CD62L as target receptor for specific gene delivery into less differentiated human T lymphocytes

Chimeric antigen receptor (CAR)-expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype compositions. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe in this study a novel receptor-targeted lentiviral vector (LV) named 62L-LV that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4+ and 50% of CD8+ primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered the binding of 62L-LV particles to T cells nor impacted their transduction. The incubation of 2 days of activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in a leukemia tumor mouse model. The data proved that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selections of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.


Suppl. Table 2:
Plasmid compositions for lentiviral vector production in suspension cells.
A) Parental HT1080 cells and HT1080 cells genetically modified to express human CD62L (HT1080CD62L) were stained with a VioBlue labeled αCD62L antibody (blue dotted line) and analyzed by flow cytometry.Unstained cells served as control (grey).B) The indicated GFP-encoding vector stocks were produced in HEK293T producer cells in 12-well format as described before by (Hartmann  et al., 2018).100 µL supernatant containing vector particles was used for transduction of the indicated target (HT1080CD62L or HT1080αHis) and non-target cells (HT1080), respectively.4x10 5 cells for transduction had been seeded one day before.Four days post transduction cells were analyzed by flow cytometry for GFP expression (x-axis).One representative data set is shown.
A/B) PBMC from three different donors were isolated and activated before they were stained with a CD3-specific antibody coupled to FITC (clone BW264/56, Miltenyi Biotec) as well as a CD62L-specific antibody (clone 145/15, Miltenyi Biotec) coupled to PE-Vio770 (donor 1) or VioBlue (donor 2 and donor 3).Percentages of CD3-positive cells (B) as well as CD62L-positive cells among CD3-positive cells (A) are reported.Gating was done according to Suppl.Fig. 15.C) PBMC from three different donors were isolated and activated.Cells were analyzed for CD3, CD19 and CD14 expression at the day of isolation (before activation) as well as two and three days post activation (dpa) by flow cytometry.Percentages of stained cells are shown.Individual results as well as means with SD are plotted.Gating was done according to Suppl.Fig. 15 with the exception that singlets were not only gated for CD3+ but also for CD19+ and CD14+ cells, respectively.
Suppl.Suppl.Suppl. Figure 9: Gene transfer activity of 62L-LV in the presence of sCD62L.
PBMC were activated and cultivated in TCM for two days before 62L-LV was added in presence of Vectofusin-1.Directly before transduction 4x10 4 PBMC were seeded in medium containing the indicated concentrations of sCD62L obtained from a 6-day PBMC culture.Flow cytometry analysis was performed 5 days after transduction.Percentages of transgene positive T cells (solid line) and the MFI for transgene expression (dotted line) are indicated.
Suppl.The consecutive gatings are illustrated by the blue arrows on representative dot plots of PBMC from one donor 5 days post-transduction with 62L-LV without Vectofusin.The cell population was gated for viability, total cells (PBMC), twice for singlets and CD3 + cells.From the CD3 + gate, CD8 + and CD4 + cells were gated.From the CD3 + , CD8 + and CD4 + gates, ΔLNGFR-positive cells were further gated, respectively (used for CAR expression in PBMC mediated by 62L-LV or VSV-LV as depicted in Figure 2A and Suppl.Figure 4).For phenotyping as depicted in Figure 2 and Suppl.Figure 7, ΔLNGFRpositive cells were further gated for CD45RA vs. CD62L, respectively.To discriminate between Tn and Tscm cells, CD45RO was gated.

Figure 4 :
Comparison of gene transfer rates into PBMC by 62L-LV and VSV-LV.4x10 4 activated human PBMC obtained from seven donors were transduced with 5 µl or 10 µl of 62L-LV or 5 µl VSV-LV and then cultivated in presence of IL-2 or IL-7/IL-15 for 9 to 12 days.Use of the transduction enhancer Vectofusin-1 is indicated by +V1.A) Percentages of total CD3-positive cells within the evaluated transduced and untransduced PBMC 9 days post transduction.B-C) Flow cytometry was performed to determine the fractions of ΔLNGFR-positive cells on CD3-positive cells (B) and on CD3-negative cells (C).Individual results from four (C) or seven (B) different donors in two (C) or four (B) independent experiments and means with standard deviation (SD) are plotted.Statistical analysis was performed using unpaired t-test.D) Representative dot plots of 62L-LV and VSV-LV transduced PBMC.Numbers in the individual gates refer to the percentage of ΔLNGFR-positive cells among CD3-negative cells (upper left gates) and CD3-positive cells (upper right gates), respectively.E) Median fluorescent intensity (MFI) of ΔLNGFR-positive cells on CD3-positive (left panel), CD4positive (middle panel) and CD8-positive (right panel) cells.Individual results from five different donors in two independent experiments and means with standard deviation (SD) are plotted.Statistical analysis was performed using unpaired t-test.Suppl.Figure 5: Percentages of CD4 and CD8 cells within untransduced and transduced PBMC at various time points Percentages of CD4+ and CD8+ activated T cells of 4 different healthy donors isolated from buffy coat with or without transduction.Cells were gated according to Suppl.Fig. 15 for the proportion of CD4 and CD8 cells at the indicated time points.A/B) CD4 and CD8 percentage of untransduced cells at the day of transduction (day 0) (A) and over a cultivation period of 12 days after transduction (B).C) Proportion of CD4 (left) and CD8 (right) cells for untransduced, 62L-LV transduced and VSV-LV transduced PBMC analyzed 5, 9 and 12 days post-transduction.Suppl.Figure 6: Monitoring of cultivated PBMC transduced with 62L-or VSV-LV.Characterization of activated PBMC incubated with 62L-LV or VSV-LV in the absence of the transduction enhancer Vectofusin-1 (V1) as depicted in Figure 2B.The respective gating strategy can be found in Suppl.Fig. 15.A) Representative dot plots of PBMC from one donor 5 days posttransduction with 62L-LV (left) or VSV-LV (right) gated for ΔLNGFR vs. CD62L cells among CD8 + (top) or CD4 + cells (bottom).B) The total percentage of CD62L+ cells for the CD4 + (left) and CD8 + (right) fractions over time.C) The total percentage of ΔLNGFR+ cells for the CD4 + (left) and CD8 + (right) fractions over time.Statistics and replica are as described for Figure 2B (B-C).0007 (***) <0.0001 (****) <0.0001 (****) <0.0001 (****) <0.0001 (****) <0.0001 (****

