Efficient derivation of chimeric-antigen receptor-modified TSCM cells

Abstract

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (T_SCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-T_SCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified T_SCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring T_SCM phenotypes and oligopotent capabilities. These in-vitro expanded T_SCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-T_SCM cells may facilitate improved adoptive immunotherapy.

Keywords: CAR; HIV-1; TSCM; adoptive immunotherapy; gene therapy.

Figure 1

Derivation of gene-marked CD8+ T cells harboring T_SCM-surface phenotype under different stimulation conditions. Freshly isolated human PBMCs were separated for CD8+ T_N cells using an EasySep™ Human Naïve CD8+ T Cell Enrichment kit. Cells were stimulated for 2 days with the condition as in (A), followed by transduction with lentiviral vector encoding EGFP as a transduction marker. Cells were cultured for an additional 12 days in the presence of 5 ng/mL of IL-7 and IL-15, and cell-surface marker profiles were analyzed by flow cytometry. (A) Summary for derivation of T_SCM cells procedure. (B) Flow cytometry analyses of CD8+ T_N cells genetically marked by EGFP with no antibody stimulation. (C) % positivity of EGFP-marked cells (Top bars) and fold changes in cell number following 14 days of culture (Bottom bars). (D) Flow cytometry analyses of EGFP-engineered CD8+ T_N cells. All experiments were repeated at least three times. Error bars in (C) show the standard deviation of a data set. One representative experiment is shown for (B, D).

Figure 2

Prolonged culture increases % population of CD8+ T cells harboring T_SCM-surface phenotype with maintaining oligopotency Freshly isolated CD8+ T_N cells were transduced with lentiviral vector encoding EGFP following stimulation with various antibody conditions shown in Figure 1A . The cells were cultured for an additional 12 days (A, Day14) or 26 days (B, Day 28) in the presence of 5 ng/mL of IL-7 and IL-15. A half million of cells in A was co-stimulated by 0.5 μg/mL anti-CD3 and 2.0 μg/mL anti-CD28 antibodies and further cultures for 14 days (C, 2^nd stimulation). Cells were staining for CD45RA, CD45RO, CCR7, CD62L, CD95 and CD122, and surface marker of EGFP-marked cells were analyzed by flow cytometry. Each cell number of EGFP-marked cells with T_N, T_SCM, T_CM, T_EM, and T_EMRA phenotypes was plotted. Experiments were repeated three times. Error bars show the standard deviation of a data set.

Figure 3

Induced differentiation of Triple-CD4ζ modified CD8+ T cells harboring T_SCM-surface phenotype with anti-CD3 and CD28 co-stimulation. (A) Triple-CD4ζ or EGFP-modified CD8+ T cells were co-stimulated with 0.5 µg/mL of anti-CD3 and 2.0 µg/mL of CD28 antibodies for 2 days (2^nd stimulation) at day14 post-1^st stimulation. The cells were further cultured for an additional 12 days in the presence of 5 ng/mL of IL-7 and IL-15. Cell-surface profiles of gene-marked CD8+ T cells were analyzed by flow cytometry. Each cell number of EGFP- or Triple-CD4ζ-marked cells with T_N, T_SCM, T_CM, T_EM, and T_EMRA phenotypes was plotted with or without 2^nd stimulation. Experiments were repeated three times. Error bars show the standard deviation of a data set. (B) The cells were plated at 5 x 10⁴ cells/100 μL in a 96-well plate. The same number of TagBFP-labeled Jurkat cells (ΔKS, non-target control) and mCherry-labeled Jurkat cells constitutively expressing HIV-1_HXBC2 envelope protein (HXBC2, target cells) were added to the wells and incubated for 4 or 16 hours. Total numbers of each population were determined by MACSQuant, and relative cytotoxicity of target cells relative to non-target cells was determined. -: unstimulated, +: 2^nd stimulated. Experiments were repeated three times. Error bars show the standard deviation of a data set. Cytotoxicity assays were performed in biological triplicate.

Figure 4

Triple-CD4ζ CAR-modified CD8+ T cells harboring T_SCM-surface phenotype eliminate tumor cells expressing HIV-1 envelope proteins in a xenograft mouse model. Two million mStrawberry-labeled CD19+ TF228.1.16 cells expressing envelope protein from HIV-1_BH10 or mCherry-labeled Jurkat cells expressing envelope protein from HIV-1_HXBC2 (HXBC2), were mixed with Matrigel at a 1:1 ratio and subcutaneously engrafted to the left or right hind limbs of NOD-SCID mice from ventral side, respectively (n = 4). Freshly isolated CD8+ T_N cells were stimulated for 2 days with 0.5 µg/mL of anti-CD3 antibody in T-cell medium, followed by transduction with a lentiviral vector encoding either Triple-CD4ζ or FMC63-IgG4ζ. Following 26 days of culture in the presence of 5 ng/mL of IL-7 and IL-15, cells corresponding to 5 x 10⁵ CAR-modified cells were intravenously injected via the retro-orbital vein on day 14 post-engraftment of TF228.1.16 and HXBC2. Biofluorescence images (A) and the weight of xenograft tumors (B) were obtained on day 42 post-engraftment (on day 28 post-transplant of CAR cells). (B) Blue or orange bars: average weight of xenograft tumors from TF228.1.16 (left limb, blue bars) or HXBC2 (right limb, orange bars) on day 42 post-engraftment. Experiments were repeated three times with similar results. Two representative animals from each group were shown.