PMC

(a) CD4⁺ and CD8⁺ T cells expressing each of the CD19 CARs were sorted for EGFRt expression and stimulated with anti-Strep-tag II or anti-Strep-tag II/CD28 mAb–coated microbeads. After 48 h of stimulation, expression of the CD25 activation marker was determined by flow cytometry. Unstimulated cells (medium) were used as controls.
(b) Growth curves of Strep-tag CAR-T cells. FACS sorted EGFRt⁺ CD19 CAR-T cells (CD8⁺ and CD4⁺) were cultured with anti-Strep-tag II or anti-Strep-tag II/CD28 mAb coated microbeads in CTL media containing IL-2 (30–50 U/ml) and IL-15 (2 ng/ml) for 9 days. Aliquots of T cells were removed from the cultures for counting on days 3, 6, and 9 and the fold-increase in cell number determined. The data show the mean fold expansion obtained in three experiments with T cells from different donors.
(c) Stimulation of Strep-tag CAR-T cells with anti-Strep-tag II/CD28 beads induces selective outgrowth of transduced cells. CD8⁺ and CD4⁺ T cells were transduced with CD19 1ST/4-1BBζ and 1ST/CD28ζ CARs and 10 days later stimulated with anti-Strep-tag II/CD28 microbeads plus IL-2 or cultured with IL-2 alone for 9 additional days. The percentage of Strep-tag II positive cells at day 0 (before stimulation) and day 9 (after stimulation) were measured by flow cytometry. The results are representative of three experiments.
(d) Anti-tumor activity of CD19 1ST/4-1BBζ or 1ST/CD28ζ CAR-T cells in Raji-ffluc-bearing NSG mice. 2.5×10⁶ CD19 CAR-T cells expanded with anti-CD3/CD28 beads or with anti-Strep-tag II/CD28 microbeads, and control non-transduced T cells were formulated in a CD8:CD4 ratio of 1:1 and infused into cohorts of NSG mice 7 days after inoculation with 0.5×x10⁶ Raji/ffluc tumor cells. Tumor progression and distribution were evaluated by serial bioluminescence imaging.

(a) Enrichment of Strep-tag CAR-T cells containing 1, 2, or 3 Strep-tag II sequences in the spacer region using StrepTactin coated beads on the automated T-CATCH device. Flow cytometric analysis of the frequency of Strep-tag CAR-T cells before and after enrichment using anti-Strep-tag II staining. Data is representative of six experiments using T cells from 3 donors.
(b) Yield of Strep-tag CAR-T cells after T-CATCH enrichment. Yield was determined by the absolute numbers of Strep-tag CAR-T cells in the enriched fraction divided by the absolute numbers of Strep-tag CAR-T cells in the starting population. Data is derived from four experiments and expressed as means ± SD. Statistical analysis was performed using the Student’s t test. *P<0.05.
(c) Experimental scheme for adoptive transfer of Strep-tag CAR-T cells enriched by StrepTactin selection or using EGFR mAb. CD8⁺ and CD4⁺ T cells were stimulated in independent cultures with anti-CD3/CD28 microbeads and transduced with the CD19-3ST/41BBζ CAR. Cultures were established at different times so that T cell administration into tumor bearing mice occurred simultaneously. Anti-CD3/CD28 beads were removed at day 5 in all groups and CAR-T cells were prepared for inoculation into mice either by selection on the T-CATCH at day 8, FACS sorting for EGFRt⁺ cells on day 10, or FACS sorting of EGFRt⁺ cells followed by 8 days of culture on irradiated CD19⁺ LCL cells with IL2 to remove residual bound anti EGFR mAb.
(d) Flow cytometric analysis of CD19 CAR expression using Strep-tag II staining of CD8⁺ and CD4⁺ CAR-T cells before enrichment, after T-CATCH purification, after EGFR mAb sorting, and after EGFR mAb sorting followed by culture.
(e) NSG mice engrafted 7 days earlier with 0.5×10⁶ Raji/ffluc were treated with a total dose of 2.5×10⁶ CAR-T cells selected by T-CATCH, EGFR sorting or EGFR sorting followed by culture and formulated in a CD4:CD8 ratio of 1:1. Tumor progression and distribution were evaluated by serial bioluminescence imaging after injection of luciferin substrate.
(f) Persistence of CD19 CAR-T cells in each cohort of NSG/Raji mice. Flow cytometric analysis of CD4⁺ and CD8⁺ CAR T cells in the peripheral blood of each group of mice after staining with CD45, CD8, CD4 and EGFR Ab at different time points after T cell infusion. The frequency of CAR-T cells is presented as percentage of live peripheral blood cells.
(g) Survival of mice treated with different CAR-T cell products or with non-transduced T cells depicted as Kaplan-Meier curves. The data in d-g are representative of two independent experiments.

