Figure Legends

Figure 1: Basic characterization of 62L-LV.

A) Physical properties of 62L-LV vector stocks. Three independently produced 62L-LV stocks were analyzed for size (left panel) and for particle concentration (right panel) by nanoparticle tracking analysis (filled bar; technical triplicates) or p24-ELISA (open bar, biological replicates). Means and standard deviations (SD) are depicted.B ) 62L-LV stocks were titrated on HT1080αHiscells or activated human PBMC. Individual results and means with standard error (SEM) are plotted. Statistical significance was determined by using unpaired t-test. C ) Target and non-target HT1080 cells were incubated with 2.5 µL 62L-LV stock for 4 days or left untransduced. Antibody staining for ΔLNGFR allowed detection of transduced cells by flow cytometry. Left panel: Representative dot plots of one vector stock. Right panel: Percent ΔLNGFR expressing HT1080 cells after transduction with seven different vector stocks. Dashed lines indicate baseline ΔLNGFR levels on each individual cell line. Individual results as well as means with SD are plotted. Statistical testing was performed by using ordinary 1-way ANOVA. WT = parental HT1080 cells.

Figure 2: CAR gene delivery to primary lymphocytes by 62L-LV

Activated PBMC were incubated with 62L-LV (blue dots) or VSV-LV (orange dots). A)  Transduction rates in the presence (+V1) or absence (-V1) of Vectofusin-1. Left panel: Representative dot plots of 62L-LV transduced PBMC pre-gated for CD3+ cells. Right panel: Results for PBMC from seven different donors in four independent experiments analyzed 9 - 12 days post-transduction. For VSV-LV, no V1 was applied. Individual results and means with standard deviation (SD) are plotted. Statistical analysis was performed by using paired t-test for the comparison of 62L-LV +/-V1 and by using unpaired t-test for the comparison of 62L-LV and VSV-LV. B) The total percentage of ΔLNGFR+ cells expressing CD62L is displayed for the CD4+ (left) and CD8+ (right) fractions. Transduction was performed in absence of V1 for 62L-LV and VSV-LV, respectively. Cells transduced with either vector were tested for significant differences at each analysis time point individually by Fisher’s least significant difference (LSD) test. Mean with standard error (SEM) of 8 individual experiments is plotted.
Figure 3: Selectivity in 62L-LV binding to primary T lymphocytes .
Activated PBMC incubated either with the CD62L-specific antibody (blue bars) or with the CD45-specific antibody (grey bars) at the indicated concentrations before PBS (open bars) or 62L-LV vector particles (filled bars) were added for 30 min at 4°C. A ) Fluorophore-labelled αCD62L and αCD45 antibodies were used to determine the staining intensity of CD62L and CD45 on activated PBMC at the indicated concentrations by flow cytometry. Mean fluorescent intensities (MFI) are shown. B ) PBMC were pre-incubated with biotin-labelled antibodies before addition of 62L-LV particles. After vector incubation, cells were stained with fluorophore coupled αCD3 and αLNGFR antibodies to allow detection of vector bound T cells by flow cytometry. Background MFI (dashed line) is determined from samples incubated with PBS (w/o) instead of 62L-LV accumulated for all antibody concentrations. Means of MFI and standard deviations (SD) are depicted. Statistical testing was performed by using 2-way ANOVA.

Figure 4: Shed CD62L does not influence vector binding.

A ) Accumulation of sCD62L in the supernatant of PBMC. Frozen PBMC were thawed, activated and cultivated in presence of IL-7 and IL-15. The complete supernatant of one well was collected at the indicated day and used for sCD62L quantification (left panel). The amounts of sCD62L present in three independent cultures at day 6 are shown in the right panel. Individual results, means and standard deviation (SD) are depicted. B ) 62L-LV (blue) or VSV-LV (orange) particles were incubated with fresh or frozen supernatant containing sCD62L (day 6 harvest) or cell medium (TCM) only. Mixture of vector stock and supernatant was incubated with activated PBMC for 30 min at 4°C. Flow cytometry was performed analyzing content of vector bound T cells by staining with fluorophore coupled αCD3 and αLNGFR antibodies. Dot plots of vector bound T cells are depicted in the right panel. Percentage of vector bound cells is indicated. ΔLNGFR intensity [MFI] of vector bound cells in 3 - 10 independent experiments is shown in the left panel. Individual results and means with SD are plotted. Statistical testing was performed by using 2-way ANOVA.

Figure 5: Antitumoral activity of CAR T cells generated with 62L-LV.

A) Experimental setting. PBMC were activated for two days prior to 24h incubation with 62L-LV, VSV-LV or PBS (control) and injected i.v. into NSG mice. Nalm6 cells were injected at day 4 post adoptive cell transfer and their growth was followed by bioluminescence imaging (BLI).B) Monitoring for tumor load by BLI at the indicated days after adoptive cell transfer. Ventral images of each mouse are depicted.C) Total body flux quantified at the indicated time points for the 62L-LV group (blue), the VSV-LV group (orange) and the control (grey). Mean with standard error (SEM) is plotted. Dotted line represents background signal of mice without imaging substrate. Ordinary 1-way ANOVA was used to determine statistics. D) Cells isolated from blood and organs of each mouse were analyzed by flow cytometry for viable, CD45 negative, CD19 and EBFP double-positive Nalm6 cells. The percentage of Nalm6 positive cells of all viable cells is depicted. Individual results and means with standard error are plotted.

Figure 6: Expression of ΔLNGFR+ in human CD4+ or CD8+ T cells

A) Presence of ΔLNGFR+ cells on human CD4+ or CD8+ T cells in blood determined by flow cytometry on day 3, 10 and 17 of the experiment. Gating on viable human CD45+, CD3+ and respective lineage marker positive cells was performed. Only data with at least 20 events in CD4+ or CD8+ T cell gate are shown. Mean with standard error (SEM) are depicted. B-E)Presence of human CD45+ cells (left), ΔLNGFR+ human CD4+ (middle) or ΔLNGFR+ human CD8+T cells (right) in spleen (B ), blood (C ), bone marrow (D ) and liver (E ) as determined by flow cytometry at final analysis. Individual results and mean with standard deviation (SD) are depicted. Unpaired t-tests were performed to determine statistics.