Tumor Antigen-Primed Dendritic Cell-Derived Exosome Synergizes with Colony Stimulating Factor-1 Receptor Inhibitor by Modulating the Tumor Microenvironment and Systemic Immunity.

Dendritic cell-derived exosomes (Dex) have overcome the disadvantages associated with dendritic cell (DC) vaccines, such as cost effectiveness, stability, and sensitivity to the systemic microenvironment. However, in clinical trials, Dex failed to provide satisfactory results because of many reasons, including inadequate maturation of DC as well as the immunosuppressive tumor microenvironment (TME). Hence, culturing DCs in the presence of a maturation cocktail showed an induced expression of MHCs and co-stimulatory molecules. Additionally, targeting the colony stimulating factor-1 (CSF-1)/CSF-1 receptor (CSF-1R) signaling pathway by a CSF-1R inhibitor could deplete tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) which are responsible for immunosuppressive TME. Hence, in this study, mDexTA were isolated from bone marrow-derived DC cultured in the presence of a novel maturation cocktail and tumor antigen. mDexTA showed elevated expression of major histocompatibility complexes (MHCs) and co-stimulatory molecules and was found capable of activating naïve DC and T cells in vitro more efficiently when compared to imDexTA isolated from immature DCs. In addition, PLX-3397, a small molecule inhibitor of CSF-1/CSF-1R, was used in combination to enhance the antitumor efficacy of mDexTA. PLX-3397 showed dose-dependent toxicity against bone marrow-derived macrophages (BMDMs). In the B16-F10 murine melanoma model, we found that the combination treatment delayed tumor growth and improved survival compared to the mice treated with mDexTA alone by enhancing the CD8 T cells infiltration in TME. mDexTA when combined with PLX-3397 modulated the TME by shifting the Th1/Th2 toward a dominant Th1 population and depleting the TAMs and MDSCs. Interestingly, PLX-3397-induced FoxP3 expression was diminished when it was used in combination with mDexTA. Combination treatment also induced favorable systemic antitumor immunity in the spleen and lymph node. In conclusion, our findings provide insights into the synergy between mDexTA-based immunotherapy and PLX-3397 as the combination overcame the disadvantages associated with monotherapy and offer a therapeutic strategy for the treatment of solid tumors including melanoma.

Ponatinib delays the growth of solid tumours by remodelling immunosuppressive tumour microenvironment through the inhibition of induced PD-L1 expression.

BACKGROUND: Therapeutic modalities including chemo, radiation, immunotherapy, etc. induce PD-L1 expression that facilitates the adaptive immune resistance to evade the antitumour immune response. IFN-γ and hypoxia are some of the crucial inducers of PD-L1 expression in tumour and systemic microenvironment which regulate the expression of PD-L1 via various factors including HIF-1α and MAPK signalling. Hence, inhibition of these factors is crucial to regulate the induced PD-L1 expression and to achieve a durable therapeutic outcome by averting the immunosuppression. METHODS: B16-F10 melanoma, 4T1 breast carcinoma, and GL261 glioblastoma murine models were established to investigate the in vivo antitumour efficacy of Ponatinib. Western blot, immunohistochemistry, and ELISA were performed to determine the effect of Ponatinib on the immunomodulation of tumour microenvironment (TME). CTL assay and flow cytometry were such as p-MAPK, p-JNK, p-Erk, and cleaved caspase-3 carried out to evaluate the systemic immunity induced by Ponatinib. RNA sequencing, immunofluorescence and Western blot analysis were used to determine the mechanism of PD-L1 regulation by Ponatinib. Antitumour immunity induced by Ponatinib were compared with Dasatinib. RESULTS: Here, Ponatinib treatment delayed the growth of tumours by inhibiting PD-L1 and modulating TME. It also downregulated the level of PD-L1 downstream signalling molecules. Ponatinib enhanced the CD8 T cell infiltration, regulated Th1/Th2 ratio and depleted tumour associated macrophages (TAMs) in TME. It induced a favourable systemic antitumour immunity by enhancing CD8 T cell population, tumour specific CTL activity, balancing the Th1/Th2 ratio and lowering PD-L1 expression. Ponatinib inhibited FoxP3 expression in tumour and spleen. RNA sequencing data revealed that Ponatinib treatment downregulated the genes related to transcription including HIF-1α. Further mechanistic studies showed that it inhibited the IFN-γ and hypoxia induced PD-L1 expression via regulating HIF-1α. Dasatinib was used as control to prove that Ponatinib induced antitumour immunity is via PD-L1 inhibition mediated T cell activation. CONCLUSIONS: RNA sequencing data along with rigorous in vitro and in vivo studies revealed a novel molecular mechanism by which Ponatinib can inhibit the induced PD-L1 levels via regulating HIF-1α expression which leads to modulation of tumour microenvironment. Thus, our study provides a novel therapeutic insight of Ponatinib for the treatment of solid tumours where it can be used alone or in combination with other drugs which are known to induce PD-L1 expression and generate adaptive resistance.

