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. 2016 Jul 28;128(4):519-28.
doi: 10.1182/blood-2015-11-683847. Epub 2016 May 25.

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies

Marianna Sabatino  1 , Jinhui Hu  2 , Michele Sommariva  3 , Sanjivan Gautam  2 , Vicki Fellowes  2 , James D Hocker  2 , Sean Dougherty  2 , Haiying Qin  4 , Christopher A Klebanoff  2 , Terry J Fry  4 , Ronald E Gress  2 , James N Kochenderfer  2 , David F Stroncek  1 , Yun Ji  2 , Luca Gattinoni  2
Affiliations

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies

Marianna Sabatino et al. Blood. .

Abstract

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8(+)CD62L(+)CD45RA(+) naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3β inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR-modified CD8(+) TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8(+) T cells generated with clinical protocols currently under investigation, CD19-CAR-modified CD8(+) TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR-modified CD8(+) TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

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Figures

Figure 1
Figure 1
Enrichment of naive CD8+ T cells by Fab-streptamer technology. (A) Flow cytometry analyses of fresh human PBMCs from a healthy donor (HD) prior to and after sequential enrichment of CD8+, CD62L+, and CD45RA+ cells with Fab multimers conjugated with Strep-Tactin–functionalized magnetic beads. Living lymphocytes in the respective positive and negative fractions of each selection step are shown. Data are shown after gating on live cells. Numbers indicate the percentage of cells in each gate. (B) Percentage of CD8+CD62L+CD45RA+ T cells prior to and after each selection step from 6 HDs; mean value ± standard error of the mean (SEM) is indicated. (C) Percentage yields of the target CD8+CD62L+CD45RA+ T cells from 6 HD; mean value ± SEM is indicated.
Figure 2
Figure 2
CD8+ T-cell subset composition of CD19-CAR–modified standard and TSCM-enriched products. (A) Flow cytometry analyses of cells from a representative healthy donor (HD) at different steps of the 9-day manufacturing processes for the generation of CD19-CAR–modified standard and TSCM-enriched products. Data are shown after gating on live cells (left panels) or live CD3+ T cells (right panels). Numbers indicate the percentage of cells in each gate. (B) Fold expansion of CD19-CAR–modified T cells generated under standard and TSCM-enriched culture conditions. Data represent results from 6 HD; mean value ± SEM is indicated (*P < .05; Wilcoxon matched-pairs signed rank test). (C) Percentage of CD19-CAR–transduced T cells in standard and TSCM-enriched products. Data represent results from 6 HDs; mean value ± SEM is indicated. (D) Percentage of CD8+ T-cell subsets in the CD19-CAR–modified standard and TSCM-enriched products. TSCM were defined as CD3+CD8+CD45RO−CCR7+CD45RA+CD62L+CD27+CD95+ cells; TCM as CD3+CD8+CCR7+CD45RO+; TEM as CD3+CD8+CCR7−CD45RO+; TTE as CD3+CD8+CCR7−CD45RO−; other cells as CD3+CD8+CCR7+CD45RO− not displaying the full TSCM phenotype. Data representing results from 6 HDs are shown as box-and-whisker plots extending to minimum and maximum values. Bands inside the box represent median values. (E) Theoretical number of CD19-CAR+CD8+ TSCM obtainable from stimulation of 1 × 108 PBMCs (standard products) or Fab-streptamer–enriched CD8+ TN (TSCM-enriched products). Data represent results from 6 HDs (*P < .05; Wilcoxon matched-pairs signed rank test).
Figure 3
Figure 3
