Extended Data Fig. 6: The production of antisense lentiviral vector in the presence of NovB2 and Tax does not impact the activity levels of transduced T cells. | Nature Biomedical Engineering

Extended Data Fig. 6: The production of antisense lentiviral vector in the presence of NovB2 and Tax does not impact the activity levels of transduced T cells.

From: A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumour-targeting receptor

Extended Data Fig. 6: The production of antisense lentiviral vector in the presence of NovB2 and Tax does not impact the activity levels of transduced T cells.

(a) Schematic of antisense lentiviral vector encoding the anti-PSMA (Gene A) and anti-CD19 (Gene B) CARs under the PGK promoter along with mCherry under 6xNFAT. (b) Transduction efficiency of CD4+ and CD8+ primary T cells using lentiviral supernatant produced in absence or presence of both NovB2 and Tax. Bar graphs show the mean + /- S.E.M. of percentage of CAR+ T cells. Data shown are for T cells from n = 3 human donors and symbols represent individual donors. (c) Evaluation of mCherry expression (total red area/μm2) by activated anti-PSMA and (d) Evaluation of mCherry expression (total red area/μm2) by activated anti CD19 CAR-T cells upon co-culture with PSMA + PC3-PIP tumour cells (panel c ns=0,4921 aPSMA versus aPSMA Tax-NovB2, ****P < 0,0001 aPSMA versus UTD, P = 0,0525 aPSMA Tax-NovB2 versus UTD); (panel d ns=0,9671 aCD19 versus aCd19 Tax-NovB2, *P < 0,0361 aCD19 versus UTD,**P = 0,0121 aCD19 Tax-NovB2 versus UTD). Values for the IncuCyte assay are the mean ± S.E.M. for T cells from n = 3 human donors. Statistical significance was assessed using a Two-Way ANOVA and Post-hoc Tukey test. (e) Schematic of antisense lentiviral vectors encoding the anti-PSMA or anti-CD19 CARs (Gene A) and luciferase as gene cargo (Gene B). The CARs are expressed under the PGK promoter and luciferase under 6xNFAT. (f) Induction of luciferase in anti-CD19 CAR-T cells upon 24 h co-culture with PC3-CD19+ tumour cells. Bar graph represents mean + /- S.E.M. of luminescence (counts) measured by HIDEX. Data are for T cells from n = 3 human donors. (ns=0,5563 aCD19 versus aCD19 Tax-NovB2). (g) Schematic of CAR-T cell transfer study in PC3-CD19 tumour bearing mice. (h) Caliper tumour volume measurements over days. Values are the mean + /- S.E.M. for n = 6 mice per group. Statistical significance was determined by Two-way ANOVA (Day12 ns=0,46 aCD19 versus aCd19 Tax-NovB2, ***P < 0,001 aCD19 versus UTD,***P < 0,001 aCD19 Tax-NovB2 versus UTD.

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