The data in dCf are representative of three experiments. (g) Fold-change in expression of selected cytokine genes in CD19-1ST/4-1BB CAR-T cells after infusion to Raji tumor bearing and non-tumor bearing NSG mice. were inserted on one or both sides of the Strep-tag II and are indicated by the zigzag line.(b,c) Incorporating Strep-tag II into a TCR does not impair surface expression or tetramer binding. CD8+ T cells were transduced with either an unmodified NY-BR-1-specific TCR (NBT1) or a Strep-tag II version of NBT1 (NBT1-Strep-tag), and analyzed for surface expression of the TCR V22 chain of NBT1 and Strep-tag II. (c) Transduced T cells were enriched for V22+ cells, expanded, and analyzed for NY-BR-1-multimer binding. Data are representative of two independent experiments. (d,e,f) Strep-tag II TCRs are functional. T cells transduced with the wild-type NBT1 TCR and with the NBT1 strep-tag TCR were tested for cytotoxicity (d), cytokine production (e), and proliferation against T2 cells alone or loaded with NY-BR-1 peptide. Untransduced CD8+ T cells were used as a control for cytotoxicity and cytokine assays. The dose of NY-BR-1 peptide in (e) and (f) is 0.1 ng/ml. Data are representative of three independent experiments and error bars represent mean SD. NIHMS745560-supplement-1.jpg (507K) GUID:?61439063-EDE9-429F-9F50-AE0E43E4F2EC 2: Supplementary Figure 2. Altering spacer length with Strep-tag II can optimize CAR function (a) Comparison of cytokine production by CD8+ T cells expressing CD19 4-1BB CARs with various IgG4 Fc spacer lengths (Hinge only: Hi; Hinge-CH3: Hi-CH3; Hinge-CH2-CH3: Hi-CH2-CH3). CAR-T cells were co-cultured with CD19+ Raji cells (2:1 ratio) for 24h and supernatants were analyzed using the Luminex Multiplex platform. The data are derived from three independent experiments using T cells from different donors. Data are expressed as means SD and normalized such that the mean cytokine release by T cells expressing the CD19-CAR Hi-CH2-CH3 spacer is designated as 1. Statistical analysis was performed using the Students t test. * P<0.05(b, c) Cytokine production by CD8+ T cells expressing CD19 4-1BB (b) or CD28 CARs (c) encoding 1, 2, or 3 Strep-tag II sequences in the spacer domain compared to T cells expressing the identical CD19 CARs with an IgG4 hinge (Hi) only spacer domain. The assays were performed as described in (a). Data from three independent experiments are expressed as mean SD normalized to the cytokine release by T cells expressing the CD19-Hi CAR. Statistical analysis was performed using the Students t test. * P<0.05 (d) Proliferation of T cells expressing CD19Hi-4-1BB, CD19 Hi-CD28 or CD19 Strep-tag CARs with 4-1BB and CD28 intracellular signaling domains. T cells were labeled with CFSE, stimulated with CD19+ Raji tumor cells (solid grey) or medium only (white), and analyzed for CFSE dilution 5 days after stimulation. ata are representative of three independent experiments NIHMS745560-supplement-2.jpg (303K) GUID:?DAD9A777-1B78-4AF2-9F04-062FFDC45A4C 3: Supplementary Figure 3. Detection of Strep-tag CAR-T cells in human blood (a) Anti-Strep-tag II mAb and anti EGFR staining of human peripheral blood mononuclear cells. Mononuclear cells isolated from human blood by Ficoll gradient centrifugation were stained with anti-CD3, anti-EGFR, anti-Strep-tag II mAbs and analyzed by flow cytometry. The data shows staining of CD3+ and CD3? populations with the respective mAbs.(b) 2105 or 2104 CD19 CAR-T cells containing Hi only, 1ST and 3ST spacers were spiked into 2106 PBMCs, which were then stained with anti-CD3 and anti- EGFR (top panels) or anti-Strep-tag II (bottom panels) mAbs and analyzed by flow cytometry. (c) 2105 or 2104 CD19 CAR-T cells containing Hi only, 1ST and 3ST spacers were spiked into 200 l human blood. The blood was then lysed using red cell lysis buffer and the cells were stained and analyzed as described in (b). NIHMS745560-supplement-3.jpg (610K) GUID:?92126FDD-86A4-40FA-9847-7EA4C2E12507 4: Supplementary Figure 4. Anti-Strep tag II mAb coated microbeads selectively activate and expand Strep-tag CAR-T cells independent of scFv specificity and co-stimulatory domain (a) T cells transduced with CD19-Hi, CD19-1ST, CD19-2ST, CD19-3ST CARs containing a CD28 signaling domain were sorted for EGFRt expression, stimulated with anti-Strep-tag II or anti-Strep-tag II/CD28 mAb coated microbeads or left unstimulated SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 (medium) and analyzed for CD25 expression.(b) T cells transduced with R12-Hi, R12-1ST, R12-2ST, R12-3ST CARs containing a 4-1BB signaling domain were SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 sorted for EGFRt expression, stimulated with anti-Strep-tag II or anti-Strep-tag II/CD28 mAb coated microbeads, Flt1 or left unstimulated (medium) and analyzed for CD25 expression. The data in a,b are representative of three experiments. (c) Growth curves of CD8+ Strep-tag CAR-T cells. FACS sorted EGFR+ CD19-1ST/CD28 and R12-1ST/4-1BB CAR-T cells were cultured with anti-Strep-tag II or anti-Strep-tag II/CD28 mAb coated microbeads in CTL media containing IL2 (50 U/ml) and IL15 (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 SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 the mean fold expansion in three experiments with T cells from different donors. NIHMS745560-supplement-4.jpg (266K) GUID:?C380B6FE-14C5-43AF-91EC-8389C05B4C28 5: Supplementary Figure 5..