Effector CD8+ T cells generated under TSCM-enriched culture condition are polyfunctional. (A) Concentration of cytokine in the supernatant of CD19-CAR–modified T cells after 16-hour coculture with CD19+ SUDHL4 cells or CD19− CCRF-CEM cells. Values are shown after subtraction of background cytokine release values obtained after coculture with the CCRF-CEM. Data representing results from 6 healthy volunteer donors (HDs) are shown as box-and-whisker plots extending to minimum and maximum values. Bands inside the box represent median values (*P < .05; Wilcoxon matched-pairs signed rank test). (B) Intracellular cytokine staining of CD19-CAR–modified standard and TSCM-enriched products from a representative HD after coculture with CD19+ SUDHL4 cells. Data are shown after gating on live CD3+CD8+ cells. Numbers indicate the percentage of cells in each quadrant. (C) Mean fluorescence intensity of cytokines produced by CCR7− effector CD8+ T cells within standard and TSCM-enriched products after coculture with CD19+ SUDHL4 cells. Data representing results from 6 HDs are shown as box-and-whisker plots extending to minimum and maximum values. Bands inside the box represent median values (*P < .05; Wilcoxon matched-pairs signed rank test). (D) Intracellular cytokine staining of CD19-CAR–modified standard and TSCM-enriched products from a representative HD after coculture with CD19+ SUDHL4 cells. Data are shown after gating on live CD3+CD8+CCR7− cells. Numbers indicate the percentage of cells in each quadrant. (E) Pie charts depicting the quality of the cytokine response in CCR7− effector CD8+ T cells from 6 HDs after coculture with CD19+ SUDHL4 cells. Values are determined by the Boolean combination of gates identifying IFN-γ+, IL-2+, TNF-α+, and CD107a+ cells. Numbers indicate cell percentages. (F) Percentage of polyfunctional CCR7− effector CD8+ T cells from 6 HDs after coculture with CD19+ SUDHL4 cells (*P < .05; Wilcoxon matched-pairs signed rank test).
Figure 4
Figure 4
CD19-CAR–modified TSCM have a transcriptome profile similar to their naturally occurring counterpart. (A) Hierarchical clustering of CD19-CAR–modified standard and TSCM-enriched products and naturally occurring CD8+ T-cell subsets from GSE23321 performed using a 900 gene list from Gattinoni et al. Red and blue colors indicate increased and decreased expression of 900 differentially regulated genes described by Gattinoni et al. Each column represents a sample and each row, a gene. (B) Principal component analysis (PCA) of the 900 differentially expressed genes described by Gattinoni et al in CD19-CAR–modified standard and TSCM-enriched products and naturally occurring CD8+ T-cell subsets from Gattinoni et al. (C). Gene set enrichment analysis (GSEA) on transcriptomes of CD19-CAR–modified standard and TSCM-enriched products using genes upregulated or downregulated in TSCM relative to TEM retrieved from Gattinoni et al as gene sets. (D) Schematic representation of the glycolytic pathway. In blue font, glycolytic enzymes and molecules whose genes were downregulated in CD19-CAR–modified TSCM-enriched cells compared with standard products (P < .05). In gray font, glycolytic enzymes not differentially expressed. DHAP, dihydroxyacetone phosphate; S/N, signal/noise.
Figure 5
Figure 5
CD19-CAR–modified TSCM exhibit enhanced metabolic fitness. (A) ECAR of CD19-CAR–modified standard and TSCM-enriched products from a representative healthy donor (HD) under basal culture conditions and in response to the indicated molecules. Data are shown as mean values ± SEM. (B) Glycolytic response by CD19-CAR–modified standard and TSCM-enriched products of 6 HDs following glucose administration. ECAR values are shown after subtraction of basal ECAR measurements (*P < .05; Wilcoxon matched-pairs signed rank test). (C) OCR of CD19-CAR–modified standard and TSCM-enriched products from a representative HD under basal culture conditions and in response to the indicated molecules. Data are shown as mean values ± SEM. (D) SRC (maximal OCR − basal OCR) of CD19-CAR–modified standard and TSCM-enriched products of 6 HD. 2-DG, 2-Deoxyglucose.
Figure 6
Figure 6
CD19-CAR–modified TSCM mediate long-lasting antitumor responses. (A) In vivo bioluminescent imaging, (B) radiance, and (C) survival of NSG mice bearing systemic NALM6-GL leukemia xenografts after adoptive transfer of CD19-CAR–modified standard or TSCM-enriched CD8+ T cells (2.5 × 105) in conjunction with intraperitoneal injections of recombinant human IL-15 every other day (****P < .0001; log-rank [Mantel-Cox]; n = 5). Data shown are representative of 2 independent experiments. d, day.
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