(a) Analysis of CD19 CAR expression. Primary human CD8⁺ T cells were transduced with epHIV7 lentiviral vectors encoding a CD19-Hi CAR or CD19 CARs with Strep-tag in various extracellular locations. Each CAR contained a 4-1BB/CD3ζ intracellular signaling domain and EGFRt downstream of a T2A element. Transduced cells were sorted for EGFRt⁺ cells by fluorescence-activated cell sorting (FACS), and purity confirmed by staining with anti-EGFR (grey – top panels). Cell surface expression of the Strep-tag CARs was evaluated by staining with anti-Strep-tag II antibodies (grey - bottom panels). Non-transduced cells (white) served as controls for staining. (b) Cytolytic activity of CD19-Hi and Strep-tag 4-1BBζ CAR-T cells. After sorting for EGFRt expression, CD8⁺ T cells transduced with each of the CARs were tested for lysis of CD19⁺ Raji lymphoma and K562 leukemia transduced with CD19 (K562/CD19) or ROR1 (K562/ROR1) at various effector/target (E:T) ratios.
(c) IFN-γ and IL2 production by CD19-Hi and Strep-tag 4-1BBζ CAR-T cells 24h after stimulation with K562/CD19 and K562/ROR1. PMA/Ionomycin treated T cells were used as a positive control. The data in a–c is representative of three experiments with CD8⁺ T cells from different donors.
(d) Cohorts of NSG mice were inoculated with 0.5×10⁶ firefly luciferase expressing CD19⁺ Raji lymphoma cells (Raji-ffluc) via tail vein injection and treated 7 days after tumor inoculation with 2.5×10⁶ CD19 4-1BBζ CAR-T cells with Hi, 1ST, and 3ST spacers, respectively. CAR-T cells were formulated in a CD8:CD4 ratio of 1:1. Tumor progression and distribution were evaluated by serial bioluminescence imaging.
(e) Tracking CAR-expressing T cells in vivo by staining with anti-Strep-tag II mAb. Blood obtained from mice 8 days after the T cell infusions was stained with anti-human CD45, CD8, CD4, anti-Strep-tag II and anti-EGFR mAbs, and analyzed by flow cytometry. Expression of Strep-tag II and EGFRt on CD45⁺ CD8⁺ and CD45⁺ CD4⁺ T cells is shown.
(f) Kinetics of expansion and contraction of CD19 CAR-T cells in the blood after adoptive transfer to NSG mice bearing Raji tumors. The mean frequency of CD45⁺CD8⁺ EGFRt⁺ and CD45⁺CD4⁺ EGFRt⁺ human T cells in blood of the mice (n=5) of each group at various times after the T cell infusion is shown. The data in d–f are representative of three experiments.
(g) Fold-change in expression of selected cytokine genes in CD19-1ST/4-1BBζ CAR-T cells after infusion to Raji tumor bearing and non-tumor bearing NSG mice. CAR-T cells were sorted 2 days after infusion from blood, bone marrow and spleen after staining with anti-EGFR or anti-Strep-tag mAb. Gene expression was analyzed using a human common cytokine PCR array. The mean fold change values of cytokine genes in the sorted CAR-T cells from NSG/Raji vs sorted CAR-T cells from non-tumor bearing NSG mice were calculated. Samples were run in triplicate and data are presented as the mean fold increase ± SD.
(h) Cytokine production by CD8⁺ T cells expressing CD19-1ST/4-1BBζ CARs 2 days after stimulation with Raji cells in vitro. Supernatants of CAR-T cells co-cultured with CD19⁺ Raji cells (intra-assay triplicates) for 48 h were pooled together and analyzed using the Luminex Multiplex platform to validate the production of cytokines that were upregulated in CAR-T cells in NSG mice bearing Raji tumors. PMA/Ionomycin treated and non-treated T cells were used as positive and negative control, respectively. The results are representative of biological replicates.