Enhancing CAR-T cells: unleashing lasting impact potential with phytohemagglutinin activation in in vivo leukemia model.

Chimeric antigen receptor T (CAR-T) cell therapy holds great promise as an innovative immunotherapeutic approach for cancer treatment. To optimize the production and application of CAR-T cells, we evaluated the in vivo stability and efficacy capacities of CAR-T cells developed under different conditions. In this study, CAR-T cells were activated using Phytohemagglutinin (PHA) or anti-CD3&anti-CD28 and were compared in an in vivo CD19+B-cell cancer model in mouse groups. Our results demonstrated that CAR-T cells activated with PHA exhibited higher stability and anti-cancer efficacy compared to those activated with anti-CD3&anti-CD28. Specifically, CAR19BB-T cells activated with PHA exhibited continuous proliferation and long-term persistence without compromising their anti-cancer efficacy. Kaplan-Meier survival analysis revealed prolonged overall survival in the CAR-T cell-treated groups compared to the only tumor group. Furthermore, specific LTR-targeted RT-PCR analysis confirmed the presence of CAR-T cells in the treated groups, with significantly higher levels observed in the CAR19BB-T (PHA) group compared to other groups. Histopathological analysis of spleen, kidney, and liver tissue sections indicated reduced inflammation and improved tissue integrity in the CAR-T cell-treated groups. Our findings highlight the potential benefits of using PHA as a co-stimulatory method for CAR-T cell production, offering a promising strategy to enhance their stability and persistence. These results provide valuable insights for the development of more effective and enduring immunotherapeutic approaches for cancer treatment. CAR-T cells activated with PHA may offer a compelling therapeutic option for advancing cancer immunotherapy in clinical applications.

A novel EGFR inhibitor, HNPMI, regulates apoptosis and oncogenesis by modulating BCL-2/BAX and p53 in colon cancer.

BACKGROUND AND PURPOSE: Colorectal cancer (CRC) is the second most lethal disease, with high mortality due to its heterogeneity and chemo-resistance. Here, we have focused on the epidermal growth factor receptor (EGFR) as an effective therapeutic target in CRC and studied the effects of polyphenols known to modulate several key signalling mechanisms including EGFR signalling, associated with anti-proliferative and anti-metastatic properties. EXPERIMENTAL APPROACH: Using ligand- and structure-based cheminformatics, we developed three potent, selective alkylaminophenols, 2-[(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl]phenol (THTMP), 2-[(1,2,3,4-tetrahydroquinolin-1-yl)(4-methoxyphenyl)methyl]phenol (THMPP) and N-[2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl]indoline (HNPMI). These alkylaminophenols were assessed for EGFR interaction, EGFR-pathway modulation, cytotoxic and apoptosis induction, caspase activation and transcriptional and translational regulation. The lead compound HNPMI was evaluated in mice bearing xenografts of CRC cells. KEY RESULTS: Of the three alkylaminophenols tested, HNPMI exhibited the lowest IC50 in CRC cells and potential cytotoxic effects on other tumour cells. Modulation of EGFR pathway down-regulated protein levels of osteopontin, survivin and cathepsin S, leading to apoptosis. Cell cycle analysis revealed that HNPMI induced G0/G1 phase arrest in CRC cells. HNPMI altered the mRNA for and protein levels of several apoptosis-related proteins including caspase 3, BCL-2 and p53. HNPMI down-regulated the proteins crucial to oncogenesis in CRC cells. Assays in mice bearing CRC xenografts showed that HNPMI reduced the relative tumour volume. CONCLUSIONS AND IMPLICATIONS: HNPMI is a promising EGFR inhibitor for clinical translation. HNPMI regulated apoptosis and oncogenesis by modulating BCL-2/BAX and p53 in CRC cell lines, showing potential as a therapeutic agent in the treatment of CRC.

Lentiviral Micro-dystrophin Gene Treatment into Late-stage mdx Mice for Duchenne Muscular Dystrophy Disease.

