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supplemental Figure 1 A T cell T1 anti-ny-eso-117-16/hla-a*:1 CDζ CH/CH scfv B T cell T1 anti-ny-eso-117-16/hla-a*:1 CDζ CH/CH scfv C T cell BW1/6 anti-cea CDζ CH/CH scfv

supplemental Figure 1.79.9.87 8.67.16 67.9 9.1 1.7 TEFF TEFF 1.19 1.1..6 6.8. 1.79.17 7.6.6.. 1.89 7.9 CDRA higg CDRA 67.8 19. 1.6 1.71 6.16 1.6 CD7 CCR7 CD7.66 7. 7.19 8.8 1. 9.86 8.6 1.16 late TEM early TEM TCM.88 16.6 8.9.7 1.19.96 9. 11.11

supplemental Figure A B anti-ny-eso-1-t1-/cdζ anti-ny-eso-1-t1-cdζ CCR7+CD-CD7+-+ CCR7+CD-CD7+-- CCR7+CD-CD7+++ CCR7+CD-CD7++- CCR7+CD-CD7--+ CCR7+CD-CD7--- CCR7+CD-CD7-++ CCR7+CD-CD7-+- CCR7+CD+CD7+-+ CCR7+CD+CD7+-- CCR7+CD+CD7+++ CCR7+CD+CD7++- CCR7+CD+CD7--+ CCR7+CD+CD7--- T CM CCR7+CD+CD7-++ CCR7+CD+CD7-+- CCR7-CD-CD7+-+ CCR7+CD-CD7+-+ CCR7+CD-CD7+-- CCR7+CD-CD7+++ CCR7+CD-CD7++- CCR7+CD-CD7--+ CCR7+CD-CD7--- T CM CCR7+CD-CD7-++ CCR7+CD-CD7-+- CCR7+CD+CD7+-+ CCR7+CD+CD7+-- CCR7+CD+CD7+++ CCR7+CD+CD7++- CCR7+CD+CD7--+ CCR7+CD+CD7--- T CMRA CCR7+CD+CD7-++ CCR7+CD+CD7-+- CCR7-CD-CD7+-+ T RVE CCR7-CD-CD7+-- CCR7-CD-CD7+++ T RVE CCR7-CD-CD7++- CCR7-CD-CD7--+ CCR7-CD-CD7--- early T EM CCR7-CD-CD7-++ late T EM CCR7-CD-CD7-+- CCR7-CD+CD7+-+ T eff /T RVE CCR7-CD+CD7+-- CCR7-CD+CD7+++ T eff /T RVE CCR7-CD+CD7++- CCR7-CD+CD7--+ CCR7-CD+CD7--- T EMRA CCR7-CD+CD7-++ T EMRA CCR7-CD+CD7-+- T CMRA CCR7-CD-CD7+-- CCR7-CD-CD7+++ T RVE CCR7-CD-CD7-+- CCR7-CD+CD7+-+ CCR7-CD-CD7++- CCR7-CD-CD7--+ CCR7-CD-CD7--- T RVE CCR7-CD-CD7-++ early T EM late T EM CCR7-CD+CD7+-- CCR7-CD+CD7+++ T eff /T RVE CCR7-CD+CD7-++ CCR7-CD+CD7-+- CCR7-CD+CD7++- CCR7-CD+CD7--+ CCR7-CD+CD7--- T eff /T RVE T EMRA T EMRA % of non-transduced CD8+ T cells % of transduced CD8+ T cells

****** ** supplemental Figure T CM CCR7+CD-CD7-++ T CMRA CCR7+CD+CD7-++ T RVE T RVE T eff /T RVE CCR7-CD+CD7++- CCR7-CD+CD7-++ early T EM CCR7-CD-CD7-++ late T EM CCR7-CD-CD7-+- T eff /T RVE CCR7-CD+CD7+-- T EMRA CCR7-CD+CD7-+- T EMRA CCR7-CD-CD7++- CCR7-CD-CD7+-- *** *** 1 MFI of CD8+ anti-ny-eso-1-t1-/cdζ

supplemental Figure A 8 T-1B LST-17 B 6 % lysis 6 IFNγ (pg/ml).:1.:1 1:1 :1 T-1B LS-17T ratio (anti-cea-bw-/cdζ : target) re-directed anti-cea-bw-/cdζ : target (:1)

supplemental Figure 6 A B x 6 6 x 6 6x human IgE (ng/ml) 1 6 8 days after intravenious injection of U66 C D 6. 86.7 counts.7 1.. 89.9.8.8 9.8. human IgG HLA class I 6. CCR7.6 6.6.1 91.9 1.

Supplementary Figures Figure S1: Schematic presentation of re-directed T cells including the different CARs: A: Re-directed T cells recognizing NY-ESO-1 17-16 peptide without co-stimulatory domain (anti-ny-eso-1-t1-cdζ); B: Re-directed T cells recognizing NY-ESO-1 17-16 peptide with co-stimulatory moiety (anti-ny-eso-1-t1-/cdζ); C: Re-directed T cells recognizing CEA with co-stimulatory domain (anti-cea-bw-/cdζ ). Figure S: Representative phenotypical characterization of CD8 + T cells expressing the anti-ny- ESO-1-T1-/CDζ CAR by multi-parametric flow cytometry. Pre-gated CCR7 + or CCR7 - / anti-ny-eso-1-t1-/cdζ re-directed T cells were arrayed in a CDRA vs. CD7 - parameter dot-plot. Cells in each of the four quadrants were then further subdivided by gating through / dot-plots. Figure S: Comparison of non-transduced (A) and transduced (B) subpopulations of anti-ny- ESO-1- CD8 + T cells. No statistically significant differences were detected between the subpopulations of CAR - and CAR + T cells (11 individual donors). T CM = central memory T cell, T EM = effector memory T cells, T EFF = effector T cells, T EMRA = effector memory T cell reexpressing CDRA, T eff = effector T cells. Figure S: Comparison of the anti-ny-eso-1-t1-/cdζ expression in different subpopulations of re-directed T cells. There was no statistically significant difference in the anti- NY-ESO-1-T1-/CDζ expression between central- and effector memory re-directed CD8+ T cells (11 individual donors). Figure S: Representative experiment showing antigen-specific cytolysis and IFN release of anti-cea-bw-/cdζ re-directed CD8 + T cells. Anti-CEA-BW-/CDζ re-directed T cells were cultured with stable-transfected T cells expressing HLA-A*:1 restricted NY- ESO-1 peptides 17-16 (T-1B) and the tumor cell line LS-17T which expresses CEA at different effector to target ratios. Figure S6: Xenograft model of intravenously injected U66 HLA-A*:1/NY-ESO-1 positive myeloma cells ( =* 7 cells, = 7 cells =6* 6 cells). After injection of different doses of U66 cells sera concentrations of secreted human IgE were measured by ELISA (A). 6 days after injection U66 cells accumulated in the bone marrow (B). HLA class I positive cells were analysed by flow cytometry indicating the presence of U66 cells in the bone marrow (C). Anti-

NY-ESO-1-T1-/CDζ re-directed CD8 + T cells were found up to days after transfer in the peripheral blood of % of mice (representative dot plots of four individual mice, D).

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