Multifaceted Nano-DEV-IL for Sustained Release of IL-12 to Avert the Immunosuppressive Tumor Microenvironment and IL-12-Associated Toxicities.

Interleukin-12 (IL-12) demonstrates potent antitumor activity by enhancing Th1/Th2 response, facilitating cytotoxic T-cell (CTL) recruitment into tumors, inhibiting tumor angiogenesis, and depleting immunosuppressive cells in the tumor microenvironment (TME). Despite having encouraging preclinical and some clinical results, further development of IL-12 is limited because dose-limiting toxicity is observed in early clinical trials with systemic administration of recombinant human IL-12. Hence, strategies aiming to lower the toxicity and to improve response rates are unmet needs. In this study, IL-12 was encapsulated in extracellular vesicles derived from mature dendritic cells (DEVs) activated with tumor antigens. IL-12-encapsulated DEVs (DEV-IL) delayed the growth of murine glioblastoma by facilitating the recruitment of CD8 T-cells, NK-cells, and DCs and effectively depleting immunosuppressive cells in the TME. DEV-IL shifted the Th1/Th2 ratio toward dominating Th1 cytokines which further led to the inhibition of angiogenesis. In addition, DEV-IL also modulated systemic immunity by enhancing CTL activity and the levels of proinflammatory cytokines in the spleen. Interestingly, DEV-IL did not impart hepatic and immunotoxicity which was observed with free IL-12 administration. Hence, our study established DEV-IL as a potent platform for the sustained delivery of cytokines and could be a promising immunotherapeutic strategy for the treatment of cancer.

Repurposing Ponatinib as a PD-L1 Inhibitor Revealed by Drug Repurposing Screening and Validation by In Vitro and In Vivo Experiments.

Cancer treatment by inhibiting the PD-1/PD-L1 pathway using monoclonal antibodies has made great advances as it showed long-lasting antitumor responses in a wide range of cancers. However, antibodies exhibit several disadvantages, which include low permeability, immune-related adverse effects, complex synthetic procedures, and high treatment costs. Hence, small-molecule inhibitors can be used as alternatives; however, no small molecule with in vivo activity has been reported. In addition, there are many challenges in developing a new drug, including the timeline and escalating cost. Therefore, repurposing an approved drug offers advantages over the development of an entirely new drug. Herein, we identify an FDA-approved small-molecule drug, Ponatinib, as a PD-L1 inhibitor via virtual drug screening of the ZINC database. Ponatinib showed stable binding with PD-L1, with the highest binding energy among all of the screened FDA-approved drugs. The binding of Ponatinib with PD-L1 was supported by a fluorescence quenching assay and immunofluorescence study. Further, we compared the in vivo antitumor efficacy of Ponatinib with a commercially available anti-PD-L1 antibody in the murine melanoma model. Ponatinib was found to be more efficient in delaying tumor growth than the anti-PD-L1 antibody. Furthermore, Ponatinib also reduced the expression of PD-L1 in tumors and increased the T-cell population. Interestingly, splenocytes isolated from Ponatinib-treated mice showed enhanced cytotoxic T-cell (CTL) activity against B16-F10 cells. However, Ponatinib itself did not have any direct toxic effect on cancer cells in vitro. These findings suggest that Ponatinib can be used as a potent small-molecule inhibitor of PD-L1 to overcome the disadvantages associated with antibodies.

SARS-CoV-2 Spike Protein-Activated Dendritic Cell-Derived Extracellular Vesicles Induce Antiviral Immunity in Mice.

The onset and spread of the SARS-CoV-2 virus have created an unprecedented universal crisis. Although vaccines have been developed against the parental SARS-CoV-2, outbreaks of the disease still occur through the appearance of different variants, suggesting a continuous need for improved and effective therapeutic strategies. Therefore, we developed a novel nanovesicle presenting Spike protein on the surface of the dendritic cell-derived extracellular vesicles (DEVs) for use as a potential vaccine platform against SARS-CoV-2. DEVs express peptide/MHC-I (pMHC-I) complexes, CCR-7, on their surface. The immunogenicity and efficacy of the Spike-activated DEVs were tested in mice and compared with free Spike protein. A 1/10 Spike equivalent dose of DEVs showed a superior potency in inducing anti-Spike IgG titers in blood of mice when compared to dendritic cells or free Spike protein treatment. Moreover, DEV-induced sera effectively reduced viral infection by 55-60% within 15 days of booster dose administration. Furthermore, a 1/10 Spike equivalent dose of DEV-treated mice was found to be equally effective in inducing CD19+CD38+ T-cells in the spleen and lymph node; CD8 cells in the bone marrow, spleen, and lymph node; and CD4+CD25+ T-cells in the spleen and lymph node after 90 days of treatment. Thus, our results support the immunogenic nature of DEVs, demonstrating that a low dose of DEVs induces antibodies to inhibit SARS-CoV-2 infection in vitro, therefore warranting further investigations.