AIM: Duchenne Muscular Dystrophy (DMD) results in a deficiency of dystrophin expression in patient muscle fibers, leading to progressive muscle degeneration. Treatment of DMD has undertaken current transformation with the advancement of novel gene therapy and molecular biology techniques, which are secure, well-tolerated, and effective therapeutic approaches. INTRODUCTION: DMD gene therapies have mainly focused on young DMD patients as in vivo animal model trials have been performed in 0-1-month DMD mice. However, it has not yet been answered how micro-dystrophin encoding lentiviral treatment affects Dystrophin expression and DMD symptoms in 10-month mdx mice. METHODS: We planned to integrate the micro-Dystrophin gene sequence into the muscle cells by viral transfer, using micro-Dystrophin-encoding lentivirus to reduce the dystrophic pathology in late-stage dmd mice. The histopathological and physiological-functional regeneration activities of the lentiviralmicro- Dystrophin gene therapy methods were compared, along with changes in temporal Dystrophin expression and their functionality, toxicity, and gene expression level. RESULTS: Here, we showed that the micro-dystrophin transgene transfers intramuscularly and intraperitoneally in late-stage dmd-mdx-4cv mice restored dystrophin expression in the skeletal and cardiac muscle (p <0.001). Furthermore, motor performance analysis, including hanging and tracking tests, improved statistically significantly after the treatment (p <0.05). CONCLUSION: Consequently, this study suggests that patients in the late stages of muscular dystrophy can benefit from lentiviral micro-dystrophin gene therapies to present an improvement in dystrophic muscle pathology.

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice.

The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A and smallpox proved to be reliable approaches for immunization for prolonged periods. In this study, a gamma-irradiated inactivated virus vaccine does not require an extra purification process, unlike the chemically inactivated vaccines. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral RNA copy per dose) of OZG-38.61.3 was initially determined in BALB/c mice. This was followed by testing the immunogenicity and protective efficacy of the vaccine. Human ACE2-encoding transgenic mice were immunized and then infected with the SARS-CoV-2 virus for the challenge test. This study shows that vaccinated mice have lowered SARS-CoV-2 viral RNA copy numbers both in oropharyngeal specimens and in the histological analysis of the lung tissues along with humoral and cellular immune responses, including the neutralizing antibodies similar to those shown in BALB/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates.

COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2 + mice.

Caffeic acid attenuates gastric mucosal damage induced by ethanol in rats via nitric oxide modulation.

Anti-oxidant and anti-inflammatory properties of caffeic acid (CA) have been reported recently. In this study, the therapeutic effects of CA on ethanol-induced ulcer and the roles of nitric oxide and cholinergic pathways in these effects were investigated. Ulcer was induced by ethanol via oral gavage. Ulcer induced rats were treated with either vehicle (ulcer group) or CA (100, 250 or 500 mg/kg, per oral gavage). Macroscopic evaluation showed that 250 mg/kg CA was the effective dose. To elucidate the action mechanism of CA, 10 mg/kg l-NAME or 1 mg/kg atropine sulfate was administered to 250 mg/kg CA treated groups. All rats were decapitated 1 h after ulcer induction and gastric samples were scored macroscopically and microscopically, and analyzed for myeloperoxidase (MPO), malondialdehyde (MDA), and glutathione (GSH) levels. ANOVA test was used for statistical analyses. Macroscopic and microscopic damage scores, MDA levels and MPO activity were increased while GSH levels were decreased in ulcer group. Treatment with 250 mg/kg and 500 mg/kg CA reduced macroscopic and microscopic damage scores, decreased MPO activity and MDA levels, and preserved the depleted glutathione significantly. l-NAME administration before CA treatment elevated MDA levels, MPO activity and depleted glutathione. However, atropine sulfate had no effect on biochemical parameters. We conclude that CA ameliorates ethanol-induced gastric mucosal damage, and NO pathway contributes to this effect. On the other hand, there is a lack of evidence for the contribution of the muscarinic cholinergic system.

Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

Objective: Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey. Materials and Methods: We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors' and ALL/NHL patients' peripheral blood mononuclear cells. Results: We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice. Conclusion: This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.