Contributions
Abstract: EP939
Type: E-Poster Presentation
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
Multiple myeloma (MM) is a plasma cell malignancy that remains incurable for most patients and, thus, new therapies are urgently needed. Oncolytic viruses are a promising new class of therapeutics that provide tumor-targeted therapy. In the context of developing a novel therapeutic approach for MM, we generated an IFNβ-expressing lentiviral vector (LV), pseudotyped with the measles virus HF glycoproteins that target myeloma cells through the binding to CD46 and SLAM receptors.
Aims
The purpose of this study was to assess the therapeutic efficacy of IFNβ/HF in vitro in H929 and JJN3 myeloma cell lines, as well as in primary cells from MM patients on first-diagnosis.
Methods
Production of IFNβ/HF and control GFP/HF LVs was carried out by transient co-transfection of HEK-293T cells. Vector titration and vector copy number per cell (VCN/cell) were determined by flow cytometry and qPCR, respectively. Cell survival and cytotoxicity were evaluated using Trypan blue exclusion and CCK-8 colorimetry assays, respectively. Apoptosis was estimated by Annexin V/7-AAD staining. The expression of apoptotic gene expression was determined by qPCR and Human Apoptosis Antibody Array, at mRNA and protein level, respectively. IFNβ secreted by IFNβ/HF-transduced cells was determined by ELISA, while its paracrine mechanism of action was evaluated by the Transwell® co-culture assay. Cell cycle analysis was performed with flow cytometry and propidium iodide. Lastly, serum neutralizing antibody activity was evaluated by flow cytometry on JJN3 cells transduced with GFP/HF LV, previously incubated with MM and normal donors’ serum.
Results
IFNβ/ΗF LV exhibited a mean transduction efficiency of 29.5% (H929) and 23.1% (JJN3) and a mean VCN/cell of 1.6 and 1.2, respectively. Moreover, IFNβ/HF-transduction at ΜΟΙ of 1, led to a dramatic 90.6% and 95.2% reduction of cell survival in H929 (p=0.01) and JJN3 (p=0.04) cells, respectively. Regarding apoptotic cells in H929 and JJN3 cells, a marked 90.3% (p≤0.001) and 74.1% (p≤0.001) increase was observed. Notably, the increase of Caspases 3 and 9 as well as of HtrA/Omi and Endo G proteins, both at the RNA and protein level, highlights the crucial role of the intrinsic apoptotic pathway as well as the caspase-independent pathway in the IFNβ-induced apoptosis. Moreover, a great reduction in the expression of the anti-apoptotic genes Bcl-2 and Bcl-xl was observed in IFNβ-transduced H929 and JJN3 cells. After Transwell® co-culture, IFNβ secreted by the transduced cells, was capable of significantly decreasing the viability of untransduced cells while the percentage of untransduced apoptotic cells surpassed 30% for both H929 (p=0.004) and JJN3 (p=0.01) cells. Furthermore, IFNβ/HF efficiently transduced primary cells (MOI=10) from patients with MM, decreasing cell survival up to 49.8% (p=0.0089), with a 51.8% increase of apoptosis (p=0.01) and induction of cell cycle arrest at a mean VCN/cell of 3.6. Finally, it was showed that the barrier of pre-existing immunity can be overcome using a higher MOI of 20, a positive outcome for its in vivo administration in patients vaccinated against measles virus.
Conclusion
Overall, the results of our study demonstrate that the IFNβ/HF LV can efficiently target and kill myeloma cells, providing evidence about the molecular mechanisms involved in cell death. Thus, our findings suggest that IFNβ/HF is a promising therapeutic candidate as a selective antitumor agent for the treatment of MM.
Keyword(s): Gene therapy, Lentiviral vector, Multiple myeloma
Abstract: EP939
Type: E-Poster Presentation
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
Multiple myeloma (MM) is a plasma cell malignancy that remains incurable for most patients and, thus, new therapies are urgently needed. Oncolytic viruses are a promising new class of therapeutics that provide tumor-targeted therapy. In the context of developing a novel therapeutic approach for MM, we generated an IFNβ-expressing lentiviral vector (LV), pseudotyped with the measles virus HF glycoproteins that target myeloma cells through the binding to CD46 and SLAM receptors.
Aims
The purpose of this study was to assess the therapeutic efficacy of IFNβ/HF in vitro in H929 and JJN3 myeloma cell lines, as well as in primary cells from MM patients on first-diagnosis.
Methods
Production of IFNβ/HF and control GFP/HF LVs was carried out by transient co-transfection of HEK-293T cells. Vector titration and vector copy number per cell (VCN/cell) were determined by flow cytometry and qPCR, respectively. Cell survival and cytotoxicity were evaluated using Trypan blue exclusion and CCK-8 colorimetry assays, respectively. Apoptosis was estimated by Annexin V/7-AAD staining. The expression of apoptotic gene expression was determined by qPCR and Human Apoptosis Antibody Array, at mRNA and protein level, respectively. IFNβ secreted by IFNβ/HF-transduced cells was determined by ELISA, while its paracrine mechanism of action was evaluated by the Transwell® co-culture assay. Cell cycle analysis was performed with flow cytometry and propidium iodide. Lastly, serum neutralizing antibody activity was evaluated by flow cytometry on JJN3 cells transduced with GFP/HF LV, previously incubated with MM and normal donors’ serum.
Results
IFNβ/ΗF LV exhibited a mean transduction efficiency of 29.5% (H929) and 23.1% (JJN3) and a mean VCN/cell of 1.6 and 1.2, respectively. Moreover, IFNβ/HF-transduction at ΜΟΙ of 1, led to a dramatic 90.6% and 95.2% reduction of cell survival in H929 (p=0.01) and JJN3 (p=0.04) cells, respectively. Regarding apoptotic cells in H929 and JJN3 cells, a marked 90.3% (p≤0.001) and 74.1% (p≤0.001) increase was observed. Notably, the increase of Caspases 3 and 9 as well as of HtrA/Omi and Endo G proteins, both at the RNA and protein level, highlights the crucial role of the intrinsic apoptotic pathway as well as the caspase-independent pathway in the IFNβ-induced apoptosis. Moreover, a great reduction in the expression of the anti-apoptotic genes Bcl-2 and Bcl-xl was observed in IFNβ-transduced H929 and JJN3 cells. After Transwell® co-culture, IFNβ secreted by the transduced cells, was capable of significantly decreasing the viability of untransduced cells while the percentage of untransduced apoptotic cells surpassed 30% for both H929 (p=0.004) and JJN3 (p=0.01) cells. Furthermore, IFNβ/HF efficiently transduced primary cells (MOI=10) from patients with MM, decreasing cell survival up to 49.8% (p=0.0089), with a 51.8% increase of apoptosis (p=0.01) and induction of cell cycle arrest at a mean VCN/cell of 3.6. Finally, it was showed that the barrier of pre-existing immunity can be overcome using a higher MOI of 20, a positive outcome for its in vivo administration in patients vaccinated against measles virus.
Conclusion
Overall, the results of our study demonstrate that the IFNβ/HF LV can efficiently target and kill myeloma cells, providing evidence about the molecular mechanisms involved in cell death. Thus, our findings suggest that IFNβ/HF is a promising therapeutic candidate as a selective antitumor agent for the treatment of MM.
Keyword(s): Gene therapy, Lentiviral vector, Multiple myeloma