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. 2018 Mar 21;9:507. doi: 10.3389/fimmu.2018.00507

Figure 7.

Figure 7

Clinical-grade production of nerve growth factor receptor (NGFR)-spaced CD44v6 chimeric antigen receptor (CAR)-T cells. Primary T cells from healthy donors were stimulated with CD3/CD28-beads and transduced with RVs encoding for CD44v6 CARs spaced with NGFR wild-type long (NWL) or NGFR mutated short (NMS) (see Materials and Methods). (A) FACS plots of CAR-T cells from a representative donor (of n = 3) stained with either the anti-NGFR monoclonal antibody (mAb) C40-1457 (upper panel) or the anti-NGFR mAb ME20.4 (lower panel). NWL-isoform-spaced CAR T cells were enriched with clinical-grade anti-NGFR immuno-magnetic beads while NMS-isoform-spaced CAR T cells were stained with the PE-conjugated anti-NGFR mAb C40-1457 and enriched with anti-PE immuno-magnetic beads. (B) Schematic representation of the retroviral construct expressing the CD44v6 CAR and the thymidine kinase (TK) suicide gene. HSV-TK: Herpes Simplex Virus thymidine kinase suicide gene. CD44v6 CAR: NMS or NWL-isoform-spaced CD44v6 CAR. U3, R, U5: LTR regions. SV: SV40 early promoter. ψ: encapsidation signal. Arrows indicate promoters. (C) Purity (left) and yield (right) of CAR-T cells after enrichment (means ± SEM from n = 3 donors). Yield was determined by the absolute numbers of NGFR-spaced CAR-T cells in the enriched fraction divided by the absolute numbers of NGFR-spaced CAR-T cells in the starting population. (D) Memory phenotypes of NGFR isoform-enriched CD44v6 CAR-T cells analyzed at day 10–13 after bead stimulation (means ± SEM from n = 3 donors). TSCM, CD45RA+/CD62L+ stem memory T cells; TCM, CD45RA−/CD62L+ central memory T cells; TEM, CD45RA−/CD62L− effector memory T cells; TEMRA, CD45RA+/CD62L− effector memory RA T cells. (E) NSG mice were xeno-engrafted with THP-1 leukemic cells and, after 14 days, treated with CD44v6 CAR-T cells enriched through either NWL (n = 4) or NMS (n = 3) or with T cells expressing a control, CD19-specific CAR (CTRL, n = 5). Forty days later, mice were sacrificed and their liver weighted. THP-1 infiltrated liver weights are shown for the different cohorts (each symbol represents a single mouse and the median value for each group is reported). The dashed area depicts the range of normal liver weight from age/sex-matched normal NSG mice. A representative experiment (of n = 3) is shown. Results from a one-way ANOVA test are indicated when statistically significant (*P ≤ 0.05; **P ≤ 0.01). (F) Growth suppression of CD44v6 suicidal CAR T cells analyzed after activation with PHA and treatment with increasing concentrations of GCV (means ± SEM from a representative donor). T cells transduced with the original vector expressing the tk suicide gene and the selection marker ΔNGFR were used as comparison.