Figure 7 :
Changes in the differentiation status of CAR+ T cells upon cultivation.Differentiation status of CD4+ and CD8+ cells expressing ΔLNGFR for the indicated time points.Data belong to the experiment described in Figure 2B.Cells from three different donors transduced with either vector in two individual experiments were analyzed.Mean with standard error (SEM) is plotted.Statistical testing was performed using 2-way ANOVA with Tukey-multiple comparison.A) Fractions of Tn, Tscm, Tcm, Tem and Teff among CD4+/ΔLNGFR+ and CD8+/ΔLNGFR+ cells for all analysis time points for 62L-LV (left) and VSV-LV (right).B/C) Comparison of CD4+/ΔLNGFR+ and CD8+/ΔLNGFR+ cells for each vector (B) as well as between both vector groups (C) at day 12. D/C) Individual p values of statistical testing at the indicated time points.*p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns >0.9999, not significant.The respective gating strategy can be found in Suppl.Fig. 15.Suppl.Figure 8: Staining capacity of CD62L and CD45 antibodies.A/B) Gating strategy for antibody binding (A) and vector binding (B) as depicted in Figure 3 and Suppl.Figure 8C.The cell population is subsequently gated for total cells (PBMC), for singlets, viability, and CD3 + cells.From the CD3 + cell gate cells were gated for CD62L and CD45 antibody binding, respectively (A) or analyzed for ΔLNGFR expression (B).C) Activated PBMC were incubated either with the parental CD62L-specific antibody clone 145/15 (blue bars) or with a CD45-specific control antibody (clone 5B1, grey bars) at indicated concentrations.Both antibodies were fluorophore-labelled to allow concentration dependent detection of CD62L and CD45.Percentages of stained cells are shown.

Figure 10 :
In vitro cytotoxicity assay.A) Phenotype of CAR T cells 3 days after transduction with VSV-LV and 62L-LV, determined by CD45RA and CCR7 expression.B) CAR-specific tumor lysis of Nalm6 tumor cells.Effector and target cells were co-cultivated for 24 h at a ratio of 0.2:1.Non-transduced cells (NT) were used as control.C/D) CCR7 expression on all T cells, ΔLNGFR+ or ΔLNGFR-T cells before (C) and after (D) tumor cell killing.Individual results as well as means with standard deviation with 6 donors (A/B) and with 3 donors (C/D) measured in technical triplicates are shown, respectively.Statistical testing was performed by two-way ANOVA with Šídák's multiple comparison test (A/C/D) or with RM one-way ANOVA with Dunnett's multiple comparison (B).No significant differences between 62L-LV and VSV-LV were observed in (C) and (D).n.d (not determined); ns (not significant).Suppl.Figure 11: Characterization of injected cells.The composition of the infused cell-vector-mixtures was analyzed by flow cytometry on the day of infusion (day 1; A/B) and after 2 days of additional culture (day 3, C).Vector bound-cells (A/B) and transduced cells (C) were identified by staining with an αLNGFR antibody via flow cytometry.A) Number of cells injected into individual mice.T cells identified being ΔLNGFR positive or negative or non-T cells being CD3 negative are indicated in different colors.DN = double negative.Percentage of vector bound cells (B) or CAR+ T cells (C) among CD4 + or CD8 + T cells, which were pre-gated for CD3 + cells.Suppl.Figure 12: CAR T cell numbers in blood.Absolute numbers of CD4 + ΔLNGFR+ (left) or CD8 + ΔLNGFR+ (right) cells are shown for each mouse individually at indicated time points.Data belong to the experiment described in Figure 7. Suppl.Figure 13: Human CD4+ and CD8+ T cells in organs.Presence of human CD45+ cells (left), ΔLNGFR+ human CD4+ (middle) or ΔLNGFR+ human CD8+ T cells (right) in spleen (A), bone marrow (B) and liver (C) 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.P values are indicated when below 0.05.Data belong to the experiment described in Figure 7. Suppl.Figure 14: Phenotype and exhaustion status of human LNGFR+ T cells in organs.Frequencies of Teff, Tem, Tcm, Tn of ΔLNGFR+ human CD4+ (left) or ΔLNGFR+ human CD8+ (right) in spleen (A) and bone marrow (B) as determined by flow cytometry at final analysis.Frequencies of non-exhausted ΔLNGFR+ human CD4+ (left) or ΔLNGFR+ human CD8+ (right) in spleen (C) and bone marrow (D) as determined by double negative TIM-3 and LAG-3 expression in flow cytometry at final analysis.Individual results and mean with standard deviation (SD) are depicted.Unpaired t-tests were performed to determine statistics.P values are indicated when below 0.05.Data belong to the experiment described in Figure 7.Suppl.Figure 15: Gating strategy for CAR gene delivery and T cell phenotyping.