mouse transferrin / serotransferrin protein  (Sino Biological)

Journal: Science Advances

Article Title: Biodegradable targeted polymeric mRNA nanoparticles enable in vivo CD19 CAR T cell generation and lead to B cell depletion

doi: 10.1126/sciadv.adz1722

Figure Lengend Snippet: In standard, ex vivo CAR T cell therapy, T cells are isolated from patient blood and activated using biomaterial systems that present anti-CD3 and anti-CD28. Activated T cells are then transduced with a viral vector to permanently express a CAR. These cells are expanded for 10 to 14 days until a therapeutic dose of CAR T cells is reached before they are reinfused into the patient. In the proposed system of tPNP-mediated in vivo CAR T cell generation, NPs composed of a PBAE polymer, a functional lipid linker, and mRNA encoding a CAR construct are fabricated and combined with anti-CD3 and anti-CD28 antibodies. Upon infusion of tPNPs into a patient, these particles will target, activate, and transfect endogenous T cells to generate functional CAR T cells. In both ex vivo and in vivo systems, the final CAR T cell will then kill its target CD19 + B cell.

Article Snippet: Samples were prepared for flow cytometric analysis of CAR expression via centrifugation at 500 g for 5 min and resuspension in 100 μl of PBS with a 1:1000 dilution of Live Dead-Near IR (Thermo Fisher Scientific, catalog no. L10119 ) and a 1:250 dilution of biotinylated mouse CD19 protein (Sino Biological, catalog no. 50510-M08H-B).

Techniques: Ex Vivo, Isolation, Transduction, Plasmid Preparation, In Vivo, Polymer, Functional Assay, Construct

Journal: Science Advances

Article Title: Biodegradable targeted polymeric mRNA nanoparticles enable in vivo CD19 CAR T cell generation and lead to B cell depletion

doi: 10.1126/sciadv.adz1722

Figure Lengend Snippet: ( A ) Primary murine T cell transfection was evaluated with tPNPs in naïve cells at a dose of 100 ng of mRNA per 50,000 cells ( n = 4). Transfection, as measured by expression of an anti-CD19 CAR construct and the mean fluorescent intensity (MFI) of that CAR expression, is shown. ( B ) CAR expression in primary murine T cells was analyzed over time. T cells were cocultured with tPNPs on day 0, and CAR expression was measured after 1, 2, 3, 4, and 7 days ( n = 3). ( C and D ) T cell activation after 24 hours (h) of coculture with tPNPs was analyzed via CD69 (C) and CD25 (D) expression ( n = 3). ( E ) T cell memory phenotype after 24 hours of coculture with tPNPs was analyzed via CD44 and CD62L expression ( n = 3). Memory subsets were delineated as follows: Naïve: CD44-CD62L + , central memory–like: CD44 + CD62L + , effector memory–like: CD44 + CD62L − . ( F ) T cell proliferation after 72 hours of coculture with tPNPs was measured via CellTrace Violet (CTV) signal. Proliferation is indicated by the number of peaks in CTV signal, with each peak representing a cell division from the original parent population. Proliferation was evaluated relative to a no treatment negative control and a Dynabead positive control, which is a commercially used reagent for T cell stimulation. ( G ) T cell transfection with aCD3+aCD28 PBAE NPs was compared to transfection with aCD3+aCD28 targeted LNPs using the Moderna LNP/SM-102 ionizable lipid formulation at a dose of 100 ng of mRNA per well of a 96-well plate. Statistical analysis for all experiments was performed using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ns, not significant.

Article Snippet: Samples were prepared for flow cytometric analysis of CAR expression via centrifugation at 500 g for 5 min and resuspension in 100 μl of PBS with a 1:1000 dilution of Live Dead-Near IR (Thermo Fisher Scientific, catalog no. L10119 ) and a 1:250 dilution of biotinylated mouse CD19 protein (Sino Biological, catalog no. 50510-M08H-B).

