Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells have been shown to play a pivotal role in reducing IAV-induced pulmonary infections in preclinical models; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Here, we investigated the effects of CYNK-001, human placental hematopoietic stem cell derived NK cells that exhibited strong cytolytic activity against a range of malignant cells and expressed high levels of activating receptors, against IAV infections. In a severe IAV-induced acute lung injury model, mice treated with CYNK-001 showed a milder body weight loss and clinical symptoms, which led to a delayed onset of mortality, thus demonstrating their antiviral protection in vivo . Analysis of bronchoalveolar lavage fluid (BALF) revealed that CYNK-001 reduced proinflammatory cytokines and chemokines highlighting CYNK-001’s anti-inflammatory actions in viral induced-lung injury. Furthermore, CYNK-001-treated mice had altered immune responses to IAV with reduced number of neutrophils in BALF yet increased number of CD8+ T cells in the BALF and lung compared to vehicle-treated mice. Our results demonstrate that CYNK-001 displays protective functions against IAV via its anti-inflammatory and immunomodulating activities, which leads to alleviation of disease burden and progression in a severe IAV-infected mice model. The potential of adoptive NK therapy for IAV infections warrants clinical investigation.
Introduction: Glioblastoma Multiforme (GBM) is the most aggressive brain malignancy in adults, where the 5-year survival rate is less than 10%. Celularity Inc. has developed an allogenic, off-the-shelf and cryopreserved human placental CD34+ derived natural killer (CYNK-001) cell therapy as potential treatment for GBM. NK cell-derived exosomes are notable for their anti-tumor activity. Exosomes have been demonstrated their ability to pass through blood-brain barrier in vivo. We hypothesized that engineering exosomes to deliver TGF-β short interfering RNA (siRNA) would reduce TFG-β expression, thus enhance the anti-tumor activity of CYNK-001 against GBM cells. This study aims to investigate if (i) CYNK-001-derived exosomes (CYNK-Exo) have anti-tumor effects against GBM, (ii) CYNK-Exo loaded with TGF-β siRNA (siCYNK-Exo) can effectively knockdown TGF-β expression in tumor cells, and (iii) siCYNK-Exo and CYNK-001 have a synergistic effect against GBM cells. Methods: CYNK-Exo were isolated from CYNK-001 conditioned media by one-step sucrose cushion ultracentrifugation and characterized by nano-tracking analysis (NTA) and on-bead flow cytometry. Cytotoxicity of CYNK-Exo against tumor cells was measured by real-time impedance-based xCELLigence assay. To measure uptake of siRNA, Cy3-tagged siRNA was loaded into CYNK-Exo and quantified by flow cytometry. Knockdown of TGF-β was measured by RT-qPCR and ELISA. Results: CYNK-Exo showed a median size of 117.4 nm by NTA and expressed the exosomal-characteristic CD9 and CD81, as well as NK cell-characteristic CD56, CD226 and CD11a biomarkers. CYNK-Exo displayed cytotoxicity against GBM cell line U251 in a time- and dose- dependent manner. U251 cells treated with siCYNK-Exo showed efficient uptake with 97.7% Cy3+ cells. RT-qPCR results demonstrated 78.6% reduction in TGF-β mRNA expression and ELISA results demonstrated 71.8% reduction in TGF-β secretion. Similar findings were demonstrated on LN-229 GBM cell line. To test if knocking down TGF-β could reduce tumor immunosuppression, U251 cells were treated with siCYNK-Exo followed by CYNK-001. Enhanced cytotoxicity against U251 was demonstrated with combination treatment when compared to that with CYNK-001 alone: 73.9% vs. 36.5% respectively. Compared to CYNK-001 alone, combination siCYNK-Exo and CYNK-001 treatment not only increased the secretion of the proinflammatory cytokines GM-CSF, IFN-γ, and TNF-α by CYNK-001 cells, but also enhanced the production of perforin, granzyme A and granzyme B, which are typically inhibited in NK cells by TGF-β. Conclusion: Our results demonstrated anti-tumor activity of CYNK-Exo against GBM cells; efficient TGF-β knockdown in GBM cells by siCYNK-Exo; and synergistic anti-tumor activity of TGF-β siCYNK-Exo in combination with CYNK-001 against GBM cells. Our data support a potential combination therapy using CYNK-001 and TGF-β siCYNK-Exo for GBM treatment. Citation Format: Marina Gergues, Veronica Farag, Alex Hariri, Qian Ye, Yuechao Zhao, Lin Kang, Robert Hariri, Shawn He. Developing CYNK-001-derived exosomes to deliver TGF-β siRNA for GBM immunotherapy enhancement. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5102.
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