated from the marine sponge Dysidea family. We could show that bromoxib comprised strong cytotoxicity in different leukemia and lymphoma cell lines (such as HL60, HPBALL, Jurkat, K562, KOPTK1, MOLT4, SUPB15 and Ramos), but also in solid tumor cell lines (such as glioblastoma cell lines SJ-GBM2 and TP365MG). Bromoxib activated the mitochondrial death pathway as evidenced by the rapid translocation of Bax to the mitochondria and the subsequent mitochondrial release of Smac. Accordingly, bromoxib-induced apoptosis was blocked in caspase 9 deficient Jurkat cells and Jurkat cells overexpressing the antiapoptotic protein Bcl-2. In addition, we could show that bromoxib functioned as an uncoupler of the electron transport chain with similar rapid kinetics as CCCP in terms of dissipation of the mitochondrial membrane potential (ΔΨm), processing of the dynamin-like GTPase OPA1 and subsequent fragmentation of mitochondria. Beyond that, bromoxib strongly abrogated ATP production via glycolysis as well as oxidative phosphorylation (OXPHOS) by targeting electron transport chain complexes II, III, and V (ATP-synthase) in Ramos lymphoma cells. Thus, bromoxib’s potential to act on both cytosolic glycolysis and mitochondrial respiration renders it a promising agent for the treatment of leukemia and lymphoma.

DDEL-02. PRECLINICAL EFFICACY OF IL13RΑ2-TARGETING POLYPEPTIDE-DRUG CONJUGATE (SELF-DEPOTTM ONCOPDC) IN GLIOBLASTOMA AND DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are currently incurable cancers. Systemic chemotherapy shows limited efficacy because of the intact blood-brain barrier. Convection-enhanced delivery (CED) offers promise by concentrating high doses of chemotherapeutics directly within these tumors. However, drugs administered via CED often face challenges in confirming precise target delivery and are quickly cleared by increased interstitial fluid flow and drug efflux transporters. This highlights the urgent need for innovative drugs that demonstrate target specificity and prolonged retention capabilities. Intrinsically disordered polypeptides (IDPs), derived from tropoelastin, self-assemble into water-insoluble structures with extended retention properties triggered by body temperature. IL13Rα2, differentially amplified in U87MG GBM and SF8628 DIPG cells, serves as a target for therapeutic intervention. An IL13Rα2-targeting IDP (XM161) was developed by incorporating IL13Rα2 binding sequences into the IDP domains. XM161 demonstrated high selectivity in binding to IL13Rα2, with Kd values of 1,786 nM for IL13Rα1 and 12.8 nM for IL13Rα2. Conjugation of XM161 with SN38, a potent topoisomerase I inhibitor, resulted in XM161-SN38, which remained in a dissolved state up to 50°C but formed insoluble aggregates above 27°C in the presence of cerebrospinal fluid. XM161-SN38 exhibited strong cytotoxicity against U87MG and SF8628 cells, with IC50 values of 14.2 nM and 0.48 nM, respectively. Importantly, XM161-SN38 demonstrated efficacy in overcoming Temozolomide resistance in T98G GBM cells, with IC50 values of 3.35 nM compared to 10.87 nM for unconjugated SN38. In orthotopic xenograft models established in BALB/cSlc-nu/nu male mice using U87MG-Luc2 cells, three doses (3 x 0.95 μg SN38 equivalents) of XM161-SN38 delivered via CED were well tolerated and significantly extended median survival to over 65 days, providing a notable survival benefit compared to untreated controls (42 days) and Topotecan-treated group (42.5 days). These findings validate the feasibility and therapeutic potential of XM161-SN38 for GBM treatment. Ongoing studies are underway to investigate the safety and pharmacokinetics of XM161-SN38 in tumor-naive mouse brains.

Peripheral Blood-Derived PD-1/CD28-CD19 CAR-Modified PD-1+ T-Cell Therapy in Patients with Solid Tumors.

T cells expressing programmed cell death 1 (PD-1) in the peripheral blood (PB) of patients with tumors possess therapeutic potential; however, the immunosuppressive, PD-1-triggered signaling pathway and limited proliferative capacity of PD-1+ T cells present challenges to their therapeutic application. Here, we observed no discernible distinction between PD-1+ and PD-1- T cells in terms of clonal overlap. However, CD8+PD-1+ T cells from PB and tumor tissues exhibited tighter clustering based on clone size. Single-cell RNA sequencing analysis showed that PD-1+ T cells from PB highly expressed cytotoxicity-related genes and were enriched for T-cell activation-related pathways compared with PD-1- T cells from PB or tumor tissues. Consistent with this, PB-derived PD-1+ T cells exhibited strong cytotoxicity toward autologous tumor cells and tumor cell lines. To augment PD-1+ T-cell activity against solid tumors in vivo, we introduced a PD-1/CD28 fusion receptor combined with a CD19 chimeric antigen receptor into PD-1+ T cells, which were then expanded in vitro. The modified PD-1+ T cells exhibited superior proliferation and antitumor abilities in vitro. In addition, four patients with cancer were infused with autologous PD-1/CD28-CD19 chimeric antigen receptor PD-1+ T cells. None of these patients experienced severe side effects, and one patient with melanoma achieved a complete response that was maintained for 6.7 months. The three other patients had stable disease. Collectively, these results suggested that cell therapy with modified PB-derived PD-1+ T cells is both safe and effective, and it may constitute a promising treatment strategy for patients with cancer.

PD-1+CD8+ T Cell-Mediated Hepatocyte Pyroptosis Promotes Progression of Murine Autoimmune Liver Disease.

The specific mechanisms underlying effector pathways in autoimmune liver disease remain enigmatic and therefore constructing appropriate murine models to investigate disease pathogenesis becomes critical. A spontaneous severe murine model of autoimmune liver disease has been previously established in dnTGFβRII Aire-/- mice, exhibiting disease phenotypes that resemble both human primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). The data suggests that auto-reactive liver-specific CD8+ T cells are the primary pathogenic cells in liver injury. In this study, these data are advanced through the use of both single-cell sequencing and extensive in vitro analysis. The results identify a specific expanded pathogenic subset of PD-1+CD8+ T cells in the liver, exhibiting strong functional activity and cytotoxicity against target cells. Depletion of PD-1+CD8+ T cells using CAR-T cells effectively alleviates the disease. GSDMD-mediated pyroptosis is found to be aberrantly activated in the livers of model mice, and treatment with a GSDMD-specific inhibitor significantly inhibits disease progression. In vitro experiments reveal that PD-1+CD8+ T cells can induce the pyroptosis of hepatocytes through elevated production of granzyme B and perforin-1. These results provide a novel explanation for the cytotoxic activity of pathogenic liver PD-1+CD8+ T cells in autoimmune liver diseases and offer potential therapeutic targets.