Techniques: Transfection, Expressing, Construct, Activation Assay, Negative Control, Positive Control, Cell Stimulation, Formulation

Journal: Science Advances

Article Title: Biodegradable targeted polymeric mRNA nanoparticles enable in vivo CD19 CAR T cell generation and lead to B cell depletion

doi: 10.1126/sciadv.adz1722

Figure Lengend Snippet: ( A ) Schematic of in vivo CAR-mediated B cell depletion study. Ten micrograms of tPNPs delivering anti-CD19 CAR-encoding mRNA was administered intravenously to C57BL/6 mice ( n = 5). Mice were bled 12 and 36 hours after NP administration. B cell depletion was measured in blood cells and systemic cytokine secretion was measured in the plasma. Mice were euthanized at the 36-hour time point, and spleens were isolated and processed to measure B cell depletion. ( B to D ) B cell depletion was measured in the blood at 12 hours (B) and 36 hours (C), as well as the spleen at 36 hours (D). Significant CD19 and CD20 B cell killing was elicited by aCD3 and aCD3+aCD28 CAR tPNPs compared to uncoated CAR NPs and control aCD3+aCD28 luciferase mRNA NPs. ( E ) Systemic secretion of cytokines, IFN-γ, TNF-α, IL6, IL-10, CCL3, and granulocyte-macrophage colony-stimulating factor (GM-CSF), was measured in the plasma at 12 and 36 hours. For (B) to (D), individual statistical analysis for depletion of CD19 + or CD20 + cells in each organ was performed using a one-way ANOVA with Tukey’s multiple comparisons test. For (E), individual statistical analysis for secretion of each cytokine at the 12- or 36-hour time point was performed using a one-way ANOVA with Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Samples were prepared for flow cytometric analysis of CAR expression via centrifugation at 500 g for 5 min and resuspension in 100 μl of PBS with a 1:1000 dilution of Live Dead-Near IR (Thermo Fisher Scientific, catalog no. L10119 ) and a 1:250 dilution of biotinylated mouse CD19 protein (Sino Biological, catalog no. 50510-M08H-B).

Techniques: In Vivo, Clinical Proteomics, Isolation, Control, Luciferase

Journal: Science Advances

Article Title: Biodegradable targeted polymeric mRNA nanoparticles enable in vivo CD19 CAR T cell generation and lead to B cell depletion

doi: 10.1126/sciadv.adz1722

Figure Lengend Snippet: ( A ) Schematic of in vivo CAR-mediated B cell depletion kinetics study. Ten micrograms of tPNPs delivering anti-CD19 CAR-encoding mRNA was administered intravenously to C57BL/6 mice ( n = 5). Mice were bled on day 0 before tPNP administration to establish a baseline level of B cells in the blood. After aCD3+aCD28 CAR tPNP administration, the mice were bled on days 1, 2, 4, and 7, with B cell depletion being measured in blood cells at each time point. ( B ) B cell depletion was found to be transient, with depletion peaking on day 1 [(B) right] and decreasing over time. ( C ) Schematic of in vivo multiple CAR-tPNP dosing B cell depletion study. Ten micrograms of tPNPs delivering anti-CD19 CAR-encoding mRNA was administered intravenously to C57BL/6 mice ( n = 4) on either days 0 and 1 or on days 0 and 3. Mice blood and spleens were harvested on day 4, and B cell depletion was compared to a baseline PBS-treated control mouse. ( D ) Potent B cell depletion was observed in the peripheral blood and ( E ) the spleen on D4. ( F ) Representative immunohistochemistry (IHC) stains of spleens treated with tPNPs across different dosing regimens. B cells are stained in blue, and T cells are stained in purple. All IHC stains are shown at 2.5× magnification; scale bar, 1 mm, and all samples can be found in fig. S12. For (D) and (E), statistical analysis for depletion of CD19 + B cells in the blood and spleen, respectively, was performed using a one-way ANOVA with Tukey’s multiple comparisons test, * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Samples were prepared for flow cytometric analysis of CAR expression via centrifugation at 500 g for 5 min and resuspension in 100 μl of PBS with a 1:1000 dilution of Live Dead-Near IR (Thermo Fisher Scientific, catalog no. L10119 ) and a 1:250 dilution of biotinylated mouse CD19 protein (Sino Biological, catalog no. 50510-M08H-B).

Techniques: In Vivo, Control, Immunohistochemistry, Staining